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High Level Expression and Dimer Characterization of the S100 EF-hand Proteins, Migration Inhibitory Factor-related Proteins 8 and 14

High Level Expression and Dimer Characterization of the S100 EF-hand Proteins, Migration... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 20, Issue of May 15, pp. 12427–12435, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. High Level Expression and Dimer Characterization of the S100 EF-hand Proteins, Migration Inhibitory Factor-related Proteins 8 and 14* (Received for publication, November 20, 1997) Michael J. Hunter‡ and Walter J. Chazin§ From the Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037 The phenotypical and functional heterogeneity of dif- nomenclature, MRP8 and MRP14 are designated as S100A8 ferent macrophage subpopulations are defined by dis- and S100A9, respectively (14). Other names for the MRP8/ crete changes in the expression of two S100 calcium- MRP14 complex include calprotectin (15), leukocyte-derived binding proteins, migration inhibitory factor-related protein (L1) light and heavy chains (16), p8/p14 (17), calgranu- proteins (MRPs) 8 and 14. To further our understanding lin A/B (9), and the cystic fibrosis antigen (11, 18). Some evi- of MRP8 and MRP14 in the developmental stages of dence suggests that MRP14 alone is the cystic fibrosis antigen inflammatory responses, overexpression of the MRPs (19), and murine MRP8 has been referred to as the murine was obtained through a combination of a T7-based ex- chemotactic cytokine (CP-10) (20). pression vector and the Escherichia coli BL21 (DE3) cell It has been suggested that MRP8 and MRP14 form a het- line. An efficient, two-step chromatographic protocol erodimeric complex in a calcium-dependent manner (21). How- was then developed for rapid, facile purification. Exten- ever, once this complex is formed, it is stable upon the addition sive biophysical characterization and chemical cross- of EDTA (22). It is not clear whether these proteins have linking experiments show that MRP8 and MRP14 form independent biological functions or whether their function is oligomers with a strong preference to associate as a strictly dependent on heterocomplex formation. Although the heterodimer. Heteronuclear NMR experiments indicate general consensus is that protein function is dependent on that a specific well packed dimer is formed only in heterodimer formation, studies also support the possibility that equimolar mixtures of the two proteins. Our results sug- these proteins may have individual functions. For example, gest that there is a unique complementarity in the in- MRP14 is shown to be expressed independently of MRP8 in terface of the MRP8/MRP14 complex that cannot be fully acutely inflamed tissues (23–25). reproduced in the MRP8 and MRP14 homodimers. Like a number of other S100 proteins (S100a, S100D, S100E, S100L, p11, CAPL, calcyclin/2A9, and psoriasin), the genes encoding MRP8 and MRP14 have been located on chromosome Macrophages, which belong to the mononuclear phagocyte 1q21 (11, 26, 27). Recently, it has been shown that the struc- system, form a heterogeneous cell population with different developmental and functional stages. The specific pathophysi- ture of the gene loci encoding S100 proteins is highly conserved (28). Although the primary protein sequences are similar, there cal situation dictates whether macrophages will perform cyto- toxic, endocytosic, or secretory functions. The phenotypical and is less similarity at the nucleotide level, with the greatest divergence in the untranslated regions. Since S100 proteins functional heterogeneity of different macrophage subpopula- tions are defined by discrete changes in the expression of two exhibit very specific patterns of expression, it has been sug- gested that these untranslated regions may be involved in S100 calcium-binding proteins, migration inhibitory factor-re- lated proteins (MRPs) 8 and 14 (1–3). Granulocytes, mono- directing the expression of specific S100 proteins in specific cell types (28). Analysis of the amino acid sequences of MRP8 and cytes/macrophages, neutrophils, and keratinocytes have been shown to express MRP8 and MRP14, suggesting that expres- MRP14 indicate that each protein is composed of two EF-hand domains characteristic of the S100 calcium-binding protein sion is tightly regulated during granulocyte differentiation (4 – 9). In addition, elevated serum levels of MRP8 and MRP14 family (27, 29, 30). S100 proteins are characterized by rela- tively low molecular masses, generally on the order of 10 kDa, have been found in patients suffering from a number of inflam- matory disorders including cystic fibrosis, rheumatoid arthri- and a unique consensus sequence in their calcium-binding sites making them a diverse, multifaceted family of proteins (31). tis, and chronic bronchitis (10 –13), suggesting possible extra- cellular roles for these proteins. In the new S100 protein They have been shown to play roles in cell cycle progression, regulation of intracellular phosphorylation events, and cy- toskeletal membrane interactions, as well as extracellular * This research was supported in part by National Institutes of events such as the stimulation of glial proliferation, prolactin Health Grant PO1 GM48495 (to W. J. C.). The costs of publication of secretion, and neuronal differentiation (30, 32). this article were defrayed in part by the payment of page charges. This MRP8 and MRP14 have molecular masses of 11 and 14 kDa article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. and are composed of 93 and 114 amino acids, respectively. A ‡ Recipient of a postdoctoral fellowship from the National Arthritis number of possible functions for these proteins have been pro- Foundation. posed including differentiation of myeloid lineage cells, regula- § Faculty Research Fellow of the American Cancer Society. To whom tion of intracellular calcium levels in neutrophils, inhibition of correspondence should be addressed: Dept. of Molecular Biology (MB- 9), The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, casein kinase I and II activity, and a zinc-mediated biostatic CA 92037. Tel.: 619-784-9860; Fax: 619-784-9985; E-mail: chazin@ activity (19, 21, 33, 34). The extended carboxyl-terminal tail of scripps.edu. MRP14 makes it the largest member of the S100 family, and it The abbreviations used are: MRP, migration inhibitory factor-re- is believed that MRP14 represents the regulatory unit of this lated protein; PCR, polymerase chain reaction; HPLC, high pressure liquid chromatography; HSQC, heteronuclear single quantum coherence. unique heterodimeric complex. Studies have shown that the This paper is available on line at http://www.jbc.org 12427 This is an Open Access article under the CC BY license. 12428 Expression and Characterization of MRP8 and MRP14 and sequencing of mutants were performed as described for the wild- penultimate amino acid of MRP14, Thr , is phosphorylated in type proteins. vivo (8, 35) and that the phosphorylation event triggers the Protein Expression—Overexpression of the gene products was translocation of this protein to the cytoskeleton (36). Possible achieved in E. coli strain BL21(DE3). For unlabeled protein, cells were functions for the tail of MRP14 may include neutrophil immo- grown at 37 °C in (23 YT) media supplemented with ampicillin to a bilization (37), regulation of cytoskeletal translocation of the final concentration of 100 mg/ml. 500-ml cultures were inoculated with MRP8/MRP14 complex (36), and inhibition of the onset of the 5 ml of an overnight culture grown in the same media. Cells were allowed to grow another 18 –24 h before harvesting. Since the pET1120 intrinsic coagulation cascade (38). The only function assigned plasmid contains no lac repressor gene, protein expression was not to MRP8 to date is the potent chemotactic activity assigned to tightly controlled, and the system was leaky enough to produce large linker region of murine MRP8 (20, 39 – 41). amounts of protein in rich media without the need for induction. To further our understanding of the roles MRP8 and MRP14 For N-labeled protein, cells were grown at 37 °C in M9 minimal play in