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Structure of nucleotide‐binding domain 1 of the cystic fibrosis transmembrane conductance regulator

Structure of nucleotide‐binding domain 1 of the cystic fibrosis transmembrane conductance regulator The EMBO Journal (2004) 23, 282–293 & 2004 European Molecular Biology Organization All Rights Reserved 0261-4189/04 | | THE THE www.embojournal.org EMB EMB EMBO O O JO JOU URN R NAL AL Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator 1, 1 Hal A Lewis *, Sean G Buchanan , Introduction 1 1 Stephen K Burley , Kris Conners , Mark Cystic fibrosis (CF) is the most prevalent lethal, autosomal- 1 2 Dickey , Michael Dorwart , Richard recessive genetic disease among Caucasians. CF patients have 1 1 3 Fowler , Xia Gao , William B Guggino , severely reduced life expectancies, largely because of chronic 4 5 Wayne A Hendrickson , John F Hunt , pulmonary damage. The root cause of CF is in defective cystic 1 1 Margaret C Kearins , Don Lorimer , Peter C fibrosis transmembrane conductance regulator (CFTR) 3 1 Maloney , Kai W Post , Kanagalaghatta R (Riordan et al, 1989). Human CFTR is a 1480 residue, multi- 1 1 Rajashankar , Marc E Rutter , J Michael domain, integral membrane protein that regulates chloride 1 1 Sauder , Stephanie Shriver , Patrick H ion flow across the cell membrane. It is a member of the ATP- 2 2 binding cassette (ABC) transporter superfamily of proteins Thibodeau , Philip J Thomas , Marie 1 1 1 and consists of two membrane-spanning domains (MSDs), Zhang , Xun Zhao and Spencer Emtage two nucleotide-binding domains (NBDs), and a regulatory 1 2 Structural GenomiX Inc., San Diego, CA, USA, Department of region (R) arranged in the order MSD1–NBD1–R–MSD2– Physiology, University of Texas Southwestern Medical Center, Dallas, NBD2 (Figure 1). The NBDs of ABC transporters are typified TX, USA, Department of Physiology, School of Medicine, The Johns Hopkins University, Baltimore, MD, USA, Department of Biochemistry by a consensus ATP-binding region, which encompasses two and Molecular Biophysics, Howard Hughes Medical Institute, Columbia Walker motifs (A and B regions), a highly conserved region University, New York, NY, USA and Department of Biological Sciences, called the signature sequence (LSGGQ), plus other conserved Columbia University, New York, NY, USA functional features identified as the Q- and H-loops, named respectively for glutamine and histidine residues involved in Cystic fibrosis transmembrane conductance regulator ATP recognition and hydrolysis. The most common CF muta- (CFTR) is an ATP-binding cassette (ABC) transporter that tion is the deletion of CFTR phenylalanine 508 (DF508), functions as a chloride channel. Nucleotide-binding do- which is located in NBD1. In total, 70% of CF alleles have main 1 (NBD1), one of two ABC domains in CFTR, also DF508 and 90% of CF patients have at least one copy of this contains sites for the predominant CF-causing mutation deletion. A better understanding of the structure and function and, potentially, for regulatory phosphorylation. We have of NBD1 and the role of Phe508 may accelerate the develop- determined crystal structures for mouse NBD1 in unli- ment of new approaches to the treatment of CF. ganded, ADP- and ATP-bound states, with and without Atomic-level structural information has been obtained for phosphorylation. This NBD1 differs from typical ABC components of several ABC transporter systems. Complete domains in having added regulatory segments, a foreshor- bacterial transmembrane transporter proteins MsbA (Chang tened subdomain interconnection, and an unusual nucleo- and Roth, 2001; Chang, 2003) and BtuCD (Locher et al, 2002) tide conformation. Moreover, isolated NBD1 has have been analyzed at modest resolution, showing similar undetectable ATPase activity and its structure is essen- associations between NBD and MSD domains but markedly tially the same independent of ligand state. Phe508, which different overall architectures. High-resolution X-ray struc- is commonly deleted in CF, is exposed at a putative NBD1- tures have also been determined for several prokaryotic NBDs transmembrane interface. Our results are consistent with (Schmitt and Tampe, 2002), most recently HlyB (Schmitt et al, a CFTR mechanism, whereby channel gating occurs 2003) and GlcV (Verdon et al, 2003), and for one eukaryotic through ATP binding in an NBD1–NBD2 nucleotide sand- NBD, TAP1 (Gaudet and Wiley, 2001). All such NBDs have a wich that forms upon displacement of NBD1 regulatory common fold characterized by two subdomains: one contains segments. an F1-like ATP-binding core plus an ABC-specific antiparallel The EMBO Journal (2004) 23, 282–293. doi:10.1038/ b region and the other an ABC-specific a-helical domain sj.emboj.7600040; Published online 18 December 2003 (Karpowich et al, 2001). The F1-like portion contains the Subject Categories: structural biology; molecular biology of primary determinants of nucleotide binding; the antiparallel disease b portion adds interactions to the base and ribose groups; and Keywords: ABC transporter; CFTR; crystal structure; NBD1 the ABC signature sequence of the a-helical domain from a dimer mate completes productive coordination of the ATP b- and g-phosphate groups of the nucleotide (Figure 1). Typical ABC transporters are thought to function as labile dimers in which coupling of ATP hydrolysis to movements in transmembrane segments drives the translocation of relevant *Corresponding author. Structural GenomiX Inc., 10505 Roselle St., San entities across the membrane. The Rad50 DNA repair Diego, CA 92121, USA. Tel.: þ 1 858 228 1555; Fax: þ 1 858 457 4533; enzyme, a remote homolog of ABC proteins, provided the E-mail: hal_lewis@stromix.com first structural model for the dimer state (Hopfner et al, 2000), and similar dimers form in BtuCD (Locher et al, Received: 23 September 2003; accepted: 25 November 2003; Published online: 18 December 2003 2002) and in a catalytically impaired E171Q variant of 282 The EMBO Journal VOL 23 NO 2 2004 &2004 European Molecular Biology Organization | | Structure of CFTR NBD1 HA Lewis et al solubility of such NBD1 constructs from 10 organisms (human, mouse, baboon, macaque, sheep, rabbit, frog, sal- mon, killifish, and dogfish). The high-level production of soluble proteins (45 mg/l) that are nonaggregated in solu- tion was limited to a narrow globular domain definition (N-terminus: residues 385–391; C-terminus: residues 670– 680). Optimal recombinant protein was obtained from mouse CFTR with an expression construct spanning residues 389–673. Attempts with human NBD1 have not yet suc- ceeded. We refer to the resulting mouse proteins as mNBD1 and mNBD1-P for the unphosphorylated and phosphorylated forms, respectively. Dynamic light scattering (DLS) and ana- lytical gel filtration chromatography (GFC) measurements indicated that purified, recombinant mNBD1 was both mono- disperse and monomeric. This protein was used for all of the structural studies reported here. A mutated version, K464A, which was expected to have reduced ATP binding, was also cloned and purified in the same manner and was used for Figure 1 Domain organization of CFTR. The five domains of CFTR ATP-binding measurements (see below). are shown. Also indicated is a putative nucleotide-binding domain association in which the ATP-binding site of one NBD is opposed by mNBD1 crystallization and structure determination the signature sequence of the other NBD. Inactivity at the NBD1 Both native (S-Met) and selenomethionyl (Se-Met) mNBD1 ATP-binding site is indicated by Ser residues in place of the catalytic Glu and His in addition to His residues substituted for the Gln and crystallized in two morphologies under similar conditions: central Gly in the NBD2 signature sequence. parallelepipeds in space group P42 2 and tetragonal bipyra- mids in space group I4 22. The structure of mNBD1 bound to Mg-AMP.PNP was initially determined by multiple MJ0796 when complexed with ATP (Smith et al, 2002). The isomorphous replacement with anomalous scattering nucleotides in this symmetric dimer are sandwiched at the (MIRAS) phasing from Ta Br -soaked I4 22 crystals. This 6 12 1 interface between protomers such that the LSGGQ residues was subsequently improved using single-wavelength anom- from one complete the binding interactions of nucleotide in alous diffraction (SAD) data from Se-Met mNBD1 P42 2 the apposing protomer. The putative functional NBD ‘dimer’ crystals. Most of the protein sequence could be built into in CFTR is believed to be intramolecular and necessarily the SAD-generated experimental electron density map, the asymmetric since ‘hemichannel’ constructs produced wild- primary exception being residues 413–428, which were not type activity when expressed together but not separately well visualized (exact boundaries are given in Table I for each (Ostedgaard et al, 1997; Chan et al, 2000). refined molecule). The initial structure of mNBD1 was sub- The boundaries of CFTR NBD1 have been a matter of some sequently used to solve the I4 22 structure in the presence of controversy. It has been suggested to begin in the span from Mg-ATP by molecular replacement and to determine isomor- residue 373 (Wang et al, 2002) to residue 441 (Bianchet et al, phous P42 2 structures in the presence of Mg-ATP, Mg-ADP, 1997) at the N-terminus and to end in the span from residue and in the absence of nucleotide. The conformation of 586 (Riordan et al, 1989) to residue 684 (Bianchet et al, 1997) mNBD1 is essentially the same in all crystal forms irrespec- at the C-terminus (human CFTR numbering, used throughout tive of the identity of the bound nucleotide and also in each of this paper). A compelling functional definition based on the the four copies in the P42 2 crystals and two copies in the coexpression of severed and deleted CFTR constructs gave I4 22 crystal. Each mNBD1 molecule is associated in a four- boundaries within 433–633 for NBD1 (Chan et al, 2000). fold symmetric, head-to-tail ring structure that recurs three Many different expression constructs have been used in times in the P42 2 lattice and once in the I4 22 lattice. 1 1 studies of NBD1 function, sometimes yielding conflicting results. Indeed, several aspects of CFTR NBD1 function Overall structure of CFTR NBD1 remain poorly characterized. The most important outstand- The mNBD1 domain has a core tertiary structure similar to ing issues pertain to ATP binding and hydrolysis, conforma- NBDs from other ABC transporters, but this core is modified tional adaptability in the domain, the effects of NBD1 with major additions and deletions. Figure 2B shows a phosphorylation, and the structural consequences of dis- topology diagram of mNBD1, indicating through color coding ease-causing mutations, in particular DF508. We have deter- the subdomains and those regions of mNBD1 that show mined the crystal structure of murine NBD1 to shed light significant differences from other ABC structures. on these vital mechanistic issues. Secondary structural elements in common with most known ABC structures are given conventional designations (S1, S2, S3, H1, etc.) and additional elements found in Results mNBD1 are denoted with lowercase letters (H1b, H1c, S6b, mNBD1 expression and purification etc.). The three-dimensional course of the polypeptide chain Given the uncertainty in the globular domain definition of is shown as a ribbon diagram in Figure 3A, where the CFTR NBD1, we chose to clone and express in Escherichia coli structural elements are colored in the same code as in many constructs in parallel covering residues from 363 to Figure 2B and key elements in the binding of ATP are also 686. A pan-genomic approach was employed, testing the identified. Figure 3B shows a worm diagram of mNBD1 in &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 2 2004 283 | | Structure of CFTR NBD1 HA Lewis et al 284 The EMBO Journal VOL 23 NO 2 2004 &2004 European Molecular Biology Organization | | Table I Data collection and refinement statistics Space group Resolution (A) Nucleotide Completeness (%) R (%) Redundancy I/s(I) sym (overall/outer shell) (overall/outer shell) (overall/outer shell) (overall/outer shell) mNBD1 data collection mNBD1+AMP.PNP P42 2 33.0–2.50 AMP.PNP 99.9/99.6 12.2/35 14.1/13.6 5.4/1.8 mNBD1-P+ATP P42 2 24.0–2.35 ATP 97.6/92.4 9.0/46 4.6/2.4 14.7/2.1 mNBD1 apo P42 2 38.9–2.20 None 93.2/95.1 7.6/44 5.1/4.1 19.8/3.1 mNBD1+ATP P42 2 39.2–2.20 ATP 98.5/92.7 7.3/50 7.0/5.7 27.7/4.0 mNBD1+ADP P42 2 36.5–2.55 ADP 100.0/99.4 7.7/35 9.8/9.9 8.0/1.9 mNBD1+ATP (2) I4 22 49.7–3.00 ATP 100.0/100.0 7.3/98 8.2/8.3 27.6/2.0 Resolution (A) RR Waters RMSD bond length RMSD bond angles Average B-factors free ˚ ˚ (A) (1) (A ) Refinement statistics mNBD1+AMP.PNP (1Q3 H) 33.0–2.50 0.215 0.266 658 0.008 1.9 36.4 mNBD1-P+ATP (1R0Z) 24.0–2.35 0.221 0.258 304 0.015 1.5 48.0 mNBD1 apo (1R0W) 38.9–2.20 0.231 0.262 423 0.021 2.0 30.8 mNBD1+ATP (1R0X) 39.2–2.20 0.234 0.266 378 0.021 2.0 39.1 mNBD1+ADP (1R0Y) 36.0–2.55 0.207 0.257 195 0.015 1.7 45.6 mNBD1+ATP (2) (1R10) 30.0–3.00 0.228 0.265 0 0.012 1.3 80.6 Ramachandran distribution Molecule A Molecule B Molecule C Molecule D Core Allowed Disallowed Residues modeled mNBD1+AMP.PNP 390–412, 429–670 389–412, 429–670 388–412, 429–670 390–412, 92.0% 7.9% 0.0% 429–670 mNBD1-P+ATP 390–413, 420–670 389–412, 420–670 388–413, 420–670 390–407, 430–670 91.2% 8.7% 0.1% mNBD1 apo 390–413, 429–670 389–413, 429–670 388–413, 430–670 390–412, 430–670 92.4% 7.5% 0.1% mNBD1+ATP 390–411, 429–670 389–411, 429–670 388–411, 430–670 390–411, 430–670 91.2% 8.7% 0.0% mNBD1+ADP 388–413, 429–671 388–411, 428–670 388–412, 430–670 390–412, 430–671 89.9% 9.9% 0.1% mNBD1+ATP (2) 391–412, 429–670 391–412, 429–670 — — 85.5% 14.5% 0.0% R ¼ S S |I (hkl)/I(hkl)S|/S S I (hkl), where I is the intensity of the observation and /IS is the mean intensity of the reflection. R ¼ S||F ||F ||/S|F |, where F and F are the observed and sym hkl i i hkl i i i o c o o c calculated structure-factor amplitudes. R is the same as R except calculated for 5% of the data randomly omitted from the refinement. RMSD is root-mean-square deviation. Average B-factors are free reported for all nonhydrogen atoms. The values in parenthesis following the dataset names are the Protein Data Bank identifiers for the respective structures. Structure of CFTR NBD1 HA Lewis et al Figure 2 Sequence alignment and topology diagram of mNBD1. (A) Sequence alignment of human and mouse NBD1 and NBD2 with NBD domains from other ABC transporters. A blue background indicates b-strands while pink indicates a-helices in known structures. Solid circles mark the locations of common CF-causative mutations, solid triangles the locations of deletion mutations, and P indicates where phosphorylation by PKA was observed in the mNBD1–P structure. Residues with high sequence conservation in ABC domains are highlighted in blue bold font. Red bold font indicates residues that significantly vary from this conservation. The secondary structure of mNBD1 is indicated graphically above the residue numbering row and is color coded by subdomain as in Figure 2B. (B) Topology diagram of mNBD1. The F1-type ATP-binding core subdomain is shown in gold, the ABC a-subdomain in cyan, and the ABC b-subdomain in green. Regions of mNBD1 that are different from previous ABC structures are shown in gray. Circles indicate the positions of 3 helices. &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 2 2004 285 | | Structure of CFTR NBD1 HA Lewis et al which the thickness of the trace is proportional to the B- factors of the C atoms, thereby reflecting potential mobility of the polypeptide backbone. Regions of highest mobility are near the N- and C-termini and at the inserted and partially disordered segment between S1 and S2. B-factors are com- monly elevated near termini and in some loops, but there may be special relevance here in relation to the putative NBD1/NBD2 interface (see below). The core structure of mNBD1-ATP most closely resembles that of TAP1-ADP with a root-mean-square deviation (r.m.s.d.) of 1.9 A for 176 C atoms spanning a region with 26% sequence identity. MJ0796-ATP is next closest overall (2.4 A r.m.s.d. over the same span; 30% sequence identity). This alignment omits the regions between S1 and S2, be- tween S4 and S6, and beyond S10, which exhibit conforma- tion differences between ABC structures. Figure 2A presents a structure-based sequence alignment of mNBD1 with the ABC domains of some known structures and with selected se- quences from human ABC transporters, including human CFTR. These comparisons should help to clarify the discus- sions of CFTR function. Four major structural features distinguish mNBD1 from other ABC NBDs. This is evident in Figure 3C, where mNBD1 is superimposed onto three representative ABC structures. First, mNBD1 contains an insertion of about 35 residues between b-strands S1 and S2. This insertion is composed of two short a-helices (denoted H1b and H1c) separated by a flexible linker region that was not observed in the electron density map (residues 413–428, red dotted line in Figure 3A). The N-terminal b-strand S1 is common to all ABC domain structures, and a conserved aromatic residue near its C-terminus (corresponding to Trp401 in CFTR) stacks against the adenine base in previously reported structures of ABC domains complexed with nucleotides. The insertion leads to an altered binding geometry for the base and ribose in mNBD1 and includes a segment (415–432) that can be deleted while preserving function (Chan et al, 2000). Second, mNBD1 lacks a 14–27 residue region between b-strands S4 and S6 (residues 485–488) that typically con- tains an additional b-strand and an a-helix (denoted S5 and Figure 3 Structural fold of mNBD1. (A) Stereo ribbon diagram of mNBD1. ATP is shown in ball and stick representation. The sub- H2 in Figure 2A; located lower left in Figure 3C). Third, domains are color coded as in Figure 2B. The dotted red line relative to the prokaryotic ABC structures, mNBD1 is trun- indicates residues missing from the mNBD1 model. (B) Stereo cated between helices H3 and H4 (lower right of Figure 3C). worm diagram of mNBD1. The worm thickness is indicative Finally, the C-terminus of mNBD1 includes a long a-helix of the relative B-factor of the residues ranging from 18 A (thinnest) to 65 A (thickest). Color coding of subdomains is according to (H9b) that is not present in other ABC domain structures. The Figure 2B. (C) Stereo C diagram of the superposition of represen- hydrophobic face of this amphipathic a-helix packs against tative ABC domain structures onto mNBD1. TAP1 (thin red), a hydrophobic surface formed primarily by the preceding MJ0796 (thin green), and HisP (thin yellow) structures superim- a-helix H9. The H9b segment of the polypeptide chain has posed onto mNBD1 (thick blue). Superposition based on least- squares alignments of the F1-type core and ABC-specific antiparallel been considered to be part of the R domain as it contains two b-subdomains. (D) Backbone structure of mNBD1 illustrating posi- potentially regulatory phosphorylation sites, Ser660 and tions of phosphorylation and CF-causative mutations. Left: mNBD1 Ser670 (Chen et al, 2000). However, the structure and our is seen in the same orientation and subdomain colorization as in expression experiments lead us to think of H9b as an integral Figure 3A. Right: the same structure rotated 801 toward the viewer. Helices of regulatory segments are drawn as ribbons; the remaining component of the mNBD1 fold; at a minimum, the associa- polypeptide chain is a worm drawing. ATP is shown as ball and tion is favorable. stick. Ser422, Ser659, Ser660, and Ser670 side chains are shown in purple. Residues 420–428 become ordered upon phosphorylation Preparation, crystallization, and structure (solid red). The remaining residues of the structure that were not modeled (414–419) are indicated as red dots. Side chains are shown determination of triphospho-mNBD1 at sites of common CF-causative mutations (Ala455, Gly480, Ile506, Phosphorylation of CFTR, particularly in the R domain, is Ile507, Ser549, Gly551, Ala559, Arg560, Tyr569, and Asp648 colored thought to regulate channel opening and closing, and protein yellow; Phe508 in green). The di-acidic code residues (D565 and kinase A (PKA) plays an important role in this process D567) are in gold. (Ostedgaard et al, 2001). There are 21 PKA phosphorylation motifs (Kennelly and Krebs, 1991) in human CFTR. About 286 The EMBO Journal VOL 23 NO 2 2004 &2004 European Molecular Biology Organization | | Structure of CFTR NBD1 HA Lewis et al one-quarter of these putative phosphorylation sites are lo- rotated away due to a backbone g torsion angle of gauche þ cated in mNBD1 (Ser422, Ser489, Ser519, Ser557, Ser660, and as opposed to trans, which is seen in other ABC domain Ser670), and the murine sequence presents an additional PKA structures (Figure 4B). As a result, the adenine base of the site at Ser659. To identify CFTR NBD1 residues available for nucleotide does not stack against an aromatic side chain. PKA phosphorylation in vitro, we incubated mNBD1 with Instead, the position of the usual stacking aromatic residue PKA and analyzed the results using phosphopeptide mapping (Tyr11 in MJ0796) is taken up by Trp401, which is in close by mass spectrometry. Residues Ser422, Ser659, Ser660, and contact with the ATP ribose (3.3 A). Phe430, which is in the Ser670 were seen to be phosphorylated with Ser659 to a S1–S2 insertion, makes an ‘edge-to-face’ interaction with the lesser extent of only about one-quarter. adenine base. Leu409 makes contacts approaching from the The phosphorylated form of the protein, mNBD1-P, crystal- opposite side of the ring. No hydrogen bonding contacts are lized isomorphously with the unmodified protein and we observed with the adenine. were able to determine its structure. Those portions of In the phosphorylated structure, mNBD1-P, residues 420– mNBD1 that are well-ordered in the unphosphorylated state 428 contribute additional contacts to the adenine base of the are unchanged in the mNBD1-P structure, but the presence of nucleotide (Figure 4C). Val428 is in van der Waals contact phosphorserine 422 confers order on residues 420–428 (solid with the adenine as is Glu425, which stacks partially under- red ribbon segment in Figure 3D) in the inserted loop where a neath the base. Together with Phe430, these residues serve to phosphate group is visible on the side chain of Ser422. In grip the nucleotide base. A hydrogen bond between the side addition, phosphorylation is evident at residues Ser660 and chain of His421 and the phosphate of Ser660 contributes Ser670, and partially at Ser659 (only seen in molecule A in to the conformational stabilization of this region. the asymmetric unit). Phosphorylation at Ser422 (Chang et al, ATP binding and hydrolysis in solution 1993), Ser660 (Cheng et al, 1991; Winter and Welsh, 1997), ATP binding to mNBD1 had an apparent K of 149748 mM and Ser670 (Wilkinson et al, 1997) has been observed in (Figure 5A). Previously measured values for nucleotide affi- human CFTR and each has been shown to have a specific nity have ranged from low micromolar (Yike et al, 1996; Qu effect on the activity of CFTR. Based on this evidence as well et al, 1997) to several millimolar (Ko et al, 1993), varying as discussions on the relevance of these regions to a putative with both the nucleotide analog used as well as the protein NBD1–NBD2 heterodimer (see below), we will refer to the construct. The values reported here are consistent with those S1–S2 loop and H9c structures as the regulatory insertion and of human NBD1 determined by several other groups (Yike regulatory extension, respectively. et al, 1996; Qu et al, 1997; Neville et al, 1998). ATP binding was significantly reduced by the K464A mutation, presum- Nucleotide coordination in mNBD1 crystal structures ably because electrostatic interactions with the b- and Nucleotides in the various crystal complexes with mNBD1 g-phosphates of ATP (Figure 4A) are lost. (Table I) are all bound in a very similar manner. This mode of ATP hydrolysis by mNBD1 in solution was measured in a binding has aspects in common with that in other ABC standard coupled assay system (Rosing et al, 1975). An active transporter NBDs, but the comparison is distinguished as ABC NBD ATPase, MJ0796, was included as a positive con- much by differences as similarities. Since Mg-ATP has its trol. Consistent with previous experiments (Moody et al, g-phosphate intact when cocrystallized with mNBD1, whether 2002), the MJ0796 protein hydrolyzed ATP at a rate approach- phosphorylated or not, mNBD1 is not an active ATPase as is ing 8 min , while the mNBD1 exhibited a hydrolytic rate less typical for such domains. Moreover, since the protein con- than 0.02 min in the colorimetric assays in both the unpho- formation is the same when uncomplexed or complexed with sphorylated and phosphorylated states (Figure 5B). The dis- ADP as when complexed with ATP (r.m.s.d. ¼ 0.2–0.3 A), the parity with previously published results (Ko and Pedersen, possibility is raised that nucleotide binding to the NBD1 site 1995; Duffieux et al, 2000; Annereau et al, 2003) may reflect in CFTR is not involved directly in movements thought to differences in ATP binding between mouse and human drive ABC transporter action in other cases (Yuan et al, 2001). proteins or, more likely, are due to other factors associated Canonical features of nucleotide binding in mNBD1 all with alternative constructs. involve interactions with the phosphate groups (Figure 4A). Specifically, Lys464 and Thr465 (Walker A), Asp572 (Walker Discussion B), and Gln493 (Q-loop) hydrogen bond with the phosphates 2þ and/or coordinate the Mg ion as in other ABC domain Intrinsic structural integrity structures. On the other hand, consistent with a catalytically The crystal structures of mNBD1 have features that distin- inactive site, there are also significant differences. Namely, guish them from other ABC cassette proteins. The structure is the Walker-B carboxylate residue, typically glutamate, that essentially the same independent of nucleotide state, nucleo- serves as the catalytic base in active ABC transporters tides bind in an unusual conformation at the ribose-phos- (Moody et al, 2002) is replaced by serine in NBD1 (Ser573), phate linkage, and regulatory segments are added to the core and the canonical H-loop histidine residue becomes Ser605. structure. Since mNBD1 molecules associate into head-to-tail Both of these fail to hydrogen bond with the g-phosphate as tetramers in our crystals, we have considered whether these in related complexes (Hung et al, 1998; Smith et al, 2002). lattice contacts (B900 A from each surface) might affect the The distinction from usual nucleotide binding by ABC intrinsic structure. The predominant interactions involve domains is even more pronounced for ribose and base residues from the Q-loop and a-subdomain at the ‘head’ of interactions in mNBD1. The ATP ribose is C3 -endo with an one molecule contacting H1c residues near the ATP site at the ‘anti’ w torsion angle base conformation as in other ATP- ‘tail’ of another molecule. bound ABC structures (eg MJ0796 (Smith et al, 2002) and While lattice influences cannot be ruled out, several lines HisP (Hung et al, 1998)), but the ribose-base assembly is of evidence suggest that they do not overwhelm the intrinsic &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 2 2004 287 | | Structure of CFTR NBD1 HA Lewis et al Figure 4 Close-up on ATP binding by mNBD1. (A) Canonical hydrogen bonding interactions in Mg-ATP mNBD1. Some relevant hydrogen bonds are indicated as green lines. Some residues in the foreground and background have been removed to clarify the interactions, here and in (B) and (C). (B) Differences in adenine base recognition from other ABC domains. Left: adenine stacks against Tyr11 of MJ0796 (PDB ID code 1L2T). Right: adenine of ATP makes edge-to-face interactions with Phe430 of mNBD1. (C) Added structure in phosphorylated mNBD1. The ATP molecule plus magnesium in blue, the additional mNBD1 residues observed in the phosphorylated state in white, the phosphate atoms of Ser422 and Ser660 in brown, and the remainder of the mNBD1 structure in yellow are shown. mNBD1 structure. With respect to the relative disposition of sequences of CFTR NBD1 from diverse vertebrate species are ABC a- and F1-ATPase subdomains, two facts are most compe- very similar (61–98%, pairwise identity) and divergent seg- lling: (1) The same I4 22 crystals grow spontaneously and ments are restricted to surface residues and loop regions. This equally well from mNBD1 in Mg-ATP, Mg-ADP, and nucleo- suggests that all NBD1s share the deviations from canonical tide-free states. (2) Intramolecular interactions between a-and ABC domains that distinguish mNBD1. In particular, human F1-subdomains in mNBD1 (five hydrogen bonds and 34 NBD1 should be very close in structure to mNBD1 as these residue pairs in van der Waals contact) are much more molecules are 78% identical in sequence over the span of extensive than in the head-to-tail interface (one hydrogen mNBD1 and there are no amino-acid insertions or deletions bond and 16 van der Waals pairs). With respect to the unusual (Figure 2A). It seems likely that its structure will also be ribose-base conformation of nucleotides in mNBD1, this is similar in intact CFTR, but functional interactions with the largely determined by the placement of Trp401 in the S1–H1b NBD2, MSD1, and/or R domains of CFTR (Figure 1) might segment, which is not involved in any contacts. well lead to adaptations in NBD1. Indications of potential plasticity in NBD1 come from the crystallographic measures of atomic mobility in mNBD1. Structural implications Parts of the regulatory insertion between S1 and S2 are We expect NBD1 domains from all CFTRs to have essentially sufficiently flexible that they are not seen in the density the same structure as that seen here for mNBD1. The known 288 The EMBO Journal VOL 23 NO 2 2004 &2004 European Molecular Biology Organization | | Structure of CFTR NBD1 HA Lewis et al mNBD1 (approximately residues 390–670), but the NBD1 core may well be stabilized by other interactions in situ. NBD1 phosphorylation sites are located in these ‘deletable’ segments, and perhaps they can be displaced in intact CFTR when phosphorylated such that exposed surfaces can interact productively with other domains, notably NBD2. Enzymatic activity Our results demonstrate that mNBD1 is incapable, on its own, of catalyzing significant ATP hydrolysis: ATP remains intact in mNBD1 crystal structures, and no significant hydrolysis is observed in our solution assays. This lack of activity is not unexpected from the model espoused in Figure 1 whereby NBD1–NBD2 ‘heterodimers’ are thought to mediate normal catalysis in CFTR, and likely only do so at the site where NBD2 provides the Walker A and B sequences (NBD2-based site). The catalytic base, usually glutamate, in the NBD1- based site is replaced by serine at position 573, and this putative site has other defects as well. We now know from the structures that there are no candidate replacements within NBD1 for the catalytic base. On the other hand, since the phosphate groups of ATP are otherwise coordinated in a conventional manner in mNBD1, the g-phosphate is poised for attack and it is conceivable that some unanticipated part of CFTR might confer catalytic competence. However, NBD1 in intact CFTR does not appear to be enzymatically active (Basso et al, 2003; Vergani et al, 2003). Interactions between NBD1 and NBD2 Our working hypothesis for the role of NBDs in CFTR function is that NBD1 interacts with NBD2 in the mode of a labile nucleotide sandwich, as depicted in Figure 1; ATP binding to the NBDs, and hydrolysis at NBD2, then couples through NBD–MSD associations to control the channel. This model is consistent with biochemical and structural informa- tion on homodimeric ABC transporter systems (Locher et al, Figure 5 ATP binding and hydrolysis by mNBD1. (A) ATP binding 2002; Moody et al, 2002; Smith et al, 2002; Chen et al, 2003). to mNBD1 was analyzed using a filter-binding assay. Wild type It is also consistent with ‘heterodimeric’ crosslinking between (K), K464A mutant (J), and ATP binding to the filters in the NBD1 and NBD2 in human multidrug-resistant P glycoprotein absence of protein (.) are shown. The wild-type K was calculated to be 149748 mM. (B) ATPase activity was analyzed by a coupled (Loo et al, 2002) and with tryptophan-fluorescence assays of colorimetric assay. ATPase activity for mNBD1, MJ0796 (LolD), and direct interactions for human CFTR between NBD1-R (373– background are shown as a function of changes in NADH absor- 859), a construct that encompasses the entire span of our bance at 340 nm. Inset: ATP hydrolysis rates were calculated to be mNBD1, and NBD2 (1151–1476) (Wang et al, 2002). Finally, it less than 0.02 min for the mNBD1, as compared to the positive control, MJ0796, which was 7.9 min . is consistent with electrophysiological studies of CFTR func- tion (Vergani et al, 2003), which indicate that normal channel opening requires Mg-ATP binding at both nucleotide sites and that normal channel closing follows ATP hydrolysis in the NBD2-based site. In order to pursue the hypothesis, we maps and B-factors are elevated for the ordered parts, H1b developed homology models for CFTR NBD2 and for the and H1c, of the insertion (Figure 3B). Similarly, B-factors are NBD1–NBD2 ‘heterodimer’. also well above average for the helices H8–H9b. These We have docked our mNBD1 structure and our NBD2 elements are candidates for displacement by interactions homology model into a hypothetical NBD1–NBD2 ‘hetero- with other CFTR domains or other regulatory events. dimer’ constructed with reference to the structure of the Evidence that this may indeed happen comes in comparing E171Q mutant MJ0796 homodimer (Smith et al, 2002). This our results with results from the coexpression of severed model, in what is expected to be the universal dimerization CFTR constructs (Chan et al, 2000). Wild-type conductance mode of ABC transporters, exhibits severe main-chain steric levels were observed from two-chain CFTR channels con- clashes between NBD1 and NBD2 (Figure 6). Specifically, the structed such that either 415–432 (the flexible loop in our inserted loop region (405–436) and C-terminal a-helix (H9b) structures) or 634–667 (H8–H9b region) was omitted, sug- are thrust directly into the opposing NBD2 model. There are gesting that these features may not be vital for CFTR function. several ways in which this conflict might be resolved. One We were unable to generate stable NBD1 proteins from possibility is that there is a conformational change in NBD1 constructs any narrower than the ordered confines of upon interdomain association, whereby the clashing regions &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 2 2004 289 | | Structure of CFTR NBD1 HA Lewis et al Figure 6 Worm figure of putative NBD1–NBD2 interaction. NBD1 is color coded as in Figure 2B. NBD2 is in red. The NBD1 worm thickness is proportional to the backbone B-factor. The figure at the right is rotated 901 toward the viewer relative to the left figure. are moved out of the way. The inserted loop and H9b 1990), perhaps due to improper folding (Qu and Thomas, extension have elevated B-factors (Figures 3B and 6) and 1996) and instability of CFTR (Lukacs et al, 1993), which is the correspondingly greater atomic mobilities may indicate detected by the protein degradation machinery in the endo- hinged conformational flexibility. Moreover, sites of phos- plasmic reticulum (ER) and lysosomes (Gelman and Kopito, phorylation in mNBD1-P are all located on the S1–S2 inser- 2002). The limited number of DF508 molecules that are tion and on the H9b extension (Figure 3D), and these retained form active chloride channels, but with altered clashing segments might possibly be displaced when phos- gating properties (Dalemans et al, 1991). phorylated to take up favorable interactions with other parts Phe508 lies exposed on the surface of mNBD1, positioned of CFTR, thereby promoting and maintaining NBD associa- at the start of a loop between helices H3 and H4. This site is tion. This hypothesis is consistent with data from R-domain distant (420 A) from either ATP in the ‘heterodimer’ model, deletions, which release CFTR gating inhibitions upon phos- suggesting that DF508 is unlikely to affect ATP binding phorylation (Rich et al, 1991) to give opening kinetics similar directly. The side chain of Phe508 is in the vicinity of to phosphorylated wild-type CFTR but with less stable open Arg560, Trp496, and Met498, but it makes only a single van channels (Csanady et al, 2000). der Waals contact, with Met498. Thus, Phe508 does not appear to play a critical role in stabilizing mNBD1 structure. Interactions between NBD1 and MSD1 Moreover, its main-chain contacts are only local, so accom- Associations between the NBD and MSD domains are ex- modation of the deletion through loop reorganization seems pected to provide the conduit of communication from nucleo- plausible. Disruptions are expected to be substantial never- tide-dependent conformational changes in NBD domains to theless. One possible impact of DF508 might be on accessi- the gating of transmembrane channels in ABC transporters. bility to the di-acidic code proposed to be involved in The structures of other ABC transporters (Chang and Roth, membrane protein transport out of the ER (Barlowe, 2003). 2001; Locher et al, 2002; Chang, 2003) provide an insight into Another impact may be on the NBD1–MSD1 interface into the nature of these associations. In each case, a-domain and which Phe508 is expected to be buried (see above). CF Q-loop portions of an NBD interact with cytoplasmic exten- mutations concentrate in the a-subdomain or contacting sions from the transmembrane portion of one MSD–NBD sites, and disruptive consequences are evident from the exclusively. We constructed homology models of CFTR structure for some. MSD1–NBD1 based on our structure of mNBD1 docked onto the corresponding region of the MsbA and BtuCD Materials and methods structures (not shown). The surface of NBD1 that would be at the interface with mMSD1 overlaps with a surface that is Protein preparation and phosphorylation buried into the head-to-tail rings in mNBD1 crystal lattices. Mouse CFTR NBD1 (residues 389–673; human numbering) was expressed in E. coli as an N-terminal His –Smt3 fusion protein This surface includes the hydrophobic side chains from 6 (Mossessova and Lima, 2000) using a pET26b-derived expression Phe494, Trp496, Met498, Val540, and Phe508, the most vector. Cells were harvested by centrifugation and lysed by common CF mutation site. sonication. Protein was initially purified by nickel ion affinity chromatography, followed by proteolytic removal of the tag using Ulp1 and GFC. A final nickel ion affinity step was employed to CF mutations in NBD1 remove traces of the His –Smt3 affinity tag. Final yields averaged The majority of sites of CF-causing missense mutations occur 4 mg/l with greater than 98% purity. The purified protein was in NBD1, primarily in its a-subdomain, and the locations in concentrated to 5–10 mg/ml in buffer containing 150 mM NaCl, the mNBD1 structure of the most common of these (A455E, 12.5% glycerol, 2 mM DTT, 2 mM ATP, and 50 mM Tris pH 7.5. Se- G480C, I506T, DI507, DF508, S549N, S549R, G551D, A559T, Met containing protein was expressed and purified using similar procedures. R560T, Y569D, and D648V; Bobadilla et al, 2002) are shown To generate phosphorylated mNBD1, 1000 U of bovine PKA in Figure 3D. The deletion of Phe508, DF508, is by far the (Sigma P2645-1KU) was incubated with 5 mg of mNBD1 at a most prevalent. This mutation leads to reduced CFTR trans- concentration of 1 mg/ml at 41C for 4 h, and then concentrated to port to, and retention in, the plasma membrane (Cheng et al, 5–10 mg/ml. Mass spectrometric characterization verified phos- 290 The EMBO Journal VOL 23 NO 2 2004 &2004 European Molecular Biology Organization | | Structure of CFTR NBD1 HA Lewis et al phorylation of three or four sites (full modification: Ser422, Ser660, buffer lacking ATP (50 mM Tris–HCl pH 7.6, 150 mM NaCl, 2 mM DTT, 12.5% glycerol) using Bio-Rad Micro Bio-Spin 6 chromato- and Ser670; partial, approximately 25% modification: Ser659). graphy columns following the manufacturer’s protocols. mNBD1 Protein characterization protein (375 pmol) was incubated with increasing concentrations of Matrix-assisted laser desorption ionization and electrospray ioniza- an Mg-a P-ATP mixture for 45 min at room temperature in a final tion mass spectrometric analyses confirmed the identity of the reaction volume of 20 ml. The protein was then applied to Millipore purified proteins and estimated purity at greater than 95%. DLS HA 0.45 mm filters that were prewetted with buffer on a 12-position measurements using a DynaPro-MS800 instrument made at protein Millipore vacuum manifold. The samples were washed three times concentrations up to 8 mg/ml showed a single monomeric species. with 2 ml of ice-cold buffer supplemented with 40 mM MgCl and Analytical gel filtration at a protein concentration of 4 mg/ml then counted on a Beckman LS-6500 scintillation counter. yielded similar results. ATP hydrolysis by NBD1 Crystallization, data collection, structure determination, ATP hydrolysis by mNBD1 was measured using a colorimetric assay and refinement described previously (Rosing et al, 1975). ATP hydrolysis was Purified mNBD1 at 5–10 mg/ml was subjected to initial hanging- measured via a coupled reaction where NADH is converted to drop vapor-diffusion screens with more than 500 different NAD in a one-to-one ratio with ADP production. The reaction was precipitant conditions at two temperatures (4 and 201C). From this monitored by changes in absorbance at 340 nm. In total, 1 nmol it was found that a simple precipitant consisting of 3.5–4.0 M mNBD1 was incubated in reaction buffer (100 mM Tris pH 8.0, sodium acetate with no additional buffer or salt and pH adjusted to 50 mM KCl, 2 mM MgCl , 0.2 mM EDTA, 4 mM Mg-ATP, 1 mM PEP, 7.5 yielded diffraction-quality crystals in two forms (I4 22: 200mM NADH, 4 U/ml LDH, 8 U/ml PK) in a final volume of ˚ ˚ a ¼ 140 A, c¼ 280 A, two molecules/asymmetric unit; P42 2: 1.015 ml at 301C and monitored by changes in A on a Shimadzu ˚ ˚ a ¼ 172 A, c ¼ 110 A, four molecules/asymmetric unit). Nucleotide UV2101 spectrophotometer. MJ0796 was used as a positive control complexes were generated through cocrystallization, except in the for ATP hydrolysis. The rates of ATP hydrolysis were calculated as a NADH case of ADP in which soaking of mNBD1 crystals with 1 mM function of the changes in A using e ¼ 6.22 mM/cm. nucleotide was employed. Crystals were frozen by immersion in liquid nitrogen prior to data collection. Diffraction data were Homology modeling measured at the SGX-CAT beamline 31-ID at the Advanced Photon Homology models were built using SCWRL3 (Bower et al, 1997) Source under standard cryogenic conditions, and processed with and Modeller 6v2 (Fiser et al, 2000). Based on the assumption that either the Mosflm (CCP4, 1994) or HKL (Otwinowski and Minor, CFTR NBD1 and NBD2 can interact, a heterodimer model was 1997) software. created using CFTR mNBD1 as a template for each domain and The structure was initially determined in space group I4 22 using several NBD structures as templates for the dimer interaction, the MIRAS method. Two Ta Br derivative data sets, diffracting to 6 12 including BtuD (1L7V) and MJ0796 (1L2T). The Walker A, LSGGQ, 4.0 A, were collected at the peak energy