the developmental stages of inflammatory responses, media supplemented with basal medium Eagle vitamin solution (Life we have cloned these two proteins and developed highly effi- Technologies, Inc.), NH Cl as the sole nitrogen source, and glucose as cient expression and purification protocols. This has allowed the carbon source. Ampicillin was added to a final concentration of 100 mg/ml. 5-ml inoculants were grown overnight, and inoculated cultures for biophysical characterization by UV, CD, fluorescence, and between 0.1 and 0.5 before induction were allowed to grow to an A heteronuclear NMR spectroscopies, as well as chemical cross- with isopropyl-thio-b-D-galactoside to a final concentration of 0.05 mM. linking experiments. MRP8 and MRP14 are shown to be stable Cells were grown an additional 24 h before harvesting. globular proteins that form dimers and preferentially associate Protein Purification—Cells were harvested and inclusion bodies were as heterodimers. isolated following methods described by Sambrook et al. (42). The pellet containing the inclusion bodies was resuspended in 10 mM Tris-HCl, 8 EXPERIMENTAL PROCEDURES M urea, 20 mM dithiothreitol at pH 8.0 in a volume sufficient to com- Construction of the Expression Vectors—The MRP8 and MRP14 pletely dissolve the pellet. These protein solutions were dialyzed for 4 cDNAs were a generous gift of Professor Clemens Sorg (University of M Tris-HCl, 2.5 mM EDTA/EGTA, pH days against 2 3 4 liters of 50 m Mu ¨ nster, Germany). The primers MRP8 –59 (59-GGA ATT CCA TAT 8.0, in 3.5-kDa cut-off dialysis membrane. The dialyzed solutions were GTT GAC CGA GCT GGA GAA-39) and MRP8 –39 (59-CGG GAT CCC centrifuged at 18,000 3 g for 20 min and then filtered through a 0.2 mM ACT CTT TGT GGC TTT CTT C-39) were used to PCR amplify the acrodisc syringe filter (Gelman Sciences, Ann Arbor, MI) prior to fur- human MRP8 gene from the pMRP-8-trp expression vector, and the ther purification. primers MRP14 –59 (59-GGA ATT CCA TAT GAC TTG CAA AAT GTC The MRPs were isolated by preparative reverse-phase HPLC on a GCA G-39) and MRP14 –39 (59-CGG GAT CCT TAG GGG GTG CCC DeltaPak C column (PrepPak 500 cartridge, Waters Corp., Milford, TCC CC-39) were used to amplify the human MRP14 gene from the MA) using a gradient of 36 –51% acetonitrile, 0.1% trifluoroacetic acid pMRP-14-trp expression vector. A NdeI restriction site was engineered over 25 min. Fractions containing protein were lyophilized and then into the 59-primers, and a BamHI restriction site was engineered into dissolved in 25 mM Tris-HCl, pH 8.0, and further purified by anion the 39-primers for proper insertion of the gene into the pET1120 ex- exchange chromatography on a Perceptive Biosystems BioCAD Sprint pression vector. Amplification was conducted in a total volume of 100 ml perfusion chromatography system using a Mono-Q anion exchange col- containing 10 mM KCl, 20 mM Tris-HCl (pH 8.8 at 25 °C), 10 mM umn (Amersham Pharmacia Biotech). The protein was eluted in a (NH ) SO , 2.0 mM MgSO , 0.1% Triton X-100, 0.3 mM dNTPs, 5.0 units 4 2 4 4 buffer containing 25 mM Tris-HCl, 1.0 mM EDTA/EGTA, pH 8.0, with a of vent DNA polymerase, and 100 pmol of primers. A small amount of 0 – 0.5 M NaCl gradient at a flow rate of 5 ml/min. All buffers used in the mineral oil was added to the top of the reaction mixture to inhibit purification of wild-type proteins contained 20 mM dithiothreitol to evaporation. The reaction mixtures were heated to 95 °C for 5 min, inhibit disulfide bond formation. Fractions containing protein were followed by 25–30 cycles of 95, 60, and 75 °C for 30 s each and a final desalted and concentrated using Millipore Ultrafree-4 centrifugal filter extension at 75 °C for 2 min. devices (5-kDa cut-off). Extinction coefficients were determined from 1 PCR products were purified using a QIAquick PCR purification kit mg/ml samples of MRP8C42S and MRP14C3S by acid hydrolysis and (QIAGEN Inc., Chatsworth, CA). Full-length oligonucleotides were dou- amino acid analysis. bly digested with 20 units BamHI and 40 units NdeIfor2hat37 °C. Isoelectric Focusing—Isoelectric focusing was performed on a Mul- The digested fragments were purified on a 1.3% agarose gel and stained tiphor II isoelectric focusing system (Amersham Pharmacia Biotech) with ethidium bromide. The bands containing the MRP8 and MRP14 using Servalyt Precote polyacrylamide gels (SERVA, Heidelberg, Ger- genes were excised and purified using a QIAEX II gel extraction kit. many) and a Pharmalyte 3–10 broad pI calibration kit. Sample volumes The MRP8 and MRP14 genes were then cloned into the NdeI and were 5 ml at a protein concentration of 3 mg/ml. The maximum power BamHI sites of the pET1120 vector using standard methods (42). The was set to 4 watts, and the initial voltage was set to 200 V. Focusing pET1120 vector was created by isolating the two BamHI/PstI fragments continued until a maximum voltage of 2000 V was reached. Gels were of Novagen’s pET11 and pET20 vectors and ligating the large fragment fixed for 10 min in a 20% solution of trichloroactetic acid, stained with of pET20 digestion with the small fragment of pET11 digestion. These SERVA Blue W dye tablets for 5 min with constant shaking, and plasmids have the b-lactamase gene and multiple cloning sites down- destained in H O until background color was at an acceptable level. stream of the efficient T7-polymerase promoter but are void of the lac Isoelectric points were determined by plotting the retention versus repressor gene. Competent DH5a cells were transformed with the isoelectric point of known standards. pET1120-MRP8wt and pET1120-MRP14wt vectors and used to produce Chemical Cross-linking—The analysis of MRP8/MRP14 complex for- plasmid stocks. Proper insertion and DNA sequences were confirmed by mation was carried out by using the bis(sulfosuccinimidyl)suberate fluorescent thermal dye DNA sequencing methods (43). (Pierce) method described by Teigelkamp et al. (21). Purified recombi- Mutagenesis—To construct the MRP8C42S gene, the primers, nant MRP8 and MRP14 were diluted in phosphate-buffered saline at MRP8C42S-59 (59-CTC GGT CTC GAG CAA TTT CTT C-39) and pH 8.5 to a final concentration of 50 mM. Cross-linking was initiated by MRP8 –59 were used to PCR amplify the 59-half of the MRP8C42S gene, the addition of freshly prepared bis(sulfosuccinimidyl)suberate (20 mM and the primers MRP8C42S-39 (59-TTG CTC GAG ACC GAG TCT CCT stock solution) to a final concentration of 5 mM. Solutions were incu- CAG TAT ATC-39) and MRP8 –39 were used to PCR amplify the 39-half M Tris to bated at room temperature for 30 min before the addition of 1 of the MRP8C42S gene. The two amplified half-fragments were purified quench the reaction. Cross-linked samples were incubated with b-mer- on a 1.5% agarose gel and visualized using ethidium bromide. The captoethanol prior to visualization on SDS-polyacrylamide gel bands containing the two fragments were excised and purified using a electrophoresis. QIAEX II gel extraction kit (QIAGEN). The two purified half-fragments CD Spectroscopy—Circular dichroism experiments were carried out were used as primers for each other, and the full gene was made using on an Aviv 61DS spectropolarimeter with 1-mm path length quartz the PCR methods described above. Since this did not result in the cuvettes. Protein solutions (20 mM MRP8C42S, 20 mM MRP14C3S, and amplification of the entire gene, a small aliquot of the above reaction 10 mM MRP8C42S plus 10 mM MRP14C3S) were prepared in 1 mM mixture, which had small amounts of the full-length mutant gene, was Tris-HCl and 0.5 mM EDTA, pH 8.5. The solution was brought to 2.5 mM used as a template to PCR amplify the full-length MRP8C42S gene using MRP8 –59 and MRP8 –39 as the amplification primers. To con- CaCl , to obtain the spectra of the calcium-loaded state. Spectra were struct the MRP14C3S gene, the primers MRP14C3S-59 (59-GGA ATT collected with an average time of 3 s for each point and a step size of CCA TAT GAC TAG TAA AAT GTC GCA GCT GG-39) and MRP14 –39 0.50 nm from 200 to 260 nm. All spectra were collected in triplicate and were used with the wild-type template in one PCR step. Transformation background-corrected against a buffer blank. A perl program (least- Expression and Characterization of MRP8 and MRP14 12429 ellip.pl) was written to determine the optimal least squares fit. Data 2 21 were converted to mean residue ellipticity [u] (degrees cm dmol )by using [u] 5 u/(10 lcn), where u is the measured ellipticity, l is the cell path length in cm, c is the molar concentration of protein in mol/liter, and n is the number of residues/chain. The a-helical content was esti- mated by using the mean residue ellipticity at 222 nm (44). Protein concentrations (20 –25 mM) were determined by using the bicinchoninic acid method (Pierce). Fluorescence Spectroscopy—Measurements of the fluorescence exci- tation and emission spectra were conducted at 25 °C on a SLM- AMINCO Series 2 Spectrofluorometer equipped with a Hewlett Pack- ard Pavilion 5040 personal computer. Excitation spectra were measured from 250 to 320 nm with emission monitored at 335 nm. Scans were performed at 1 nm/s with the excitation and emission band pass set at 16 and 4 nm, respectively. Emission spectra were measured FIG.1. Expression and purification of recombinant MRP8 and from 300 to 450 nm using an excitation wavelength of 290 nm. Scans MRP14. Lanes A and B, total cell lysates from bacteria transformed were performed at 1 nm/s with the excitation and emission band pass with the pET1120-MRP8C42S and MRP14C3S expression vectors, re- spectively; lanes C–H, soluble cytosolic fractions of the cell lysate (lane set at 4 nm. Each 1.5-ml sample contained 5 mM total protein C and D), Triton X-100 fractions (lanes E and F), and 8 M urea fractions (MRP8C42S, MRP14C3S, or a 1:1 mixture) in 1 mM Tris-HCl, 0.5 mM (lanes G and H) of MRP8C42S and MRP14C3S, respectively. Lanes I EDTA at pH 8.5. For the calcium-loaded state, 1 M CaCl was added to and J, ion exchange-purified MRP8C42S and MRP14C3S, respectively. a final concentration of 1.5 mM. All samples were preequilibrated over- night prior to the fluorescence measurements. Each experiment was collected in triplicate, and a perl program (leastgen.pl) was written to fit MRP14 was cloned into the bacterial expression vector the data using a least squares algorithm. NMR Spectroscopy—Wild type and mutant protein samples were pET1120. This expression vector was previously shown to effi- dissolved and concentrated in a buffer containing 10 mM Tris-HCl, ciently overexpress the very closely related S100 protein calcy- 100 mM KCl, and 5 mM EDTA/EGTA, at pH 8.5. For NMR samples of clin. The cDNAs were engineered with 59 NdeI restriction sites wild-type protein, deuterated dithiothreitol was also added to a final and 39 BamHI restriction sites for facile insertion into the concentration of 5.0 mM. Pure H O was added to a final concentra- expression vector downstream of the efficient T7 polymerase tion of 10% for the spectrometer lock system. Spectra were recorded 15 15 promoter. All gene sequences were confirmed by standard DNA for the following samples: N-MRP8wt, N-MRP8wt 1 MRP14C3S, 15 15 15 N-MRP8C42S 1 MRP14C3S, N-MRP14wt, MRP8C42S 1 N- sequencing methods. MRP14wt, and MRP8C42S 1 N-MRP14C3S. The mixtures were MRP8 and MRP14 each contain a single cysteine residue. To made up in a 1:1.2 ratio of labeled to unlabeled protein (0.75–1.0 mM avoid problems associated with the formation of unwanted final concentration). disulfide bonds, the MRP8C42S and MRP14C3S mutant genes All NMR experiments were performed at 27 °C on a Bruker AMX500 1 15 were also constructed and inserted into the pET1120 expres- operating at 499.87 MHz for H and 50.65 MHz for N. Phase-sensitive sion vector. DNA sequencing showed that the gene encoding two-dimensional data were recorded using the method of States et al. (45). H chemical shifts were referenced to the H O peak at 4.75 ppm, MRP14C3S had a single base pair mutation (G to A) at position 15 1 and the N shifts were referenced indirectly using the H frequency of 318. However, both codons (AAG, AAA) code for the amino acid 15 1 the H O resonance (46). The N- H HSQC spectra (47) were recorded lysine, making this a silent mutation. with 16 transients/increment, a total of 128 complex points in t and 512 points in t , and spectral widths of 1500.15 Hz in v and 4000 Hz in v . 2 1 2 Protein Expression All two-dimensional data sets were processed on a Sun SPARCstation LX workstation using FELIX (version 95.0; MSI, San Diego, CA.). The expression vectors containing the cDNA for MRP8wt, Homology Model of MRP8/MRP14 —The model of apo-MRP8/ MRP8C42S, MRP14wt, and MRP14C3S were used to trans- MRP14 was generated using the Homology module of the InsightII form the protease-deficient E. coli strain BL21(DE3). In rich package (MSI, San Diego, CA), based on the low resolution structure media, BL21(DE3) cells transformed with the expression plas- of the apo state of the related S100 protein calcyclin (29). Calcyclin is mids overexpressed proteins that migrated in SDS gels with a symmetric homodimer, so the model was created with MRP8 apparent molecular masses of 8 kDa (MRP8wt, MRP8C42S) or aligned to one subunit and MRP14 aligned to the other. The auto- mated sequence alignment algorithm in the software was used to 14 kDa (MRP14wt, MRP14C3S). Overexpression of MRP8 and define the conserved regions of MRP8 and MRP14. Residues 1– 42 MRP14 proved not to be detrimental to the bacteria, presum- and 51– 88 of MRP8 were assigned to residues 1– 42 and 53–90 of ably because the proteins formed inclusion bodies (Fig. 1). calcyclin, respectively, and residues 4 – 45 and 57–96 of MRP14 were Since these plasmids do not carry the lac repressor gene, high assigned to residues 19–429 and 539–909 of calcyclin, respectively. level expression of these proteins in rich (23 YT) media was After construction of the conserved regions, the loop or “hinge” re- attained without the need for induction. However, in M9 min- gions were generated for residues 43–50 of MRP8 and residues 46 –56 of MRP14, and then the model was minimized using 3000 iterations imal media, protein expression was enhanced considerably af- of steepest descents minimization in the Discover module of InsightII. ter induction with isopropyl-thio-b-D-galactoside. In minimal During minimization, the atomic coordinates for residues 1– 42 and media, cells were grown from mid- to late log phase and in- 51– 88 of MRP8 and residues 4 – 45 and 57–96 of MRP14 were held duced with isopropyl-thio-b-D-galactoside and then grown an fixed. The v angles within the hinge regions of MRP8 (residues additional 24 h before harvesting. 43–50) and MRP14 (residues 46 –56) and the tail region of MRP14 Approximately 100 mg of crude protein could be obtained (residues 97–114) were constrained to 2180° throughout the minimi- zation process. The model was considered sufficiently minimized from 1-liter cultures in 23 YT media, whereas expression lev- when no van der Waals violations were detected. els dropped to nearly half in minimal media. Since tens of milligrams of purified, isotopically enriched protein is neces- RESULTS sary for multidimensional NMR studies, the cost effectiveness Cloning of MRP8 and MRP14 of different carbon sources on expression levels in minimal media were explored. The choice of carbon sources (glycerol or In order to obtain sufficient quantities of protein for biophys- glucose) made little difference to the overall expression levels ical characterization and structure/function studies, the cDNA in minimal media. Excess glucose was shown to increase the containing the entire coding sequence for human MRP8 and expression levels only slightly. This program is available on the World Wide Web at http://chazin.scripps.edu/wisdom. M. Lubienski, J. Glasser, and W. J. Chazin, unpublished results. 