of tantalum and used with a and Walker B motifs of NBD1 were structurally aligned with each native 3.1 A data set in phasing calculations. Cluster sites located ABC monomer using Spock (Christopher, 1998), and then CFTR with SNB (Weeks and Miller, 1999) gave phasing powers in NBD1/NBD2 was modeled simultaneously. Multiple models were MLPHARE (CCP4, 1994) of 2.12 and 1.98 for the two derivatives generated and the best model was chosen based on Modeller’s and produced experimental phases useful to 4.0 A. Two copies of a energy term, ProsaII analysis (Sippl, 1993), and visual inspection. homology model of NBD1 based on the TAP1 structure (PDB code 1JJ7) were manually placed in the density-modified (DM; CCP4, Figure preparation 1994) electron density map. Further density modification with NCS Structure figures were made with MOLSCRIPT (Kraulis, 1991) and averaging then produced better maps and allowed a more extensive Raster3D (Merritt and Bacon, 1997), except for Figures 3C, 4A, 4B, NBD1 backbone construction. and 4C which were made using Xtalview (McRee, 1999) and Figure The mNBD1 backbone from the I4 22 structure was positioned 3B and 6 which were made using Spock. by molecular replacement into the P42 2 lattice using a 2.9 A data set acquired at the selenium peak energy of an SeMet crystal. A Bijvoet-difference Fourier based on resulting model phases allowed Data deposition the identification of 21 Se sites, which were refined with MLPHARE Coordinates have been deposited in the Protein Data Bank (http:// using 2.5 A native P42 2 data. The resulting phases were NCS www.rcsb.org/pdb). Accession codes are listed in Table I. averaged to obtain a final map that was used for building the side chains and finalizing the model. The structure of NBD1 was built manually over several cycles of model building with XtalView Acknowledgements (McRee, 1999) and refinement with CNX (Brunger et al, 1998) and/ or Refmac (CCP4, 1994). Subsequent structures were determined We thank Drs E de la Fortelle and T Harris for many useful using the molecular replacement program EPMR (Kissinger et al, discussions; Dr A Verkman for supplying putative CFTR ligands 1999), and refined to convergence. PROCHECK (Laskowski et al, used in some cocrystallization efforts; L Pelletier, F Lu, K Bain, and 1993) revealed main-chain and side-chain structural parameters Drs S Antonysamy, IK Feil, J Hendle, B Noland, and F Park for their consistently better than average (overall G-values 41.0). See Table I expert contributions towards the expression, characterization, and for data collection and refinement statistics. crystallization of mNBD1; and K Schwinn for his help in making some of the figures. Use of the Advanced Photon Source was ATP binding to NBD1 supported by the US Department of Energy, Office of Science, and ATP binding was measured using a standard filter-binding assay Office of Basic Energy Sciences, under Contract No. W-31-109-Eng- (Widlak et al, 2001). Endogenous ATP added during purification 38. This work was supported by a research contract from the Cystic was first removed by exchanging (three times) the protein into Fibrosis Foundation. References Annereau JP, Ko YH, Pedersen PL (2003) Cystic fibrosis transmem- nucleotide binding domain and its role in channel gating. J Gen brane conductance regulator: the NBF1+R (nucleotide-binding Physiol 122: 333–348 fold 1 and regulatory domain) segment acting alone catalyses a Bianchet MA, Ko YH, Amzel LM, Pedersen PL (1997) Modeling 2+ 2+ 2+ Co /Mn /Mg -ATPase activity markedly inhibited by both of nucleotide binding domains of ABC transporter proteins 2+ Cd and the transition-state analogue orthovanadate. Biochem J based on a F1-ATPase/recA topology: structural model of the 371: 451–462 nucleotide binding domains of the cystic fibrosis transmem- Barlowe C (2003) Signals for COPII-dependent export from the ER: brane conductance regulator (CFTR). J Bioenerg Biomembr 29: what’s the ticket out? Trends Cell Biol 13: 295–300 503–524 Basso C, Vergani P, Nairn AC, Gadsby DC (2003) Prolonged non- Bobadilla JL, Macek Jr M, Fine JP, Farrell PM (2002) Cystic fibrosis: hydrolytic interaction of nucleotide with CFTR’s NH -terminal a worldwide analysis of CFTR mutations—correlation with in- &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 2 2004 291 | | Structure of CFTR NBD1 HA Lewis et al cidence data and application to screening. Hum Mutat 19: Kissinger CR, Gehlhaar DK, Fogel DB (1999) Rapid automated 575–606 molecular replacement by evolutionary search. Acta Crystallogr Bower MJ, Cohen FE, Dunbrack Jr RL (1997) Prediction of protein D 55: 484–491 side-chain rotamers from a backbone-dependent rotamer library: Ko YH, Pedersen PL (1995) The first nucleotide binding fold of the a new homology modeling tool. J Mol Biol 267: 1268–1282 cystic fibrosis transmembrane conductance regulator can func- Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse- tion as an active ATPase. J Biol Chem 270: 22093–22096 Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS et al. Ko YH, Thomas PJ, Delannoy MR, Pedersen PL (1993) The cystic (1998) Crystallography & NMR system: a new software suite for fibrosis transmembrane conductance regulator. Overexpression, macromolecular structure determination. Acta Crystallogr D 54: purification, and characterization of wild type and delta F508 905–921 mutant forms of the first nucleotide binding fold in fusion with CCP4 (1994) The CCP4 suite: programs for protein crystallography. the maltose-binding protein. J Biol Chem 268: 24330–24338 Acta Crystallogr D 50: 760–763 Kraulis PJ (1991) MOLSCRIPT: a program to produce both detailed Chan KW, Csanady L, Seto-Young D, Nairn AC, Gadsby DC (2000) and schematic plots of protein structures. J Appl Crystallogr 24: Severed molecules functionally define the boundaries of the 946–950 cystic fibrosis transmembrane conductance regulator’s NH(2)- Laskowski RJ, MacArthus MW, Moss DS, Thornton JM (1993) terminal nucleotide binding domain. J Gen Physiol 116: 163–180 PROCHECK: aprogram to check stereochemical quality of protein Chang G (2003) Structure of MsbA from Vibrio cholera: a multidrug structures. J Appl Crystallogr 26: 283–291 resistance ABC transporter homolog in a closed conformation. Locher KP, Lee AT, Rees DC (2002) The E. coli BtuCD structure: a J Mol Biol 330: 419–430 framework for ABC transporter architecture and mechanism. Chang G, Roth CB (2001) Structure of MsbA from E. coli: a homolog Science 296: 1091–1098 of the multidrug resistance ATP binding cassette (ABC) transpor- Loo TW, Bartlett MC, Clarke DM (2002) The ‘LSGGQ’ motif in each ters. Science 293: 1793–1800 nucleotide-binding domain of human P-glycoprotein is adjacent Chang XB, Tabcharani JA, Hou YX, Jensen TJ, Kartner N, Alon N, to the opposing walker A sequence. J Biol Chem 277: 41303– Hanrahan JW, Riordan JR (1993) Protein kinase A (PKA) 41306 still activates CFTR chloride channel after mutagenesis of Lukacs GL, Chang XB, Bear C, Kartner N, Mohamed A, Riordan JR, all 10 PKA consensus phosphorylation sites. J Biol Chem 268: Grinstein S (1993) The DF508 mutation decreases the stability of 11304–11311 cystic fibrosis transmembrane conductance regulator in the plas- Chen J, Lu G, Lin J, Davidson AL, Quiocho FA (2003) A tweezers- ma membrane. Determination of functional half-lives on trans- like motion of the ATP-binding cassette dimer in an ABC trans- fected cells. J Biol Chem 268: 21592–21598 port cycle. Mol Cell 12: 651–661 McRee DE (1999) XtalView/Xfit—a versatile program for manipu- Chen JM, Scotet V, Ferec C (2000) Definition of a ‘functional R lating atomic coordinates and electron density. J Struct Biol 125: domain’ of the cystic fibrosis transmembrane conductance reg- 156–165 ulator. Mol Genet Metab 71: 245–249 Merritt EA, Bacon DJ (1997) Raster3D photorealistic molecular Cheng SH, Gregory RJ, Marshall J, Paul S, Souza DW, White GA, graphics. Methods Enzymol 277: 505–524 O’Riordan CR, Smith AE (1990) Defective intracellular transport Moody JE, Millen L, Binns D, Hunt JF, Thomas PJ (2002) and processing of CFTR is the molecular basis of most cystic Cooperative, ATP-dependent association of the nucleotide bind- fibrosis. Cell 63: 827–834 ing cassettes during the catalytic cycle of ATP-binding cassette Cheng SH, Rich DP, Marshall J, Gregory RJ, Welsh MJ, Smith AE transporters. J Biol Chem 277: 21111–21114 (1991) Phosphorylation of the R domain by cAMP-dependent Mossessova E, Lima CD (2000) Ulp1-SUMO crystal structure and protein kinase regulates the CFTR chloride channel. Cell 66: genetic analysis reveal conserved interactions and a regulatory 1027–1036 element essential for cell growth in yeast. Mol Cell 5: 865–876 Christopher JA (1998) SPOCK: The Structural Properties Neville DC, Rozanas CR, Tulk BM, Townsend RR, Verkman AS Observation and Calculation Kit. Program Manual College (1998) Expression and characterization of the NBD1-R domain Station, TX: A&M University region of CFTR: evidence for subunit–subunit interactions. Csanady L, Chan KW, Seto-Young D, Kopsco DC, Nairn AC, Gadsby Biochemistry 37: 2401–2409 DC (2000) Severed channels probe regulation of gating of cystic Ostedgaard LS, Baldursson O, Welsh MJ (2001) Regulation of the fibrosis transmembrane conductance regulator by its cytoplasmic cystic fibrosis transmembrane conductance regulator Cl-channel domains. J Gen Physiol 116: 477–500 by its R domain. J Biol Chem 276: 7689–7692 Dalemans W, Barbry P, Champigny G, Jallat S, Dott K, Dreyer D, Ostedgaard LS, Rich DP, DeBerg LG, Welsh MJ (1997) Association of Crystal RG, Pavirani A, Lecocq JP, Lazdunski M (1991) Altered domains within the cystic fibrosis transmembrane conductance chloride ion channel kinetics associated with the delta F508 cystic regulator. Biochemistry 36: 1287–1294 fibrosis mutation. Nature 354: 526–528 Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data Duffieux F, Annereau JP, Boucher J, Miclet E, Pamlard O, Schneider collected in oscillation mode. Methods Enzymol 276: 307–326 M, Stoven V, Lallemand JY (2000) Nucleotide-binding domain 1 Qu BH, Strickland EH, Thomas PJ (1997) Localization and suppres- of cystic fibrosis transmembrane conductance regulator produc- sion of a kinetic defect in cystic fibrosis transmembrane conduc- tion of a suitable protein for structural studies. Eur J Biochem 267: tance regulator folding. J Biol Chem 272: 15739–15744 5306–5312 Qu BH, Thomas PJ (1996) Alteration of the cystic fibrosis trans- Fiser A, Do RK, Sali A (2000) Modeling of loops in protein membrane conductance regulator folding pathway. J Biol Chem structures. Protein Sci 9: 1753–1773 271: 7261–7264 Gelman MS, Kopito RR (2002) Rescuing protein conformation: Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, prospects for pharmacological therapy in cystic fibrosis. J Clin Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL et al Invest 110: 1591–1597 (1989) Identification of the cystic fibrosis gene: cloning Hopfner KP, Karcher A, Shin DS, Craig L, Arthur LM, Carney JP, and characterization of complementary DNA. Science 245: Tainer JA (2000) Structural biology of Rad50 ATPase: ATP-driven 1066–1073 conformational control in DNA double-strand break repair and Rosing J, Harris DA, Kemp Jr A, Slater EC (1975) Nucleotide-binding the ABC-ATPase superfamily. Cell 101: 789–800 properties of native and cold-treated mitochondrial ATPase. Hung LW, Wang IX, Nikaido K, Liu PQ, Ames GF, Kim SH (1998) Biochim Biophys Acta 376: 13–26 Crystal structure of the ATP-binding subunit of an ABC transpor- Schmitt L, Benabdelhak H, Blight MA, Holland IB, Stubbs MT ter. Nature 396: 703–707 (2003) Crystal structure of the nucleotide-binding domain of the Karpowich N, Martsinkevich O, Millen L, Yuan YR, Dai PL, ABC-transporter Haemolysin B: identification of a variable region MacVey K, Thomas PJ, Hunt JF (2001) Crystal structures of the within ABC helical domains. J Mol Biol 330: 333–342 MJ1267 ATP binding cassette reveal an induced-fit effect at the Schmitt L, Tampe R (2002) Structure and mechanism of ABC ATPase active site of an ABC transporter. Structure (Camb) 9: transporters. Curr Opin Struct Biol 12: 754–760 571–586 Sippl MJ (1993) Recognition of errors in three-dimensional struc- Kennelly PJ, Krebs EG (1991) Consensus sequences as substrate tures of proteins. Proteins 17: 355–362 specificity determinants for protein kinases and protein phospha- Smith PC, Karpowich N, Millen L, Moody JE, Rosen J, Thomas PJ, tases. J Biol Chem 266: 15555–15558 Hunt JF (2002) ATP binding to the motor domain from an ABC 292 The EMBO Journal VOL 23 NO 2 2004 &2004 European Molecular Biology Organization | | Structure of CFTR NBD1 HA Lewis et al transporter drives formation of a nucleotide sandwich dimer. Mol substrates: cooperation with exonuclease and DNase I. J Biol Cell 10: 139–149 Chem 276: 48404–48409 Verdon G, Albers SV, Dijkstra BW, Driessen AJM, Thunnissen Wilkinson DJ, Strong TV, Mansoura MK, Wood DL, Smith SS, AWH (2003) Crystal structures of the ATPase subunit of the Collins FS, Dawson DC (1997) CFTR activation: additive effects blucose ABC transporter from Sulfolobus solfataricus: nucleo- of stimulatory and inhibitory phosphorylation sites in the R tide-free and nucleotide-bound conformations. J Mol Biol 330: domain. Am J Physiol 273: L127–L133 343–358 Winter MC, Welsh MJ (1997) Stimulation of CFTR activity by its Vergani P, Nairn AC, Gadsby DC (2003) On the mechanism of phosphorylated R domain. Nature 389: 294–296 MgATP-dependent gating of CFTR Cl() channels. J Gen Physiol Yike I, Ye J, Zhang Y, Manavalan P, Gerken TA, Dearborn DG (1996) 121: 17–36 A recombinant peptide model of the first nucleotide-binding fold Wang W, He Z, O’Shaughnessy TJ, Rux J, Reenstra WW (2002) of the cystic fibrosis transmembrane conductance regulator: Domain–domain associations in cystic fibrosis transmem- comparison of wild-type and delta F508 mutant forms. Protein brane conductance regulator. Am J Physiol Cell Physiol 282: Sci 5: 89–97 C1170–C1180 Yuan YR, Blecker S, Martsinkevich O, Millen L, Thomas PJ, Hunt JF Weeks CM, Miller R (1999) Optimizing shake-and-bake for proteins. (2001) The crystal structure of the MJ0796 ATP-binding cassette. Acta Crystallogr D 55: 492–500 Implications for the structural consequences of ATP hydrolysis Widlak P, Li LY, Wang X, Garrard WT (2001) Action of recombinant in the active site of an ABC transporter. J Biol Chem 276: human apoptotic endonuclease G on naked DNA and chromatin 32313–32321 &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 2 2004 293 | | http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Structure of nucleotide‐binding domain 1 of the cystic fibrosis transmembrane conductance regulator