12430 Expression and Characterization of MRP8 and MRP14 FIG.2. Chemical cross-linking of MRP8 and MRP14. Purified recombinant MRP8C42S and MRP14C3S were cross-linked using the bis(sulfosuccinimidyl)suberate cross-linking agent. Lane A, solution containing both MRP8C42S and MRP14C3S; lane B, solution contain- ing only MRP8C42S; lane C, solution containing only MRP14C3S. Purification It was apparent early on that overexpression of MRP8 and MRP14 led to formation of inclusion bodies. In fact, no MRP8 (and small amounts of MRP14) was found in the cytosolic or Triton X-100 fractions after cell lysis (Fig. 1). This greatly facilitated the purification process. After treatment with Triton X-100, the inclusion bodies could be pelleted, and the recombi- nant protein could be isolated from the inclusion bodies by dissolving the pellet in 8 M urea. Spectroscopic analysis and dimerization assays (see below) indicated that dialysis of the urea solution resulted in properly folded protein. This is in agreement with experiments, which demonstrated that the protein complex was still recognized by a protein-specific anti- body after unfolding in urea and refolding (24). Purification of recombinant MRP8 and MRP14 involved preparative scale re- verse-phase HPLC followed by Mono-Q ion exchange chroma- tography. All four recombinant proteins eluted from C resin in nearly pure form with only minor amounts of contaminating proteins (data not shown). Ion exchange chromatography pro- duced essentially pure MRP8 and MRP14, with each protein exhibiting a separate and distinct elution profile. Characterization Purified recombinant MRP8 and MRP14 were found to mi- grate in SDS-polyacrylamide gel electrophoresis with apparent molecular masses of 8 and 14 kDa, respectively (Fig. 1, lanes I FIG.3. Circular dichroism spectra of MRP8 and MRP14. Shown are spectra of MRP8C42S (A), MRP14C3S (B), and a 1:1 ratio of and J). The molecular weights of MRP8wt, MRP8C42S, MRP8C42S plus MRP14C3S (C) in the presence of 0.5 mM EDTA (open MRP14wt, and MRP14C3S were confirmed by either electro- circles) and 0.5 mM EDTA, 1.0 mM CaCl (closed circles). spray or MALDI mass spectrometry and were in close agree- ment with calculated molecular weights, determined using a JavaScript program. For MRP14wt and MRP14C3S, the cal- gates (apparently trimer and tetramer). culated molecular masses were ;130 atomic mass units higher The propensity for the formation of homodimers versus het- than those determined by mass spectrometry. This discrepancy erodimers was explored in a second set of experiments. These was attributed to the absence of the initiating methionine in results showed a strong preference for the MRP8/MRP14 het- both proteins, an apparent post-translational modification. An- erodimer in all solutions containing a mixture of both proteins alytical isoelectric focusing was used to measure the isoelectric (Fig. 2, lane A), which is similar to results obtained by points of MRP8C42S (6.7) and MRP14C3S (5.5). Both of these Teigelkamp et al. (21). The formation of heterodimers was values are in excellent agreement with the published values of found to occur both in the absence or presence of Ca (data not 6.7 and 5.6, respectively, for the native MRPs (48). shown). Chemical Cross-linking—To determine the states of associ- Circular Dichroism—The far UV CD spectrum of the wild- ation of recombinant MRP8 and MRP14, chemical cross-linking type and mutant MRP8 and MRP14 proteins indicates a high experiments were employed using bis(sulfosuccinimidyl)suber- degree of a-helical content (Fig. 3), which is characteristic of ate. First, the possibility that the proteins form homodimers the EF-hand family of proteins. The assignment of secondary was explored. The Cys 3 Ser mutants were utilized for these structure and the examination of the calcium-dependent experiments because this obviated the need for stringent meth- changes were made with the Cys 3 Ser mutants, because this ods to keep the proteins reduced. The results from the cross- obviated the need for reducing agents to keep unwanted disul- linking experiments are clear in indicating that both MRP8 fides from forming. The spectra for homogeneous solutions of and MRP14 will form homodimers in homogenous solutions MRP8 and MRP14 appear quite similar to the spectra obtained (Fig. 2). MRP14 is found to form only the monomer and the for 1:1 mixtures of the two proteins. Using the method of Chen (MRP14) complex. MRP8 is found to form monomer, ho- et al. (44), an estimate of 44, 35, and 40% a-helix was obtained modimer, and a small amount of distinct, higher order aggre- for MRP8C42S alone, MRP14C3S alone, and the mixture of Expression and Characterization of MRP8 and MRP14 12431 FIG.4. Fluorescence emission spec- tra of MRP8 and MRP14. Spectra were measured from 300 to 450 nm using an excitation wavelength of 290 nm. Scans were performed at 1 nm/s with the exci- tation and emission band pass set at 4 nm each. A, MRP8C42S; B, MRP14C3S; C, 1:1 ratio of MRP8C42S plus MRP14C3S in the presence of 0.5 mM EDTA (open circles) and 0.5 mM EDTA, 1.5 mM CaCl (closed circles). Experiments were per- formed on 5 mM protein solutions in 1 mM Tris-HCl at pH 8.5. The dashed line in panel C represents the normalized addi- tions of the spectra of apo-MRP8C42S and apo-MRP14C3S, and the thin solid line represents the normalized addition of the spectra of calcium-loaded MRP8C42S and calcium-loaded MRP14C3S. MRP8C42S plus MRP14C3S, respectively, in the presence of due to the addition of reducing agents to the solution. The EDTA. emission maxima of the spectra (Fig. 4) indicate that the tryp- The spectra in Fig. 3 show that the addition of calcium ions tophan residues of both MRP8 and MRP14 are shielded from to the MRP8/MRP14 heterodimer causes only very small solvent, as the l (emission) for free tryptophan is at 350 nm. max changes in the degree of helicity. While a similar result is It is notable that all tryptophan fluorescence spectra of MRP8 obtained for the homogenous solution of MRP14, there are and MRP14 are asymmetric due to a small shoulder at ;375 surprisingly marked changes in the CD spectrum of MRP8 nm. The signal of free tryptophan is symmetric, and there is no upon the addition of Ca , with a reduction in signal intensity readily apparent explanation for this shoulder. at 222 nm corresponding to an apparent 16% loss in helical In the spectra for the homodimer of MRP8 (Fig. 4A), there is content upon calcium addition. Although similar results have a distinctive 20% drop in fluorescence emission intensity upon been reported for a few other S100 proteins (49, 50), we believe the addition of excess calcium ions. This is accompanied by a that in the case of MRP8, this observation is a consequence of small shift in l (emission) from 338 to 334 nm. In the MRP14 max calcium-induced aggregation of the protein, since higher con- homodimer, a similar 17% reduction in fluorescence emission centrations of calcium ultimately lead to precipitation. intensity is observed upon the addition of excess calcium ions Fluorescence—The presence of a single tryptophan residue in but with only a slight shift in l (emission) from 342 to 341 max both MRP8 and MRP14 provides a ready means to analyze nm (Fig. 4B). These results indicate there are distinct Ca - these proteins by fluorescence spectroscopy. Trp in MRP8 is induced changes in the microenvironment of the respective located in the middle of helix III, and Trp in MRP14 is located tryptophan residues in both homodimers. toward the COOH terminus of helix IV, both in their respective In the spectra for the 1:1 mixture of MRP8 and MRP14 (Fig. COOH-terminal EF hands. An excitation wavelength of 290 nm 4C), a significant but clearly smaller 7% drop in fluorescence was selected to avoid excitation of the four tyrosine residues in emission intensity is observed upon the addition of excess cal- MRP8 and the single tyrosine residue in MRP14. As for other cium ions, accompanied by a small shift in l (emission) from max biophysical experiments, the Cys 3 Ser mutants were utilized 338 to 337 nm. Thus, there appears to be some Ca -induced as opposed to the wild-type protein to minimize adverse affects change in the microenvironments of the two tryptophan probes. 12432 Expression and Characterization of MRP8 and MRP14 However, since the fluorescence spectra of the MRP8/MRP14 heterodimer reflect the cumulative emission from the two dif- ferent tryptophan probes, the results cannot be interpreted in a straightforward manner because it is not possible to attribute the relative contributions from each. Some insight can be ob- tained from the comparison of the spectrum of the heterodimer versus the two homodimers (Fig. 4C). The curves shown as dashed and thin solid lines were generated by adding the spectra for the MRP8/MRP8 and MRP14/MRP14 homodimers and then dividing by 2 to normalize the protein concentration. There is a very large difference in the calculated and observed fluorescence intensities, which strongly suggests that the mi- croenvironments of one or both of the two probes in the het- erodimer are different from their environments in the respec- tive homodimers. NMR Spectroscopy—The relative integrity of the MRP8 and MRP14 homodimers and the MRP8/MRP14 heterodimer were further examined by NMR spectroscopy. MRP8 has inherent solubility problems in the pH range of ;5.0 – 8.0, where most biomolecular NMR experiments are performed. This, in con- junction with the tendency of both MRPs to aggregate at the millimolar concentrations required for NMR, poses a signifi- cant challenge to NMR analysis. An extensive search of exper- imental conditions was required in order to obtain the spectra shown here. 1 15 1 The one-dimensional H NMR and two-dimensional N- H HSQC experiments on the homogeneous solutions of both MRP8 and MRP14 in the absence of calcium exhibit poor sen- sitivity, broad resonance lines, and limited spectral dispersion. Such observations are typically associated with aggregation 15 1 phenomena, limited structural stability, or conformational ex- FIG.5. 500-MHz N- H HSQC spectra of MRP8 in the het- erodimeric complex in the absence of calcium at pH 8.5 and change. In contrast to the results obtained for the isolated 15 15 27 °C. A, N-enriched MRP8wt and unlabeled MRP14C3S; B, N- proteins, the addition of MRP8 to the solution of MRP14 and of enriched MRP8C42S and unlabeled MRP14C3S. The ratio of N-en- MRP14 to the solution MRP8 result in a radical improvement riched to unlabeled protein is 1:1.2. Details on sample preparation and in resolution and signal dispersion in the spectra. Figs. 5 and 6 acquisition parameters are provided under “Experimental Procedures.” 15 1 show the N- H HSQC spectra of various mixtures of labeled position 14 of the calcium-binding loop in the NH-terminal and unlabeled MRP8 and MRP14. These spectra indicate that pseudo-EF hand (site I). Glutamic acid is highly conserved in MRP8 and MRP14 preferentially form a stable, well packed this position in S100 proteins (Fig. 8) and the EF-hand calcium- heterodimeric complex. binding protein family as a whole (51). This residue is known to The direct addition of calcium to the solutions of the ho- play an important role in calcium binding by providing a bi- modimers and the heterodimer had different effects on each. dentate ligand to the calcium ion and forming an integral part MRP8 immediately forms an insoluble precipitate in direct of a network of hydrogen bonds that serve to stabilize the proportion to the amount of Ca added to the solution. In binding loop (51). Substitution of this Glu residue is known to contrast, MRP14 remains fully soluble throughout a titration drastically reduce calcium affinity (52, 53) and alter the large up to 2 molar equivalents/mol of protein and beyond. This 21 21 Ca -induced conformational changes in the Ca sensors cal- solution gives excellent one-dimensional H and two-dimen- 15 1 modulin and troponin C (54, 55). In analogy to the S100 homo- sional N- H HSQC spectra, as shown in Fig. 7A. The mixture logue p11, which also has an aspartate at this position, we of MRP8 and MRP14 has a similar response to the addition of anticipate that the NH-terminal site of MRP8 is not likely to Ca as MRP8 alone, presumably due to precipitation of MRP8. bind Ca with appreciable affinity. However, we did discover that MRP8 can be titrated into a One of the characteristic properties of S100 proteins is their solution of fully Ca -loaded MRP14 without formation of any tendency to dimerize. A calcium-dependent association of precipitate. NMR spectra obtained on the Ca -loaded het- MRP8 and MRP14 has been reported (21, 48), suggesting the erodimer produced in this manner are clearly distinct from the preferential formation of a 1:1 complex. In fact, the Kyte and spectrum of Ca -loaded MRP14/MRP14 homodimer, strongly Doolittle hydropathy analysis (56) shown in Fig. 9 indicates suggesting that the Ca -loaded MRP8/MRP14 heterodimer is that MRP8 and MRP14 have hydropathic profiles similar to formed and that it has a unique structure (Fig. 7B). S100 proteins known to dimerize (57, 58). Both MRP8 and DISCUSSION MRP14 exhibit the same hydrophobic NH and COOH termini The experiments presented here describe the high level ex- and hydrophilic EF-hand regions (32) as calcyclin and S100b. pression and biophysical characterization of recombinant hu- These results imply that both MRP8 and MRP14 should also man MRP8 and MRP14, two members of the S100 calcium- form dimers. binding protein family. Alignment of the calcium-binding loops The absence or presence and the biological significance of of MRP8 and MRP14 with other members of the S100 family disulfide-linked covalent dimers of S100 proteins has been a show that MRP14 contains the conserved sequence determi- topic of some debate (57, 58). It is not known whether MRP8 or nants necessary for calcium-binding in sites I and II that are MRP14 form biologically relevant, disulfide-linked dimers in found in other S100 proteins. MRP8 has a standard binding solution. To examine this possibility, a homology model of the loop in site II but a significant Glu 3 Asp substitution at MRP8/MRP14 complex was assembled based on the three- Expression and Characterization of MRP8 and MRP14 12433 15 1 FIG.6. 500-MHz N- H HSQC spectra of MRP14 in the ho- modimeric and heterodimeric complexes in the absence of cal- cium at pH 8.5 and 27 °C. A, N-enriched MRP14wt and unlabeled 15 1 MRP8C42S. The inset shows the N- H HSQC spectra of the same sample of MRP14wt prior to the addition of unlabeled MRP8C42S. B, 15 1 21 15 15 FIG.7. 500-MHz N- H HSQC spectra of the Ca -loaded N-enriched MRP14C3S and unlabeled MRP8C42S. The ratio of N- MRP8/MRP14 heterodimeric and MRP14/MRP14 homodimeric enriched to unlabeled protein is 1:1.2. Details on sample preparation complexes at pH 8.5 and 27 °C. A, N-enriched MRP14C3s and and acquisition parameters are provided under “Experimental unlabeled MRP8C42S. B, N-enriched MRP14C3S. Each sample con- Procedures.” tained 10 mM Tris-HCl, 1 mM EDTA, and 5 mM CaCl . dimensional solution structure of apo calcyclin determined in this laboratory (29). Inspection of this model suggests that the with the formation of a dimeric (and not higher order) species. 42 3 distance between Cys in MRP8 and Cys in MRP14 would These results suggest a unique complementarity between make it highly unlikely that biologically relevant disulfides MRP8 and MRP14, leading to a well folded stable heterodimer. would form in the heterodimer (Fig. 10). This prompted the The comparison of the fluorescence emission spectra shown production of the two MRP mutants, MRP8C42S and in Fig. 4 strongly suggests distinct differences between the MRP14C3S, which provide a means to avoid problems associ- structures of the homodimers and the heterodimer. The low ated with unwanted disulfide bond formation, particularly dur- fluorescence intensity of Trp in the MRP14/MRP14 ho- ing biophysical characterization at high protein concentrations. modimer (Fig. 4B) suggests that it is more solvent-exposed UV, CD, fluorescence, and especially NMR experiments than is Trp in the MRP8/MRP8 homodimer (Fig. 4A). This showed that the mutant proteins are essentially identical to suggests a rationale for why the co-addition of the normalized wild type. spectra of the homodimers does not correspond to the experi- The propensity of S100 proteins to dimerize and the pairing mentally observed spectrum of the heterodimer. The sequence 88 82 82 of MRP8 and MRP14 in vivo motivated the characterization of homologs of Trp , Met in calcyclin, and Thr in S100b, are the extent to which these two proteins associate with each residues that are known to participate in the formation of a other. Chemical cross-linking experiments showed that ho- stable dimer interface (29, 59). If Trp is partially solvent- modimers (and for MRP8, specific higher order oligomers) are exposed in the homodimer but becomes buried in the well formed