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Abstract

The EMBO Journal (2004) 23, 282–293 & 2004 European Molecular Biology Organization All Rights Reserved 0261-4189/04 | | THE THE www.embojournal.org EMB EMB EMBO O O JO JOU URN R NAL AL Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator 1, 1 Hal A Lewis *, Sean G Buchanan , Introduction 1 1 Stephen K Burley , Kris Conners , Mark Cystic fibrosis (CF) is the most prevalent lethal, autosomal- 1 2 Dickey , Michael Dorwart , Richard recessive genetic disease among Caucasians. CF patients have 1 1 3 Fowler , Xia Gao , William B Guggino , severely reduced life expectancies, largely because of chronic 4 5 Wayne A Hendrickson , John F Hunt , pulmonary damage. The root cause of CF is in defective cystic 1 1 Margaret C Kearins , Don Lorimer , Peter C fibrosis transmembrane conductance regulator (CFTR) 3 1 Maloney , Kai W Post , Kanagalaghatta R (Riordan et al, 1989). Human CFTR is a 1480 residue, multi- 1 1 Rajashankar , Marc E Rutter , J Michael domain, integral membrane protein that regulates chloride 1 1 Sauder , Stephanie Shriver , Patrick H ion flow across the cell membrane. It is a member of the ATP- 2 2 binding cassette (ABC) transporter superfamily of proteins Thibodeau , Philip J Thomas , Marie 1 1 1 and consists of two membrane-spanning domains (MSDs), Zhang , Xun Zhao and Spencer Emtage two nucleotide-binding domains (NBDs), and a regulatory 1 2 Structural GenomiX Inc., San Diego, CA, USA, Department of region (R) arranged in the order MSD1–NBD1–R–MSD2– Physiology, University of Texas Southwestern Medical Center, Dallas, NBD2 (Figure 1). The NBDs of ABC transporters are typified TX, USA, Department of Physiology, School of Medicine, The Johns Hopkins University, Baltimore, MD, USA, Department of Biochemistry by a consensus ATP-binding region, which encompasses two and Molecular Biophysics, Howard Hughes Medical Institute, Columbia Walker motifs (A and B regions), a highly conserved region University, New York, NY, USA and Department of Biological Sciences, called the signature sequence (LSGGQ), plus other conserved Columbia University, New York, NY, USA functional features identified as the Q- and H-loops, named respectively for glutamine and histidine residues involved in Cystic fibrosis transmembrane conductance regulator ATP recognition and hydrolysis. The most common CF muta- (CFTR) is an ATP-binding cassette (ABC) transporter that tion is the deletion of CFTR phenylalanine 508 (DF508), functions as a chloride channel. Nucleotide-binding do- which is located in NBD1. In total, 70% of CF alleles have main 1 (NBD1), one of two ABC domains in CFTR, also DF508 and 90% of CF patients have at least one copy of this contains sites for the predominant CF-causing mutation deletion. A better understanding of the structure and function and, potentially, for regulatory phosphorylation. We have of NBD1 and the role of Phe508 may accelerate the develop- determined crystal structures for mouse NBD1 in unli- ment of new approaches to the treatment of CF. ganded, ADP- and ATP-bound states, with and without Atomic-level structural information has been obtained for phosphorylation. This NBD1 differs from typical ABC components of several ABC transporter systems. Complete domains in having added regulatory segments, a foreshor- bacterial transmembrane transporter proteins MsbA (Chang tened subdomain interconnection, and an unusual nucleo- and Roth, 2001; Chang, 2003) and BtuCD (Locher et al, 2002) tide conformation. Moreover, isolated NBD1 has have been analyzed at modest resolution, showing similar undetectable ATPase activity and its structure is essen- associations between NBD and MSD domains but markedly tially the same independent of ligand state. Phe508, which different overall architectures. High-resolution X-ray struc- is commonly deleted in CF, is exposed at a putative NBD1- tures have also been determined for several prokaryotic NBDs transmembrane interface. Our results are consistent with (Schmitt and Tampe, 2002), most recently HlyB (Schmitt et al, a CFTR mechanism, whereby channel gating occurs 2003) and GlcV (Verdon et al, 2003), and for one eukaryotic through ATP binding in an NBD1–NBD2 nucleotide sand- NBD, TAP1 (Gaudet and Wiley, 2001). All such NBDs have a wich that forms upon displacement of NBD1 regulatory common fold characterized by two subdomains: one contains segments. an F1-like ATP-binding core plus an ABC-specific antiparallel The EMBO Journal (2004) 23, 282–293. doi:10.1038/ b region and the other an ABC-specific a-helical domain sj.emboj.7600040; Published online 18 December 2003 (Karpowich et al, 2001). The F1-like portion contains the Subject Categories: structural biology; molecular biology of primary determinants of nucleotide binding; the antiparallel disease b portion adds interactions to the base and ribose groups; and Keywords: ABC transporter; CFTR; crystal structure; NBD1 the ABC signature sequence of the a-helical domain from a dimer mate completes productive coordination of the ATP b- and g-phosphate groups of the nucleotide (Figure 1). Typical ABC transporters are thought to function as labile dimers in which coupling of ATP hydrolysis to movements in transmembrane segments drives the translocation of relevant *Corresponding author. Structural GenomiX Inc., 10505 Roselle St., San entities across the membrane. The Rad50 DNA repair Diego, CA 92121, USA. Tel.: þ 1 858 228 1555; Fax: þ 1 858 457 4533; enzyme, a remote homolog of ABC proteins, provided the E-mail: hal_lewis@stromix.com first structural model for the dimer state (Hopfner et al, 2000), and similar dimers form in BtuCD (Locher et al, Received: 23 September 2003; accepted: 25 November 2003; Published online: 18 December 2003 2002) and in a catalytically impaired E171Q variant of 282 The EMBO Journal VOL 23 NO 2 2004 &2004 European Molecular Biology Organization | | Structure of CFTR NBD1 HA Lewis et al solubility of such NBD1 constructs from 10 organisms (human, mouse, baboon, macaque, sheep, rabbit, frog, sal- mon, killifish, and dogfish). The high-level production of soluble proteins (45 mg/l) that are nonaggregated in solu- tion was limited to a narrow globular domain definition (N-terminus: residues 385–391; C-terminus: residues 670– 680). Optimal recombinant protein was obtained from mouse CFTR with an expression construct spanning residues 389–673. Attempts with human NBD1 have not yet suc- ceeded. We refer to the resulting mouse proteins as mNBD1 and mNBD1-P for the unphosphorylated and phosphorylated forms, respectively. Dynamic light scattering (DLS) and ana- lytical gel filtration chromatography (GFC) measurements indicated that purified, recombinant mNBD1 was both mono- disperse and monomeric. This protein was used for all of the structural studies reported here. A mutated version, K464A, which was expected to have reduced ATP binding, was also cloned and purified in the same manner and was used for Figure 1 Domain organization of CFTR. The five domains of CFTR ATP-binding measurements (see below). are shown. Also indicated is a putative nucleotide-binding domain association in which the ATP-binding site of one NBD is opposed by mNBD1 crystallization and structure determination the signature sequence of the other NBD. Inactivity at the NBD1 Both native (S-Met) and selenomethionyl (Se-Met) mNBD1 ATP-binding site is indicated by Ser residues in place of the catalytic Glu and His in addition to His residues substituted for the Gln and crystallized in two morphologies under similar conditions: central Gly in the NBD2 signature sequence. parallelepipeds in space group P42 2 and tetragonal bipyra- mids in space group I4 22. The structure of mNBD1 bound to Mg-AMP.PNP was initially determined by multiple MJ0796 when complexed with ATP (Smith et al, 2002). The isomorphous replacement with anomalous scattering nucleotides in this symmetric dimer are sandwiched at the (MIRAS) phasing from Ta Br -soaked I4 22 crystals. This 6 12 1 interface between protomers such that the LSGGQ residues was subsequently improved using single-wavelength anom- from one complete the binding interactions of nucleotide in alous diffraction (SAD) data from Se-Met mNBD1 P42 2 the apposing protomer. The putative functional NBD ‘dimer’ crystals. Most of the protein sequence could be built into in CFTR is believed to be intramolecular and necessarily the SAD-generated experimental electron density map, the asymmetric since ‘hemichannel’ constructs produced wild- primary exception being residues 413–428, which were not type activity when expressed together but not separately well visualized (exact boundaries are given in Table I for each (Ostedgaard et al, 1997; Chan et al, 2000). refined molecule). The initial structure of mNBD1 was sub- The boundaries of CFTR NBD1 have been a matter of some sequently used to solve the I4 22 structure in the presence of controversy. It has been suggested to begin in the span from Mg-ATP by molecular replacement and to determine isomor- residue 373 (Wang et al, 2002) to residue 441 (Bianchet et al, phous P42 2 structures in the presence of Mg-ATP, Mg-ADP, 1997) at the N-terminus and to end in the span from residue and in the absence of nucleotide. The conformation of 586 (Riordan et al, 1989) to residue 684 (Bianchet et al, 1997) mNBD1 is essentially the same in all crystal forms irrespec- at the C-terminus (human CFTR numbering, used throughout tive of the identity of the bound nucleotide and also in each of this paper). A compelling functional definition based on the the four copies in the P42 2 crystals and two copies in the coexpression of severed and deleted CFTR constructs gave I4 22 crystal. Each mNBD1 molecule is associated in a four- boundaries within 433–633 for NBD1 (Chan et al, 2000). fold symmetric, head-to-tail ring structure that recurs three Many different expression constructs have been used in times in the P42 2 lattice and once in the I4 22 lattice. 1 1 studies of NBD1 function, sometimes yielding conflicting results. Indeed, several aspects of CFTR NBD1 function Overall structure of CFTR NBD1 remain poorly characterized. The most important outstand- The mNBD1 domain has a core tertiary structure similar to ing issues pertain to ATP binding and hydrolysis, conforma- NBDs from other ABC transporters, but this core is modified tional adaptability in the domain, the effects of NBD1 with major additions and deletions. Figure 2B shows a phosphorylation, and the structural consequences of dis- topology diagram of mNBD1, indicating through color coding ease-causing mutations, in particular DF508. We have deter- the subdomains and those regions of mNBD1 that show mined the crystal structure of murine NBD1 to shed light significant differences from other ABC structures. on these vital mechanistic issues. Secondary structural elements in common with most known ABC structures are given conventional designations (S1, S2, S3, H1, etc.) and additional elements found in Results mNBD1 are denoted with lowercase letters (H1b, H1c, S6b, mNBD1 expression and purification etc.). The three-dimensional course of the polypeptide chain Given the uncertainty in the globular domain definition of is shown as a ribbon diagram in Figure 3A, where the CFTR NBD1, we chose to clone and express in Escherichia coli structural elements are colored in the same code as in many constructs in parallel covering residues from 363 to Figure 2B and key elements in the binding of ATP are also 686. A pan-genomic approach was employed, testing the identified. Figure 3B shows a worm diagram of mNBD1 in &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 2 2004 283 | | Structure of CFTR NBD1 HA Lewis et al 284 The EMBO Journal VOL 23 NO 2 2004 &2004 European Molecular Biology Organization | | Table I Data collection and refinement statistics Space group Resolution (A) Nucleotide Completeness (%) R (%) Redundancy I/s(I) sym (overall/outer shell) (overall/outer shell) (overall/outer shell) (overall/outer shell) mNBD1 data collection mNBD1+AMP.PNP P42 2 33.0–2.50 AMP.PNP 99.9/99.6 12.2/35 14.1/13.6 5.4/1.8 mNBD1-P+ATP P42 2 24.0–2.35 ATP 97.6/92.4 9.0/46 4.6/2.4 14.7/2.1 mNBD1 apo P42 2 38.9–2.20 None 93.2/95.1 7.6/44 5.1/4.1 19.8/3.1 mNBD1+ATP P42 2 39.2–2.20 ATP 98.5/92.7 7.3/50 7.0/5.7 27.7/4.0 mNBD1+ADP P42 2 36.5–2.55 ADP 100.0/99.4 7.7/35 9.8/9.9 8.0/1.9 mNBD1+ATP (2) I4 22 49.7–3.00 ATP 100.0/100.0 7.3/98 8.2/8.3 27.6/2.0 Resolution (A) RR Waters RMSD bond length RMSD bond angles Average B-factors free ˚ ˚ (A) (1) (A ) Refinement statistics mNBD1+AMP.PNP (1Q3 H) 33.0–2.50 0.215 0.266 658 0.008 1.9 36.4 mNBD1-P+ATP (1R0Z) 24.0–2.35 0.221 0.258 304 0.015 1.5 48.0 mNBD1 apo (1R0W) 38.9–2.20 0.231 0.262 423 0.021 2.0 30.8 mNBD1+ATP (1R0X) 39.2–2.20 0.234 0.266 378 0.021 2.0 39.1 mNBD1+ADP (1R0Y) 36.0–2.55 0.207 0.257 195 0.015 1.7 45.6 mNBD1+ATP (2) (1R10) 30.0–3.00 0.228 0.265 0 0.012 1.3 80.6 Ramachandran distribution Molecule A Molecule B Molecule C Molecule D Core Allowed Disallowed Residues modeled mNBD1+AMP.PNP 390–412, 429–670 389–412, 429–670 388–412, 429–670 390–412, 92.0% 7.9% 0.0% 429–670 mNBD1-P+ATP 390–413, 420–670 389–412, 420–670 388–413, 420–670 390–407, 430–670 91.2% 8.7% 0.1% mNBD1 apo 390–413, 429–670 389–413, 429–670 388–413, 430–670 390–412, 430–670 92.4% 7.5% 0.1% mNBD1+ATP 390–411, 429–670 389–411, 429–670 388–411, 430–670 390–411, 430–670 91.2% 8.7% 0.0% mNBD1+ADP 388–413, 429–671 388–411, 428–670 388–412, 430–670 390–412, 430–671 89.9% 9.9% 0.1% mNBD1+ATP (2) 391–412, 429–670 391–412, 429–670 — — 85.5% 14.5% 0.0% R ¼ S S |I (hkl)/I(hkl)S|/S S I (hkl), where I is the intensity of the observation and /IS is the mean intensity of the reflection. R ¼ S||F ||F ||/S|F |, where F and F are the observed and sym hkl i i hkl i i i o c o o c calculated structure-factor amplitudes. R is the same as R except calculated for 5% of the data randomly omitted from the refinement. RMSD is root-mean-square deviation. Average B-factors are free reported for all nonhydrogen atoms. The values in parenthesis following the dataset names are the Protein Data Bank identifiers for the respective structures. Structure of CFTR NBD1 HA Lewis et al Figure 2 Sequence alignment and topology diagram of mNBD1. (A) Sequence alignment of human and mouse NBD1 and NBD2 with NBD domains from other ABC transporters. A blue background indicates b-strands while pink indicates a-helices in known structures. Solid circles mark the locations of common CF-causative mutations, solid triangles the locations of deletion mutations, and P indicates where phosphorylation by PKA was observed in the mNBD1–P structure. Residues with high sequence conservation in ABC domains are highlighted in blue bold font. Red bold font indicates residues that significantly vary from this conservation. The secondary structure of mNBD1 is indicated graphically above the residue numbering row and is color coded by subdomain as in Figure 2B. (B) Topology diagram of mNBD1. The F1-type ATP-binding core subdomain is shown in gold, the ABC a-subdomain in cyan, and the ABC b-subdomain in green. Regions of mNBD1 that are different from previous ABC structures are shown in gray. Circles indicate the positions of 3 helices. &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 2 2004 285 | | Structure of CFTR NBD1 HA Lewis et al which the thickness of the trace is proportional to the B- factors of the C atoms, thereby reflecting potential mobility of the polypeptide backbone. Regions of highest mobility are near the N- and C-termini and at the inserted and partially disordered segment between S1 and S2. B-factors are com- monly elevated near termini and in some loops, but there may be special relevance here in relation to the putative NBD1/NBD2 interface (see below). The core structure of mNBD1-ATP most closely resembles that of TAP1-ADP with a root-mean-square deviation (r.m.s.d.) of 1.9 A for 176 C atoms spanning a region with 26% sequence identity. MJ0796-ATP is next closest overall (2.4 A r.m.s.d. over the same span; 30% sequence identity). This alignment omits the regions between S1 and S2, be- tween S4 and S6, and beyond S10, which exhibit conforma- tion differences between ABC structures. Figure 2A presents a structure-based sequence alignment of mNBD1 with the ABC domains of some known structures and with selected se- quences from human ABC transporters, including human CFTR. These comparisons should help to clarify the discus- sions of CFTR function. Four major structural features distinguish mNBD1 from other ABC NBDs. This is evident in Figure 3C, where mNBD1 is superimposed onto three representative ABC structures. First, mNBD1 contains an insertion of about 35 residues between b-strands S1 and S2. This insertion is composed of two short a-helices (denoted H1b and H1c) separated by a flexible linker region that was not observed in the electron density map (residues 413–428, red dotted line in Figure 3A). The N-terminal b-strand S1 is common to all ABC domain structures, and a conserved aromatic residue near its C-terminus (corresponding to Trp401 in CFTR) stacks against the adenine base in previously reported structures of ABC domains complexed with nucleotides. The insertion leads to an altered binding geometry for the base and ribose in mNBD1 and includes a segment (415–432) that can be deleted while preserving function (Chan et al, 2000). Second, mNBD1 lacks a 14–27 residue region between b-strands S4 and S6 (residues 485–488) that typically con- tains an additional b-strand and an a-helix (denoted S5 and Figure 3 Structural fold of mNBD1. (A) Stereo ribbon diagram of mNBD1. ATP is shown in ball and stick representation. The sub- H2 in Figure 2A; located lower left in Figure 3C). Third, domains are color coded as in Figure 2B. The dotted red line relative to the prokaryotic ABC structures, mNBD1 is trun- indicates residues missing from the mNBD1 model. (B) Stereo cated between helices H3 and H4 (lower right of Figure 3C). worm diagram of mNBD1. The worm thickness is indicative Finally, the C-terminus of mNBD1 includes a long a-helix of the relative B-factor of the residues ranging from 18 A (thinnest) to 65 A (thickest). Color coding of subdomains is according to (H9b) that is not present in other ABC domain structures. The Figure 2B. (C) Stereo C diagram of the superposition of represen- hydrophobic face of this amphipathic a-helix packs against tative ABC domain structures onto mNBD1. TAP1 (thin red), a hydrophobic surface formed primarily by the preceding MJ0796 (thin green), and HisP (thin yellow) structures superim- a-helix H9. The H9b segment of the polypeptide chain has posed onto mNBD1 (thick blue). Superposition based on least- squares alignments of the F1-type core and ABC-specific antiparallel been considered to be part of the R domain as it contains two b-subdomains. (D) Backbone structure of mNBD1 illustrating posi- potentially regulatory phosphorylation sites, Ser660 and tions of phosphorylation and CF-causative mutations. Left: mNBD1 Ser670 (Chen et al, 2000). However, the structure and our is seen in the same orientation and subdomain colorization as in expression experiments lead us to think of H9b as an integral Figure 3A. Right: the same structure rotated 801 toward the viewer. Helices of regulatory segments are drawn as ribbons; the remaining component of the mNBD1 fold; at a minimum, the associa- polypeptide chain is a worm drawing. ATP is shown as ball and tion is favorable. stick. Ser422, Ser659, Ser660, and Ser670 side chains are shown in purple. Residues 420–428 become ordered upon phosphorylation Preparation, crystallization, and structure (solid red). The remaining residues of the structure that were not modeled (414–419) are indicated as red dots. Side chains are shown determination of triphospho-mNBD1 at sites of common CF-causative mutations (Ala455, Gly480, Ile506, Phosphorylation of CFTR, particularly in the R domain, is Ile507, Ser549, Gly551, Ala559, Arg560, Tyr569, and Asp648 colored thought to regulate channel opening and closing, and protein yellow; Phe508 in green). The di-acidic code residues (D565 and kinase A (PKA) plays an important role in this process D567) are in gold. (Ostedgaard et al, 2001). There are 21 PKA phosphorylation motifs (Kennelly and Krebs, 1991) in human CFTR. About 286 The EMBO Journal VOL 23 NO 2 2004 &2004 European Molecular Biology Organization | | Structure of CFTR NBD1 HA Lewis et al one-quarter of these putative phosphorylation sites are lo- rotated away due to a backbone g torsion angle of gauche þ cated in mNBD1 (Ser422, Ser489, Ser519, Ser557, Ser660, and as opposed to trans, which is seen in other ABC domain Ser670), and the murine sequence presents an additional PKA structures (Figure 4B). As a result, the adenine base of the site at Ser659. To identify CFTR NBD1 residues available for nucleotide does not stack against an aromatic side chain. PKA phosphorylation in vitro, we incubated mNBD1 with Instead, the position of the usual stacking aromatic residue PKA and analyzed the results using phosphopeptide mapping (Tyr11 in MJ0796) is taken up by Trp401, which is in close by mass spectrometry. Residues Ser422, Ser659, Ser660, and contact with the ATP ribose (3.3 A). Phe430, which is in the Ser670 were seen to be phosphorylated with Ser659 to a S1–S2 insertion, makes an ‘edge-to-face’ interaction with the lesser extent of only about one-quarter. adenine base. Leu409 makes contacts approaching from the The phosphorylated form of the protein, mNBD1-P, crystal- opposite side of the ring. No hydrogen bonding contacts are lized isomorphously with the unmodified protein and we observed with the adenine. were able to determine its structure. Those portions of In the phosphorylated structure, mNBD1-P, residues 420– mNBD1 that are well-ordered in the unphosphorylated state 428 contribute additional contacts to the adenine base of the are unchanged in the mNBD1-P structure, but the presence of nucleotide (Figure 4C). Val428 is in van der Waals contact phosphorserine 422 confers order on residues 420–428 (solid with the adenine as is Glu425, which stacks partially under- red ribbon segment in Figure 3D) in the inserted loop where a neath the base. Together with Phe430, these residues serve to phosphate group is visible on the side chain of Ser422. In grip the nucleotide base. A hydrogen bond between the side addition, phosphorylation is evident at residues Ser660 and chain of His421 and the phosphate of Ser660 contributes Ser670, and partially at Ser659 (only seen in molecule A in to the conformational stabilization of this region. the asymmetric unit). Phosphorylation at Ser422 (Chang et al, ATP binding and hydrolysis in solution 1993), Ser660 (Cheng et al, 1991; Winter and Welsh, 1997), ATP binding to mNBD1 had an apparent K of 149748 mM and Ser670 (Wilkinson et al, 1997) has been observed in (Figure 5A). Previously measured values for nucleotide affi- human CFTR and each has been shown to have a specific nity have ranged from low micromolar (Yike et al, 1996; Qu effect on the activity of CFTR. Based on this evidence as well et al, 1997) to several millimolar (Ko et al, 1993), varying as discussions on the relevance of these regions to a putative with both the nucleotide analog used as well as the protein NBD1–NBD2 heterodimer (see below), we will refer to the construct. The values reported here are consistent with those S1–S2 loop and H9c structures as the regulatory insertion and of human NBD1 determined by several other groups (Yike regulatory extension, respectively. et al, 1996; Qu et al, 1997; Neville et al, 1998). ATP binding was significantly reduced by the K464A mutation, presum- Nucleotide coordination in mNBD1 crystal structures ably because electrostatic interactions with the b- and Nucleotides in the various crystal complexes with mNBD1 g-phosphates of ATP (Figure 4A) are lost. (Table I) are all bound in a very similar manner. This mode of ATP hydrolysis by mNBD1 in solution was measured in a binding has aspects in common with that in other ABC standard coupled assay system (Rosing et al, 1975). An active transporter NBDs, but the comparison is distinguished as ABC NBD ATPase, MJ0796, was included as a positive con- much by differences as similarities. Since Mg-ATP has its trol. Consistent with previous experiments (Moody et al, g-phosphate intact when cocrystallized with mNBD1, whether 2002), the MJ0796 protein hydrolyzed ATP at a rate approach- phosphorylated or not, mNBD1 is not an active ATPase as is ing 8 min , while the mNBD1 exhibited a hydrolytic rate less typical for such domains. Moreover, since the protein con- than 0.02 min in the colorimetric assays in both the unpho- formation is the same when uncomplexed or complexed with sphorylated and phosphorylated states (Figure 5B). The dis- ADP as when complexed with ATP (r.m.s.d. ¼ 0.2–0.3 A), the parity with previously published results (Ko and Pedersen, possibility is raised that nucleotide binding to the NBD1 site 1995; Duffieux et al, 2000; Annereau et al, 2003) may reflect in CFTR is not involved directly in movements thought to differences in ATP binding between mouse and human drive ABC transporter action in other cases (Yuan et al, 2001). proteins or, more likely, are due to other factors associated Canonical features of nucleotide binding in mNBD1 all with alternative constructs. involve interactions with the phosphate groups (Figure 4A). Specifically, Lys464 and Thr465 (Walker A), Asp572 (Walker Discussion B), and Gln493 (Q-loop) hydrogen bond with the phosphates 2þ and/or coordinate the Mg ion as in other ABC domain Intrinsic structural integrity structures. On the other hand, consistent with a catalytically The crystal structures of mNBD1 have features that distin- inactive site, there are also significant differences. Namely, guish them from other ABC cassette proteins. The structure is the Walker-B carboxylate residue, typically glutamate, that essentially the same independent of nucleotide state, nucleo- serves as the catalytic base in active ABC transporters tides bind in an unusual conformation at the ribose-phos- (Moody et al, 2002) is replaced by serine in NBD1 (Ser573), phate linkage, and regulatory segments are added to the core and the canonical H-loop histidine residue becomes Ser605. structure. Since mNBD1 molecules associate into head-to-tail Both of these fail to hydrogen bond with the g-phosphate as tetramers in our crystals, we have considered whether these in related complexes (Hung et al, 1998; Smith et al, 2002). lattice contacts (B900 A from each surface) might affect the The distinction from usual nucleotide binding by ABC intrinsic structure. The predominant interactions involve domains is even more pronounced for ribose and base residues from the Q-loop and a-subdomain at the ‘head’ of interactions in mNBD1. The ATP ribose is C3 -endo with an one molecule contacting H1c residues near the ATP site at the ‘anti’ w torsion angle base conformation as in other ATP- ‘tail’ of another molecule. bound ABC structures (eg MJ0796 (Smith et al, 2002) and While lattice influences cannot be ruled out, several lines HisP (Hung et al, 1998)), but the ribose-base assembly is of evidence suggest that they do not overwhelm the intrinsic &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 2 2004 287 | | Structure of CFTR NBD1 HA Lewis et al Figure 4 Close-up on ATP binding by mNBD1. (A) Canonical hydrogen bonding interactions in Mg-ATP mNBD1. Some relevant hydrogen bonds are indicated as green lines. Some residues in the foreground and background have been removed to clarify the interactions, here and in (B) and (C). (B) Differences in adenine base recognition from other ABC domains. Left: adenine stacks against Tyr11 of MJ0796 (PDB ID code 1L2T). Right: adenine of ATP makes edge-to-face interactions with Phe430 of mNBD1. (C) Added structure in phosphorylated mNBD1. The ATP molecule plus magnesium in blue, the additional mNBD1 residues observed in the phosphorylated state in white, the phosphate atoms of Ser422 and Ser660 in brown, and the remainder of the mNBD1 structure in yellow are shown. mNBD1 structure. With respect to the relative disposition of sequences of CFTR NBD1 from diverse vertebrate species are ABC a- and F1-ATPase subdomains, two facts are most compe- very similar (61–98%, pairwise identity) and divergent seg- lling: (1) The same I4 22 crystals grow spontaneously and ments are restricted to surface residues and loop regions. This equally well from mNBD1 in Mg-ATP, Mg-ADP, and nucleo- suggests that all NBD1s share the deviations from canonical tide-free states. (2) Intramolecular interactions between a-and ABC domains that distinguish mNBD1. In particular, human F1-subdomains in mNBD1 (five hydrogen bonds and 34 NBD1 should be very close in structure to mNBD1 as these residue pairs in van der Waals contact) are much more molecules are 78% identical in sequence over the span of extensive than in the head-to-tail interface (one hydrogen mNBD1 and there are no amino-acid insertions or deletions bond and 16 van der Waals pairs). With