in solutions containing only MRP8 or MRP14. In mix- packed heterodimer, then the intensity of Trp fluorescence is tures of the two proteins, the heterodimer is greatly preferred expected to increase in the less polar environment of the het- (in a ratio of at least 10:1; Fig. 2). The uniqueness of the erodimer (47). The more highly sequestered location of Trp in heterodimer is strongly supported by the observation of (i) very the dimer interface is attributed to a greater degree of comple- poor NMR spectral features for the homogenous solutions of mentary in the packing of the hydrophobic side chains in the either MRP8 or MRP14 but far superior results for the 1:1 heterodimer versus the homodimer. The significance of this mixture (Figs. 5 and 6); (ii) no evidence for a second set of hypothesis must now be explored by the combination of site- signals in any of the NMR spectra of the heterodimer, which directed mutagenesis and more detailed structural analysis. suggests that the equilibrium concentration of homooligomers We have characterized the Ca dependence of the struc- must be less than 1:50; (iii) NMR line widths and sensitivity in tural properties of the MRP8, MRP14, and MRP8/MRP14 15 1 the two-dimensional N- H HSQC spectra that are consistent dimers. The CD spectra show that calcium addition leads to 12434 Expression and Characterization of MRP8 and MRP14 FIG.8. Sequences of the calcium-binding loops of some S100 proteins. Data are adapted from Potts et al. (29). FIG. 10. Proximity of cysteine residues in the homology model of the MRP8/MRP14 heterodimer. The structure is represented as a backbone ribbon with the MRP8 chain lighter than the MRP14 chain. 42 3 The side chains of Cys in MRP8 and Cys in MRP14 are shown in a space-filling representation. For greater clarity, residues 96 –114 of MRP14 are not displayed. in intensity is consistent with that seen for calgranulin C and FIG.9. Hydropathic character of calcyclin, S100b, MRP8, and S100b (50, 64), suggesting that there are distinct calcium- MRP14. The numbering refers to structurally conserved regions of each induced structural changes for the MRP8/MRP8, MRP14/ protein as determined from sequence analysis (29). Hydrophilicity val- MRP14, and MRP8/MRP14 dimers. The presence of Ca -in- ues were calculated using a seven-residue window size according to 15 1 2 6 42 duced changes is most clearly exemplified in the N- H HSQC Kyte and Doolittle (56) using in house software. Residues Asp –Leu 50 87 and Asp –Ala of human calcyclin (solid line) overlaid with residues spectra of the Ca -loaded MRP14/MRP14 homodimer and the 4 40 50 88 Glu –Leu and Gln –Phe of bovine S100b (hatched line). B, residues MRP8/MRP14 heterodimer (Fig. 7, A and B, respectively). Each 4 42 48 87 Glu –Cys and Lys –Ala of human MRP8 (solid line) overlaid with spectrum is distinctly different from their respective apo coun- 7 45 56 107 residues Gln –Leu and Glu –Pro of human MRP14 (hatched line). terparts (Fig. 6), which directly indicates that changes are brought on by Ca binding. The ability to obtain spectra only slight changes in helicity for the MRP14 homodimer and indicative of a stable well packed homodimer of MRP14, but not the MRP8/MRP14 heterodimer. However, there is a distinct of MRP8, is an intriguing result in light of the hypothesis that reduction in molar ellipticity in the spectrum of MRP8 upon MRP14 alone, but not MRP8 alone, has biologically relevant calcium addition, similar to that seen for other homodimeric activity in specific inflammatory events. S100 proteins (e.g. S100A3 (50) and S100b (49)). However, in In summary, the chemical cross-linking, CD, fluorescence, the case of MRP8, there are potential complications due to the and NMR analysis of MRP8 and MRP14 show that a stable well limited solubility in the presence of calcium, such that the packed heterodimeric complex is preferentially formed both in change in CD intensity could also arise from aggregation. In the absence and presence of calcium ions. These results indi- addition, changes in secondary structure upon calcium binding cate that the functionally relevant form of the MRP8 and are not expected, since there is no change in the distribution of MRP14 complex is a heterodimer. Furthermore, the corre- secondary structure seen upon Ca -loading in the three-di- sponding studies of isolated MRP14 strongly suggest that its mensional structures of calcyclin (60) and S100b (61, 62) or expression and functional roles independent of MRP8 are as- other EF-hand CaBPs (e.g. Ref. 63). sociated with a stable dimeric state. The determination of the The fluorescence data show distinct changes in emission three-dimensional solution structures of MRP8/MRP14 and intensity upon calcium addition for all three dimers. This loss MRP14/MRP14 is currently in progress in this laboratory. Expression and Characterization of MRP8 and MRP14 12435 28. Zimmer, D. B., Chessher, J., and Song, W. (1996) Biochim. Biophys. 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(1994) Protein Sci. 3, 1961–1974 197–204 27. Scha ¨ fer, B. W., and Heizmann, C. W. (1996) Trends Biochem. Sci. 21, 134 –140 64. Baudier, J., and Cole, R. D. (1989) Biochem. J. 264, 79–85 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

High Level Expression and Dimer Characterization of the S100 EF-hand Proteins, Migration Inhibitory Factor-related Proteins 8 and 14

Hunter, Michael J.; Chazin, Walter J.
Journal of Biological Chemistry – May 1, 1998
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 20, Issue of May 15, pp. 12427–12435, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. High Level Expression and Dimer Characterization of the S100 EF-hand Proteins, Migration Inhibitory Factor-related Proteins 8 and 14* (Received for publication, November 20, 1997) Michael J. Hunter‡ and Walter J. Chazin§ From the Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037 The phenotypical and functional heterogeneity of dif- nomenclature, MRP8 and MRP14 are designated as S100A8 ferent macrophage subpopulations are defined by dis- and S100A9, respectively (14). Other names for the MRP8/ crete changes in the expression of two S100 calcium- MRP14 complex include calprotectin (15), leukocyte-derived binding proteins, migration inhibitory factor-related protein (L1) light and heavy chains (16), p8/p14 (17), calgranu- proteins (MRPs) 8 and 14. To further our understanding lin A/B (9), and the cystic fibrosis antigen (11, 18). Some evi- of MRP8 and MRP14 in the developmental stages of dence suggests that MRP14 alone is the cystic fibrosis antigen inflammatory responses, overexpression of the MRPs (19), and murine MRP8 has been referred to as the murine was obtained through a combination of a T7-based ex- chemotactic cytokine (CP-10) (20). pression vector and the Escherichia coli BL21 (DE3) cell It has been suggested that MRP8 and MRP14 form a het- line. An efficient, two-step chromatographic protocol erodimeric complex in a calcium-dependent manner (21). How- was then developed for rapid, facile purification. Exten- ever, once this complex is formed, it is stable upon the addition sive biophysical characterization and chemical cross- of EDTA (22). It is not clear whether these proteins have linking experiments show that MRP8 and MRP14 form independent biological functions or whether their function is oligomers with a strong preference to associate as a strictly dependent on heterocomplex formation. Although the heterodimer. Heteronuclear NMR experiments indicate general consensus is that protein function is dependent on that a specific well packed dimer is formed only in heterodimer formation, studies also support the possibility that equimolar mixtures of the two proteins. Our results sug- these proteins may have individual functions. For example, gest that there is a unique complementarity in the in- MRP14 is shown to be expressed independently of MRP8 in terface of the MRP8/MRP14 complex that cannot be fully acutely inflamed tissues (23–25). reproduced in the MRP8 and MRP14 homodimers. Like a number of other S100 proteins (S100a, S100D, S100E, S100L, p11, CAPL, calcyclin/2A9, and psoriasin), the genes encoding MRP8 and MRP14 have been located on chromosome Macrophages, which