respect to the unusual (Figure 2A). It seems likely that its structure will also be ribose-base conformation of nucleotides in mNBD1, this is similar in intact CFTR, but functional interactions with the largely determined by the placement of Trp401 in the S1–H1b NBD2, MSD1, and/or R domains of CFTR (Figure 1) might segment, which is not involved in any contacts. well lead to adaptations in NBD1. Indications of potential plasticity in NBD1 come from the crystallographic measures of atomic mobility in mNBD1. Structural implications Parts of the regulatory insertion between S1 and S2 are We expect NBD1 domains from all CFTRs to have essentially sufficiently flexible that they are not seen in the density the same structure as that seen here for mNBD1. The known 288 The EMBO Journal VOL 23 NO 2 2004 &2004 European Molecular Biology Organization | | Structure of CFTR NBD1 HA Lewis et al mNBD1 (approximately residues 390–670), but the NBD1 core may well be stabilized by other interactions in situ. NBD1 phosphorylation sites are located in these ‘deletable’ segments, and perhaps they can be displaced in intact CFTR when phosphorylated such that exposed surfaces can interact productively with other domains, notably NBD2. Enzymatic activity Our results demonstrate that mNBD1 is incapable, on its own, of catalyzing significant ATP hydrolysis: ATP remains intact in mNBD1 crystal structures, and no significant hydrolysis is observed in our solution assays. This lack of activity is not unexpected from the model espoused in Figure 1 whereby NBD1–NBD2 ‘heterodimers’ are thought to mediate normal catalysis in CFTR, and likely only do so at the site where NBD2 provides the Walker A and B sequences (NBD2-based site). The catalytic base, usually glutamate, in the NBD1- based site is replaced by serine at position 573, and this putative site has other defects as well. We now know from the structures that there are no candidate replacements within NBD1 for the catalytic base. On the other hand, since the phosphate groups of ATP are otherwise coordinated in a conventional manner in mNBD1, the g-phosphate is poised for attack and it is conceivable that some unanticipated part of CFTR might confer catalytic competence. However, NBD1 in intact CFTR does not appear to be enzymatically active (Basso et al, 2003; Vergani et al, 2003). Interactions between NBD1 and NBD2 Our working hypothesis for the role of NBDs in CFTR function is that NBD1 interacts with NBD2 in the mode of a labile nucleotide sandwich, as depicted in Figure 1; ATP binding to the NBDs, and hydrolysis at NBD2, then couples through NBD–MSD associations to control the channel. This model is consistent with biochemical and structural informa- tion on homodimeric ABC transporter systems (Locher et al, Figure 5 ATP binding and hydrolysis by mNBD1. (A) ATP binding 2002; Moody et al, 2002; Smith et al, 2002; Chen et al, 2003). to mNBD1 was analyzed using a filter-binding assay. Wild type It is also consistent with ‘heterodimeric’ crosslinking between (K), K464A mutant (J), and ATP binding to the filters in the NBD1 and NBD2 in human multidrug-resistant P glycoprotein absence of protein (.) are shown. The wild-type K was calculated to be 149748 mM. (B) ATPase activity was analyzed by a coupled (Loo et al, 2002) and with tryptophan-fluorescence assays of colorimetric assay. ATPase activity for mNBD1, MJ0796 (LolD), and direct interactions for human CFTR between NBD1-R (373– background are shown as a function of changes in NADH absor- 859), a construct that encompasses the entire span of our bance at 340 nm. Inset: ATP hydrolysis rates were calculated to be mNBD1, and NBD2 (1151–1476) (Wang et al, 2002). Finally, it less than 0.02 min for the mNBD1, as compared to the positive control, MJ0796, which was 7.9 min . is consistent with electrophysiological studies of CFTR func- tion (Vergani et al, 2003), which indicate that normal channel opening requires Mg-ATP binding at both nucleotide sites and that normal channel closing follows ATP hydrolysis in the NBD2-based site. In order to pursue the hypothesis, we maps and B-factors are elevated for the ordered parts, H1b developed homology models for CFTR NBD2 and for the and H1c, of the insertion (Figure 3B). Similarly, B-factors are NBD1–NBD2 ‘heterodimer’. also well above average for the helices H8–H9b. These We have docked our mNBD1 structure and our NBD2 elements are candidates for displacement by interactions homology model into a hypothetical NBD1–NBD2 ‘hetero- with other CFTR domains or other regulatory events. dimer’ constructed with reference to the structure of the Evidence that this may indeed happen comes in comparing E171Q mutant MJ0796 homodimer (Smith et al, 2002). This our results with results from the coexpression of severed model, in what is expected to be the universal dimerization CFTR constructs (Chan et al, 2000). Wild-type conductance mode of ABC transporters, exhibits severe main-chain steric levels were observed from two-chain CFTR channels con- clashes between NBD1 and NBD2 (Figure 6). Specifically, the structed such that either 415–432 (the flexible loop in our inserted loop region (405–436) and C-terminal a-helix (H9b) structures) or 634–667 (H8–H9b region) was omitted, sug- are thrust directly into the opposing NBD2 model. There are gesting that these features may not be vital for CFTR function. several ways in which this conflict might be resolved. One We were unable to generate stable NBD1 proteins from possibility is that there is a conformational change in NBD1 constructs any narrower than the ordered confines of upon interdomain association, whereby the clashing regions &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 2 2004 289 | | Structure of CFTR NBD1 HA Lewis et al Figure 6 Worm figure of putative NBD1–NBD2 interaction. NBD1 is color coded as in Figure 2B. NBD2 is in red. The NBD1 worm thickness is proportional to the backbone B-factor. The figure at the right is rotated 901 toward the viewer relative to the left figure. are moved out of the way. The inserted loop and H9b 1990), perhaps due to improper folding (Qu and Thomas, extension have elevated B-factors (Figures 3B and 6) and 1996) and instability of CFTR (Lukacs et al, 1993), which is the correspondingly greater atomic mobilities may indicate detected by the protein degradation machinery in the endo- hinged conformational flexibility. Moreover, sites of phos- plasmic reticulum (ER) and lysosomes (Gelman and Kopito, phorylation in mNBD1-P are all located on the S1–S2 inser- 2002). The limited number of DF508 molecules that are tion and on the H9b extension (Figure 3D), and these retained form active chloride channels, but with altered clashing segments might possibly be displaced when phos- gating properties (Dalemans et al, 1991). phorylated to take up favorable interactions with other parts Phe508 lies exposed on the surface of mNBD1, positioned of CFTR, thereby promoting and maintaining NBD associa- at the start of a loop between helices H3 and H4. This site is tion. This hypothesis is consistent with data from R-domain distant (420 A) from either ATP in the ‘heterodimer’ model, deletions, which release CFTR gating inhibitions upon phos- suggesting that DF508 is unlikely to affect ATP binding phorylation (Rich et al, 1991) to give opening kinetics similar directly. The side chain of Phe508 is in the vicinity of to phosphorylated wild-type CFTR but with less stable open Arg560, Trp496, and Met498, but it makes only a single van channels (Csanady et al, 2000). der Waals contact, with Met498. Thus, Phe508 does not appear to play a critical role in stabilizing mNBD1 structure. Interactions between NBD1 and MSD1 Moreover, its main-chain contacts are only local, so accom- Associations between the NBD and MSD domains are ex- modation of the deletion through loop reorganization seems pected to provide the conduit of communication from nucleo- plausible. Disruptions are expected to be substantial never- tide-dependent conformational changes in NBD domains to theless. One possible impact of DF508 might be on accessi- the gating of transmembrane channels in ABC transporters. bility to the di-acidic code proposed to be involved in The structures of other ABC transporters (Chang and Roth, membrane protein transport out of the ER (Barlowe, 2003). 2001; Locher et al, 2002; Chang, 2003) provide an insight into Another impact may be on the NBD1–MSD1 interface into the nature of these associations. In each case, a-domain and which Phe508 is expected to be buried (see above). CF Q-loop portions of an NBD interact with cytoplasmic exten- mutations concentrate in the a-subdomain or contacting sions from the transmembrane portion of one MSD–NBD sites, and disruptive consequences are evident from the exclusively. We constructed homology models of CFTR structure for some. MSD1–NBD1 based on our structure of mNBD1 docked onto the corresponding region of the MsbA and BtuCD Materials and methods structures (not shown). The surface of NBD1 that would be at the interface with mMSD1 overlaps with a surface that is Protein preparation and phosphorylation buried into the head-to-tail rings in mNBD1 crystal lattices. Mouse CFTR NBD1 (residues 389–673; human numbering) was expressed in E. coli as an N-terminal His –Smt3 fusion protein This surface includes the hydrophobic side chains from 6 (Mossessova and Lima, 2000) using a pET26b-derived expression Phe494, Trp496, Met498, Val540, and Phe508, the most vector. Cells were harvested by centrifugation and lysed by common CF mutation site. sonication. Protein was initially purified by nickel ion affinity chromatography, followed by proteolytic removal of the tag using Ulp1 and GFC. A final nickel ion affinity step was employed to CF mutations in NBD1 remove traces of the His –Smt3 affinity tag. Final yields averaged The majority of sites of CF-causing missense mutations occur 4 mg/l with greater than 98% purity. The purified protein was in NBD1, primarily in its a-subdomain, and the locations in concentrated to 5–10 mg/ml in buffer containing 150 mM NaCl, the mNBD1 structure of the most common of these (A455E, 12.5% glycerol, 2 mM DTT, 2 mM ATP, and 50 mM Tris pH 7.5. Se- G480C, I506T, DI507, DF508, S549N, S549R, G551D, A559T, Met containing protein was expressed and purified using similar procedures. R560T, Y569D, and D648V; Bobadilla et al, 2002) are shown To generate phosphorylated mNBD1, 1000 U of bovine PKA in Figure 3D. The deletion of Phe508, DF508, is by far the (Sigma P2645-1KU) was incubated with 5 mg of mNBD1 at a most prevalent. This mutation leads to reduced CFTR trans- concentration of 1 mg/ml at 41C for 4 h, and then concentrated to port to, and retention in, the plasma membrane (Cheng et al, 5–10 mg/ml. Mass spectrometric characterization verified phos- 290 The EMBO Journal VOL 23 NO 2 2004 &2004 European Molecular Biology Organization | | Structure of CFTR NBD1 HA Lewis et al phorylation of three or four sites (full modification: Ser422, Ser660, buffer lacking ATP (50 mM Tris–HCl pH 7.6, 150 mM NaCl, 2 mM DTT, 12.5% glycerol) using Bio-Rad Micro Bio-Spin 6 chromato- and Ser670; partial, approximately 25% modification: Ser659). graphy columns following the manufacturer’s protocols. mNBD1 Protein characterization protein (375 pmol) was incubated with increasing concentrations of Matrix-assisted laser desorption ionization and electrospray ioniza- an Mg-a P-ATP mixture for 45 min at room temperature in a final tion mass spectrometric analyses confirmed the identity of the reaction volume of 20 ml. The protein was then applied to Millipore purified proteins and estimated purity at greater than 95%. DLS HA 0.45 mm filters that were prewetted with buffer on a 12-position measurements using a DynaPro-MS800 instrument made at protein Millipore vacuum manifold. The samples were washed three times concentrations up to 8 mg/ml showed a single monomeric species. with 2 ml of ice-cold buffer supplemented with 40 mM MgCl and Analytical gel filtration at a protein concentration of 4 mg/ml then counted on a Beckman LS-6500 scintillation counter. yielded similar results. ATP hydrolysis by NBD1 Crystallization, data collection, structure determination, ATP hydrolysis by mNBD1 was measured using a colorimetric assay and refinement described previously (Rosing et al, 1975). ATP hydrolysis was Purified mNBD1 at 5–10 mg/ml was subjected to initial hanging- measured via a coupled reaction where NADH is converted to drop vapor-diffusion screens with more than 500 different NAD in a one-to-one ratio with ADP production. The reaction was precipitant conditions at two temperatures (4 and 201C). From this monitored by changes in absorbance at 340 nm. In total, 1 nmol it was found that a simple precipitant consisting of 3.5–4.0 M mNBD1 was incubated in reaction buffer (100 mM Tris pH 8.0, sodium acetate with no additional buffer or salt and pH adjusted to 50 mM KCl, 2 mM MgCl , 0.2 mM EDTA, 4 mM Mg-ATP, 1 mM PEP, 7.5 yielded diffraction-quality crystals in two forms (I4 22: 200mM NADH, 4 U/ml LDH, 8 U/ml PK) in a final volume of ˚ ˚ a ¼ 140 A, c¼ 280 A, two molecules/asymmetric unit; P42 2: 1.015 ml at 301C and monitored by changes in A on a Shimadzu ˚ ˚ a ¼ 172 A, c ¼ 110 A, four molecules/asymmetric unit). Nucleotide UV2101 spectrophotometer. MJ0796 was used as a positive control complexes were generated through cocrystallization, except in the for ATP hydrolysis. The rates of ATP hydrolysis were calculated as a NADH case of ADP in which soaking of mNBD1 crystals with 1 mM function of the changes in A using e ¼ 6.22 mM/cm. nucleotide was employed. Crystals were frozen by immersion in liquid nitrogen prior to data collection. Diffraction data were Homology modeling measured at the SGX-CAT beamline 31-ID at the Advanced Photon Homology models were built using SCWRL3 (Bower et al, 1997) Source under standard cryogenic conditions, and processed with and Modeller 6v2 (Fiser et al, 2000). Based on the assumption that either the Mosflm (CCP4, 1994) or HKL (Otwinowski and Minor, CFTR NBD1 and NBD2 can interact, a heterodimer model was 1997) software. created using CFTR mNBD1 as a template for each domain and The structure was initially determined in space group I4 22 using several NBD structures as templates for the dimer interaction, the MIRAS method. Two Ta Br derivative data sets, diffracting to 6 12 including BtuD (1L7V) and MJ0796 (1L2T). The Walker A, LSGGQ, 4.0 A, were collected at the peak energy of tantalum and used with a and Walker B motifs of NBD1 were structurally aligned with each native 3.1 A data set in phasing calculations. Cluster sites located ABC monomer using Spock (Christopher, 1998), and then CFTR with SNB (Weeks and Miller, 1999) gave phasing powers in NBD1/NBD2 was modeled simultaneously. Multiple models were MLPHARE (CCP4, 1994) of 2.12 and 1.98 for the two derivatives generated and the best model was chosen based on Modeller’s and produced experimental phases useful to 4.0 A. Two copies of a energy term, ProsaII analysis (Sippl, 1993), and visual inspection. homology model of NBD1 based on the TAP1 structure (PDB code 1JJ7) were manually placed in the density-modified (DM; CCP4, Figure preparation 1994) electron density map. Further density modification with NCS Structure figures were made with MOLSCRIPT (Kraulis, 1991) and averaging then produced better maps and allowed a more extensive Raster3D (Merritt and Bacon, 1997), except for Figures 3C, 4A, 4B, NBD1 backbone construction. and 4C which were made using Xtalview (McRee, 1999) and Figure The mNBD1 backbone from the I4 22 structure was positioned 3B and 6 which were made using Spock. by molecular replacement into the P42 2 lattice using a 2.9 A data set acquired at the selenium peak energy of an SeMet crystal. A Bijvoet-difference Fourier based on resulting model phases allowed Data deposition the identification of 21 Se sites, which were refined with MLPHARE Coordinates have been deposited in the Protein Data Bank (http:// using 2.5 A native P42 2 data. The resulting phases were NCS www.rcsb.org/pdb). Accession codes are listed in Table I. averaged to obtain a final map that was used for building the side chains and finalizing the model. The structure of NBD1 was built manually over several cycles of model building with XtalView Acknowledgements (McRee, 1999) and refinement with CNX (Brunger et al, 1998) and/ or Refmac (CCP4, 1994). Subsequent structures were determined We thank Drs E de la Fortelle and T Harris for many useful using the molecular replacement program EPMR (Kissinger et al, discussions; Dr A Verkman for supplying putative CFTR ligands 1999), and refined to convergence. PROCHECK (Laskowski et al, used in some cocrystallization efforts; L Pelletier, F Lu, K Bain, and 1993) revealed main-chain and side-chain structural parameters Drs S Antonysamy, IK Feil, J Hendle, B Noland, and F Park for their consistently better than average (overall G-values 41.0). See Table I expert contributions towards the expression, characterization, and for data collection and refinement statistics. crystallization of mNBD1; and K Schwinn for his help in making some of the figures. Use of the Advanced Photon Source was ATP binding to NBD1 supported by the US Department of Energy, Office of Science, and ATP binding was measured using a standard filter-binding assay Office of Basic Energy Sciences, under Contract No. W-31-109-Eng- (Widlak et al, 2001). Endogenous ATP added during purification 38. This work was supported by a research contract from the Cystic was first removed by exchanging (three times) the protein into Fibrosis Foundation. References Annereau JP, Ko YH, Pedersen PL (2003) Cystic fibrosis transmem- nucleotide binding domain and its role in channel gating. J Gen brane conductance regulator: the NBF1+R (nucleotide-binding Physiol 122: 333–348 fold 1 and regulatory domain) segment acting alone catalyses a Bianchet MA, Ko YH, Amzel LM, Pedersen PL (1997) Modeling 2+ 2+ 2+ Co /Mn /Mg -ATPase activity markedly inhibited by both of nucleotide binding domains of ABC transporter proteins 2+ Cd and the transition-state analogue orthovanadate. Biochem J based on a F1-ATPase/recA topology: structural model of the 371: 451–462 nucleotide binding domains of the cystic fibrosis transmem- Barlowe C (2003) Signals for COPII-dependent export from the ER: brane conductance regulator (CFTR). J Bioenerg Biomembr 29: what’s the ticket out? Trends Cell Biol 13: 295–300 503–524 Basso C, Vergani P, Nairn AC, Gadsby DC (2003) Prolonged non- Bobadilla JL, Macek Jr M, Fine JP, Farrell PM (2002) Cystic fibrosis: hydrolytic interaction of nucleotide with CFTR’s NH -terminal a worldwide analysis of CFTR mutations—correlation with in- &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 2 2004 291 | | Structure of CFTR NBD1 HA Lewis et al cidence data and application to screening. Hum Mutat 19: Kissinger CR, Gehlhaar DK, Fogel DB (1999) Rapid automated 575–606 molecular replacement by evolutionary search. Acta Crystallogr Bower MJ, Cohen FE, Dunbrack Jr RL (1997) Prediction of protein D 55: 484–491 side-chain rotamers from a backbone-dependent rotamer library: Ko YH, Pedersen PL (1995) The first nucleotide binding fold of the a new homology modeling tool. J Mol Biol 267: 1268–1282 cystic fibrosis transmembrane conductance regulator can func- Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse- tion as an active ATPase. J Biol Chem 270: 22093–22096 Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS et al. Ko YH, Thomas PJ, Delannoy MR, Pedersen PL (1993) The cystic (1998) Crystallography & NMR system: a new software suite for fibrosis transmembrane conductance regulator. Overexpression, macromolecular structure determination. Acta Crystallogr D 54: purification, and characterization of wild type and delta F508 905–921 mutant forms of the first nucleotide binding fold in fusion with CCP4 (1994) The CCP4 suite: programs for protein crystallography. the maltose-binding protein. J Biol Chem 268: 24330–24338 Acta Crystallogr D 50: 760–763 Kraulis PJ (1991) MOLSCRIPT: a program to produce both detailed Chan KW, Csanady L, Seto-Young D, Nairn AC, Gadsby DC (2000) and schematic plots of protein structures. J Appl Crystallogr 24: Severed molecules functionally define the boundaries of the 946–950 cystic fibrosis transmembrane conductance regulator’s NH(2)- Laskowski RJ, MacArthus MW, Moss DS, Thornton JM (1993) terminal nucleotide binding domain. J Gen Physiol 116: 163–180 PROCHECK: aprogram to check stereochemical quality of protein Chang G (2003) Structure of MsbA from Vibrio cholera: a multidrug structures. J Appl Crystallogr 26: 283–291 resistance ABC transporter homolog in a closed conformation. Locher KP, Lee AT, Rees DC (2002) The E. coli BtuCD structure: a J Mol Biol 330: 419–430 framework for ABC transporter architecture and mechanism. Chang G, Roth CB (2001) Structure of MsbA from E. coli: a homolog Science 296: 1091–1098 of the multidrug resistance ATP binding cassette (ABC) transpor- Loo TW, Bartlett MC, Clarke DM (2002) The ‘LSGGQ’ motif in each ters. Science 293: 1793–1800 nucleotide-binding domain of human P-glycoprotein is adjacent Chang XB, Tabcharani JA, Hou YX, Jensen TJ, Kartner N, Alon N, to the opposing walker A sequence. J Biol Chem 277: 41303– Hanrahan JW, Riordan JR (1993) Protein kinase A (PKA) 41306 still activates CFTR chloride channel after mutagenesis of Lukacs GL, Chang XB, Bear C, Kartner N, Mohamed A, Riordan JR, all 10 PKA consensus phosphorylation sites. J Biol Chem 268: Grinstein S (1993) The DF508 mutation decreases the stability of 11304–11311 cystic fibrosis transmembrane conductance regulator in the plas- Chen J, Lu G, Lin J, Davidson AL, Quiocho FA (2003) A tweezers- ma membrane. Determination of functional half-lives on trans- like motion of the ATP-binding cassette dimer in an ABC trans- fected cells. J Biol Chem 268: 21592–21598 port cycle. Mol Cell 12: 651–661 McRee DE (1999) XtalView/Xfit—a versatile program for manipu- Chen JM, Scotet V, Ferec C (2000) Definition of a ‘functional R lating atomic coordinates and electron density. J Struct Biol 125: domain’ of the cystic fibrosis transmembrane conductance reg- 156–165 ulator. Mol Genet Metab 71: 245–249 Merritt EA, Bacon DJ (1997) Raster3D photorealistic molecular Cheng SH, Gregory RJ, Marshall J, Paul S, Souza DW, White GA, graphics. Methods Enzymol 277: 505–524 O’Riordan CR, Smith AE (1990) Defective intracellular transport Moody JE, Millen L, Binns D, Hunt JF, Thomas PJ (2002) and processing of CFTR is the molecular basis of most cystic Cooperative, ATP-dependent association of the nucleotide bind- fibrosis. Cell 63: 827–834 ing cassettes during the catalytic cycle of ATP-binding cassette Cheng SH, Rich DP, Marshall J, Gregory RJ, Welsh MJ, Smith AE transporters. J Biol Chem 277: 21111–21114 (1991) Phosphorylation of the R domain by cAMP-dependent Mossessova E, Lima CD (2000) Ulp1-SUMO crystal structure and protein kinase regulates the CFTR chloride channel. Cell 66: genetic analysis reveal conserved interactions and a regulatory 1027–1036 element essential for cell growth in yeast. Mol Cell 5: 865–876 Christopher JA (1998) SPOCK: The Structural Properties Neville DC, Rozanas CR, Tulk BM, Townsend RR, Verkman AS Observation and Calculation Kit. Program Manual College (1998) Expression and characterization of the NBD1-R domain Station, TX: A&M University region of CFTR: evidence for subunit–subunit interactions. Csanady L, Chan KW, Seto-Young D, Kopsco DC, Nairn AC, Gadsby Biochemistry 37: 2401–2409 DC (2000) Severed channels probe regulation of gating of cystic Ostedgaard LS, Baldursson O, Welsh MJ (2001) Regulation of the fibrosis transmembrane conductance regulator by its cytoplasmic cystic fibrosis transmembrane conductance regulator Cl-channel domains. J Gen Physiol 116: 477–500 by its R domain. J Biol Chem 276: 7689–7692 Dalemans W, Barbry P, Champigny G, Jallat S, Dott K, Dreyer D, Ostedgaard LS, Rich DP, DeBerg LG, Welsh MJ (1997) Association of Crystal RG, Pavirani A, Lecocq JP, Lazdunski M (1991) Altered domains within the cystic fibrosis transmembrane conductance chloride ion channel kinetics associated with the delta F508 cystic regulator. Biochemistry 36: 1287–1294 fibrosis mutation. Nature 354: 526–528 Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data Duffieux F, Annereau JP, Boucher J, Miclet E, Pamlard O, Schneider collected in oscillation mode. Methods Enzymol 276: 307–326 M, Stoven V, Lallemand JY (2000) Nucleotide-binding domain 1 Qu BH, Strickland EH, Thomas PJ (1997) Localization and suppres- of cystic fibrosis transmembrane conductance regulator produc- sion of a kinetic defect in cystic fibrosis transmembrane conduc- tion of a suitable protein for structural studies. Eur J Biochem 267: tance regulator folding. J Biol Chem 272: 15739–15744 5306–5312 Qu BH, Thomas PJ (1996) Alteration of the cystic fibrosis trans- Fiser A, Do RK, Sali A (2000) Modeling of loops in protein membrane conductance regulator folding pathway. J Biol Chem structures. Protein Sci 9: 1753–1773 271: 7261–7264 Gelman MS, Kopito RR (2002) Rescuing protein conformation: Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, prospects for pharmacological therapy in cystic fibrosis. J Clin Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL et al Invest 110: 1591–1597 (1989) Identification of the cystic fibrosis gene: cloning Hopfner KP, Karcher A, Shin DS, Craig L, Arthur LM, Carney JP, and characterization of complementary DNA. Science 245: Tainer JA (2000) Structural biology of Rad50 ATPase: ATP-driven 1066–1073 conformational control in DNA double-strand break repair and Rosing J, Harris DA, Kemp Jr A, Slater EC (1975) Nucleotide-binding the ABC-ATPase superfamily. Cell 101: 789–800 properties of native and cold-treated mitochondrial ATPase. Hung LW, Wang IX, Nikaido K, Liu PQ, Ames GF, Kim SH (1998) Biochim Biophys Acta 376: 13–26 Crystal structure of the ATP-binding subunit of an ABC transpor- Schmitt L, Benabdelhak H, Blight MA, Holland IB, Stubbs MT ter. Nature 396: 703–707 (2003) Crystal structure of the nucleotide-binding domain of the Karpowich N, Martsinkevich O, Millen L, Yuan YR, Dai PL, ABC-transporter Haemolysin B: identification of a variable region MacVey K, Thomas PJ, Hunt JF (2001) Crystal structures of the within ABC helical domains. J Mol Biol 330: 333–342 MJ1267 ATP binding cassette reveal an induced-fit effect at the Schmitt L, Tampe R (2002) Structure and mechanism of ABC ATPase active site of an ABC transporter. Structure (Camb) 9: transporters. Curr Opin Struct Biol 12: 754–760 571–586 Sippl MJ (1993) Recognition of errors in three-dimensional struc- Kennelly PJ, Krebs EG (1991) Consensus sequences as substrate tures of proteins. Proteins 17: 355–362 specificity determinants for protein kinases and protein phospha- Smith PC, Karpowich N, Millen L, Moody JE, Rosen J, Thomas PJ, tases. J Biol Chem 266: 15555–15558 Hunt JF (2002) ATP binding to the motor domain from an ABC 292 The EMBO Journal VOL 23 NO 2 2004 &2004 European Molecular Biology Organization | | Structure of CFTR NBD1 HA Lewis et al transporter drives formation of a nucleotide sandwich dimer. Mol substrates: cooperation with exonuclease and DNase I. J Biol Cell 10: 139–149 Chem 276: 48404–48409 Verdon G, Albers SV, Dijkstra BW, Driessen AJM, Thunnissen Wilkinson DJ, Strong TV, Mansoura MK, Wood DL, Smith SS, AWH (2003) Crystal structures of the ATPase subunit of the Collins FS, Dawson DC (1997) CFTR activation: additive effects blucose ABC transporter from Sulfolobus solfataricus: nucleo- of stimulatory and inhibitory phosphorylation sites in the R tide-free and nucleotide-bound conformations. J Mol Biol 330: domain. Am J Physiol 273: L127–L133 343–358 Winter MC, Welsh MJ (1997) Stimulation of CFTR activity by its Vergani P, Nairn AC, Gadsby DC (2003) On the mechanism of phosphorylated R domain. Nature 389: 294–296 MgATP-dependent gating of CFTR Cl() channels. J Gen Physiol Yike I, Ye J, Zhang Y, Manavalan P, Gerken TA, Dearborn DG (1996) 121: 17–36 A recombinant peptide model of the first nucleotide-binding fold Wang W, He Z, O’Shaughnessy TJ, Rux J, Reenstra WW (2002) of the cystic fibrosis transmembrane conductance regulator: Domain–domain associations in cystic fibrosis transmem- comparison of wild-type and delta F508 mutant forms. Protein brane conductance regulator. Am J Physiol Cell Physiol 282: Sci 5: 89–97 C1170–C1180 Yuan YR, Blecker S, Martsinkevich O, Millen L, Thomas PJ, Hunt JF Weeks CM, Miller R (1999) Optimizing shake-and-bake for proteins. (2001) The crystal structure of the MJ0796 ATP-binding cassette. Acta Crystallogr D 55: 492–500 Implications for the structural consequences of ATP hydrolysis Widlak P, Li LY, Wang X, Garrard WT (2001) Action of recombinant in the active site of an ABC transporter. J Biol Chem 276: human apoptotic endonuclease G on naked DNA and chromatin 32313–32321 &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 2 2004 293 | |

Journal

The EMBO Journal – Springer Journals

Published: Jan 28, 2004

Keywords: ABC transporter; CFTR; crystal structure; NBD1

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