belong to the mononuclear phagocyte 1q21 (11, 26, 27). Recently, it has been shown that the struc- system, form a heterogeneous cell population with different developmental and functional stages. The specific pathophysi- ture of the gene loci encoding S100 proteins is highly conserved (28). Although the primary protein sequences are similar, there cal situation dictates whether macrophages will perform cyto- toxic, endocytosic, or secretory functions. The phenotypical and is less similarity at the nucleotide level, with the greatest divergence in the untranslated regions. Since S100 proteins functional heterogeneity of different macrophage subpopula- tions are defined by discrete changes in the expression of two exhibit very specific patterns of expression, it has been sug- gested that these untranslated regions may be involved in S100 calcium-binding proteins, migration inhibitory factor-re- lated proteins (MRPs) 8 and 14 (1–3). Granulocytes, mono- directing the expression of specific S100 proteins in specific cell types (28). Analysis of the amino acid sequences of MRP8 and cytes/macrophages, neutrophils, and keratinocytes have been shown to express MRP8 and MRP14, suggesting that expres- MRP14 indicate that each protein is composed of two EF-hand domains characteristic of the S100 calcium-binding protein sion is tightly regulated during granulocyte differentiation (4 – 9). In addition, elevated serum levels of MRP8 and MRP14 family (27, 29, 30). S100 proteins are characterized by rela- tively low molecular masses, generally on the order of 10 kDa, have been found in patients suffering from a number of inflam- matory disorders including cystic fibrosis, rheumatoid arthri- and a unique consensus sequence in their calcium-binding sites making them a diverse, multifaceted family of proteins (31). tis, and chronic bronchitis (10 –13), suggesting possible extra- cellular roles for these proteins. In the new S100 protein They have been shown to play roles in cell cycle progression, regulation of intracellular phosphorylation events, and cy- toskeletal membrane interactions, as well as extracellular * This research was supported in part by National Institutes of events such as the stimulation of glial proliferation, prolactin Health Grant PO1 GM48495 (to W. J. C.). The costs of publication of secretion, and neuronal differentiation (30, 32). this article were defrayed in part by the payment of page charges. This MRP8 and MRP14 have molecular masses of 11 and 14 kDa article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. and are composed of 93 and 114 amino acids, respectively. A ‡ Recipient of a postdoctoral fellowship from the National Arthritis number of possible functions for these proteins have been pro- Foundation. posed including differentiation of myeloid lineage cells, regula- § Faculty Research Fellow of the American Cancer Society. To whom tion of intracellular calcium levels in neutrophils, inhibition of correspondence should be addressed: Dept. of Molecular Biology (MB- 9), The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, casein kinase I and II activity, and a zinc-mediated biostatic CA 92037. Tel.: 619-784-9860; Fax: 619-784-9985; E-mail: chazin@ activity (19, 21, 33, 34). The extended carboxyl-terminal tail of scripps.edu. MRP14 makes it the largest member of the S100 family, and it The abbreviations used are: MRP, migration inhibitory factor-re- is believed that MRP14 represents the regulatory unit of this lated protein; PCR, polymerase chain reaction; HPLC, high pressure liquid chromatography; HSQC, heteronuclear single quantum coherence. unique heterodimeric complex. Studies have shown that the This paper is available on line at http://www.jbc.org 12427 This is an Open Access article under the CC BY license. 12428 Expression and Characterization of MRP8 and MRP14 and sequencing of mutants were performed as described for the wild- penultimate amino acid of MRP14, Thr , is phosphorylated in type proteins. vivo (8, 35) and that the phosphorylation event triggers the Protein Expression—Overexpression of the gene products was translocation of this protein to the cytoskeleton (36). Possible achieved in E. coli strain BL21(DE3). For unlabeled protein, cells were functions for the tail of MRP14 may include neutrophil immo- grown at 37 °C in (23 YT) media supplemented with ampicillin to a bilization (37), regulation of cytoskeletal translocation of the final concentration of 100 mg/ml. 500-ml cultures were inoculated with MRP8/MRP14 complex (36), and inhibition of the onset of the 5 ml of an overnight culture grown in the same media. Cells were allowed to grow another 18 –24 h before harvesting. Since the pET1120 intrinsic coagulation cascade (38). The only function assigned plasmid contains no lac repressor gene, protein expression was not to MRP8 to date is the potent chemotactic activity assigned to tightly controlled, and the system was leaky enough to produce large linker region of murine MRP8 (20, 39 – 41). amounts of protein in rich media without the need for induction. To further our understanding of the roles MRP8 and MRP14 For N-labeled protein, cells were grown at 37 °C in M9 minimal play in the developmental stages of inflammatory responses, media supplemented with basal medium Eagle vitamin solution (Life we have cloned these two proteins and developed highly effi- Technologies, Inc.), NH Cl as the sole nitrogen source, and glucose as cient expression and purification protocols. This has allowed the carbon source. Ampicillin was added to a final concentration of 100 mg/ml. 5-ml inoculants were grown overnight, and inoculated cultures for biophysical characterization by UV, CD, fluorescence, and between 0.1 and 0.5 before induction were allowed to grow to an A heteronuclear NMR spectroscopies, as well as chemical cross- with isopropyl-thio-b-D-galactoside to a final concentration of 0.05 mM. linking experiments. MRP8 and MRP14 are shown to be stable Cells were grown an additional 24 h before harvesting. globular proteins that form dimers and preferentially associate Protein Purification—Cells were harvested and inclusion bodies were as heterodimers. isolated following methods described by Sambrook et al. (42). The pellet containing the inclusion bodies was resuspended in 10 mM Tris-HCl, 8 EXPERIMENTAL PROCEDURES M urea, 20 mM dithiothreitol at pH 8.0 in a volume sufficient to com- Construction of the Expression Vectors—The MRP8 and MRP14 pletely dissolve the pellet. These protein solutions were dialyzed for 4 cDNAs were a generous gift of Professor Clemens Sorg (University of M Tris-HCl, 2.5 mM EDTA/EGTA, pH days against 2 3 4 liters of 50 m Mu ¨ nster, Germany). The primers MRP8 –59 (59-GGA ATT CCA TAT 8.0, in 3.5-kDa cut-off dialysis membrane. The dialyzed solutions were GTT GAC CGA GCT GGA GAA-39) and MRP8 –39 (59-CGG GAT CCC centrifuged at 18,000 3 g for 20 min and then filtered through a 0.2 mM ACT CTT TGT GGC TTT CTT C-39) were used to PCR amplify the acrodisc syringe filter (Gelman Sciences, Ann Arbor, MI) prior to fur- human MRP8 gene from the pMRP-8-trp expression vector, and the ther purification. primers MRP14 –59 (59-GGA ATT CCA TAT GAC TTG CAA AAT GTC The MRPs were isolated by preparative reverse-phase HPLC on a GCA G-39) and MRP14 –39 (59-CGG GAT CCT TAG GGG GTG CCC DeltaPak C column (PrepPak 500 cartridge, Waters Corp., Milford, TCC CC-39) were used to amplify the human MRP14 gene from the MA) using a gradient of 36 –51% acetonitrile, 0.1% trifluoroacetic acid pMRP-14-trp expression vector. A NdeI restriction site was engineered over 25 min. Fractions containing protein were lyophilized and then into the 59-primers, and a BamHI restriction site was engineered into dissolved in 25 mM Tris-HCl, pH 8.0, and further purified by anion the 39-primers for proper insertion of the gene into the pET1120 ex- exchange chromatography on a Perceptive Biosystems BioCAD Sprint pression vector. Amplification was conducted in a total volume of 100 ml perfusion chromatography system using a Mono-Q anion exchange col- containing 10 mM KCl, 20 mM Tris-HCl (pH 8.8 at 25 °C), 10 mM umn (Amersham Pharmacia Biotech). The protein was eluted in a (NH ) SO , 2.0 mM MgSO , 0.1% Triton X-100, 0.3 mM dNTPs, 5.0 units 4 2 4 4 buffer containing 25 mM Tris-HCl, 1.0 mM EDTA/EGTA, pH 8.0, with a of vent DNA polymerase, and 100 pmol of primers. A small amount of 0 – 0.5 M NaCl gradient at a flow rate of 5 ml/min. All buffers used in the mineral oil was added to the top of the reaction mixture to inhibit purification of wild-type proteins contained 20 mM dithiothreitol to evaporation. The reaction mixtures were heated to 95 °C for 5 min, inhibit disulfide bond formation. Fractions containing protein were followed by 25–30 cycles of 95, 60, and 75 °C for 30 s each and a final desalted and concentrated using Millipore Ultrafree-4 centrifugal filter extension at 75 °C for 2 min. devices (5-kDa cut-off). Extinction coefficients were determined from 1 PCR products were purified using a QIAquick PCR purification kit mg/ml samples of MRP8C42S and MRP14C3S by acid hydrolysis and (QIAGEN Inc., Chatsworth, CA). Full-length oligonucleotides were dou- amino acid analysis. bly digested with 20 units BamHI and 40 units NdeIfor2hat37 °C. Isoelectric Focusing—Isoelectric focusing was performed on a Mul- The digested fragments were purified on a 1.3% agarose gel and stained tiphor II isoelectric focusing system (Amersham Pharmacia Biotech) with ethidium bromide. The bands containing the MRP8 and MRP14 using Servalyt Precote polyacrylamide gels (SERVA, Heidelberg, Ger- genes were excised and purified using a QIAEX II gel extraction kit. many) and a Pharmalyte 3–10 broad pI calibration kit. Sample volumes The MRP8 and MRP14 genes were then cloned into the NdeI and were 5 ml at a protein concentration of 3 mg/ml. The maximum power BamHI sites of the pET1120 vector using standard methods (42). The was set to 4 watts, and the initial voltage was set to 200 V. Focusing pET1120 vector was created by isolating the two BamHI/PstI fragments continued until a maximum voltage of 2000 V was reached. Gels were of Novagen’s pET11 and pET20 vectors and ligating the large fragment fixed for 10 min in a 20% solution of trichloroactetic acid, stained with of pET20 digestion with the small fragment of pET11 digestion. These SERVA Blue W dye tablets for 5 min with constant shaking, and plasmids have the b-lactamase gene and multiple cloning sites down- destained in H O until background color was at an acceptable level. stream of the efficient T7-polymerase promoter but are void of the lac Isoelectric points were determined by plotting the retention versus repressor gene. Competent DH5a cells were transformed with the isoelectric point of known standards. pET1120-MRP8wt and pET1120-MRP14wt vectors and used to produce Chemical Cross-linking—The analysis of MRP8/MRP14 complex for- plasmid stocks. Proper insertion and DNA sequences were confirmed by mation was carried out by using the bis(sulfosuccinimidyl)suberate fluorescent thermal dye DNA sequencing methods (43). (Pierce) method described by Teigelkamp et al. (21). Purified recombi- Mutagenesis—To construct the MRP8C42S gene, the primers, nant MRP8 and MRP14 were diluted in phosphate-buffered saline at MRP8C42S-59 (59-CTC GGT CTC GAG CAA TTT CTT C-39) and pH 8.5 to a final concentration of 50 mM. Cross-linking was initiated by MRP8 –59 were used to PCR amplify the 59-half of the MRP8C42S gene, the addition of freshly prepared bis(sulfosuccinimidyl)suberate (20 mM and the primers MRP8C42S-39 (59-TTG CTC GAG ACC GAG TCT CCT stock solution) to a final concentration of 5 mM. Solutions were incu- CAG TAT ATC-39) and MRP8 –39 were used to PCR amplify the 39-half M Tris to bated at room temperature for 30 min before the addition of 1 of the MRP8C42S gene. The two amplified half-fragments were purified quench the reaction. Cross-linked samples were incubated with b-mer- on a 1.5% agarose gel and visualized using ethidium bromide. The captoethanol prior to visualization on SDS-polyacrylamide gel bands containing the two fragments were excised and purified using a electrophoresis. QIAEX II gel extraction kit (QIAGEN). The two purified half-fragments CD Spectroscopy—Circular dichroism experiments were carried out were used as primers for each other, and the full gene was made using on an Aviv 61DS spectropolarimeter with 1-mm path length quartz the PCR methods described above. Since this did not result in the cuvettes. Protein solutions (20 mM MRP8C42S, 20 mM MRP14C3S, and amplification of the entire gene, a small aliquot of the above reaction 10 mM MRP8C42S plus 10 mM MRP14C3S) were prepared in 1 mM mixture, which had small amounts of the full-length mutant gene, was Tris-HCl and 0.5 mM EDTA, pH 8.5. The solution was brought to 2.5 mM used as a template to PCR amplify the full-length MRP8C42S gene using MRP8 –59 and MRP8 –39 as the amplification primers. To con- CaCl , to obtain the spectra of the calcium-loaded state. Spectra were struct the MRP14C3S gene, the primers MRP14C3S-59 (59-GGA ATT collected with an average time of 3 s for each point and a step size of CCA TAT GAC TAG TAA AAT GTC GCA GCT GG-39) and MRP14 –39 0.50 nm from 200 to 260 nm. All spectra were collected in triplicate and were used with the wild-type template in one PCR step. Transformation background-corrected against a buffer blank. A perl program (least- Expression and Characterization of MRP8 and MRP14 12429 ellip.pl) was written to determine the optimal least squares fit. Data 2 21 were converted to mean residue ellipticity [u] (degrees cm dmol )by using [u] 5 u/(10 lcn), where u is the measured ellipticity, l is the cell path length in cm, c is the molar concentration of protein in mol/liter, and n is the number of residues/chain. The a-helical content was esti- mated by using the mean residue ellipticity at 222 nm (44). Protein concentrations (20 –25 mM) were determined by using the bicinchoninic acid method (Pierce). Fluorescence Spectroscopy—Measurements of the fluorescence exci- tation and emission spectra were conducted at 25 °C on a SLM- AMINCO Series 2 Spectrofluorometer equipped with a Hewlett Pack- ard Pavilion 5040 personal computer. Excitation spectra were measured from 250 to 320 nm with emission monitored at 335 nm. Scans were performed at 1 nm/s with the excitation and emission band pass set at 16 and 4 nm, respectively. Emission spectra were measured FIG.1. Expression and purification of recombinant MRP8 and from 300 to 450 nm using an excitation wavelength of 290 nm. Scans MRP14. Lanes A and B, total cell lysates from bacteria transformed were performed at 1 nm/s with the excitation and emission band pass with the pET1120-MRP8C42S and MRP14C3S expression vectors, re- spectively; lanes C–H, soluble cytosolic fractions of the cell lysate (lane set at 4 nm. Each 1.5-ml sample contained 5 mM total protein C and D), Triton X-100 fractions (lanes E and F), and 8 M urea fractions (MRP8C42S, MRP14C3S, or a 1:1 mixture) in 1 mM Tris-HCl, 0.5 mM (lanes G and H) of MRP8C42S and MRP14C3S, respectively. Lanes I EDTA at pH 8.5. For the calcium-loaded state, 1 M CaCl was added to and J, ion exchange-purified MRP8C42S and MRP14C3S, respectively. a final concentration of 1.5 mM. All samples were preequilibrated over- night prior to the fluorescence measurements. Each experiment was collected in triplicate, and a perl program (leastgen.pl) was written to fit MRP14 was cloned into the bacterial expression vector the data using a least squares algorithm. NMR Spectroscopy—Wild type and mutant protein samples were pET1120. This expression vector was previously shown to effi- dissolved and concentrated in a buffer containing 10 mM Tris-HCl, ciently overexpress the very closely related S100 protein calcy- 100 mM KCl, and 5 mM EDTA/EGTA, at pH 8.5. For NMR samples of clin. The cDNAs were engineered with 59 NdeI restriction sites wild-type protein, deuterated dithiothreitol was also added to a final and 39 BamHI restriction sites for facile insertion into the concentration of 5.0 mM. Pure H O was added to a final concentra- expression vector downstream of the efficient T7 polymerase tion of 10% for the spectrometer lock system. Spectra were recorded 15 15 promoter. All gene sequences were confirmed by standard DNA for the following samples: N-MRP8wt, N-MRP8wt 1 MRP14C3S, 15 15 15 N-MRP8C42S 1 MRP14C3S, N-MRP14wt, MRP8C42S 1 N- sequencing methods. MRP14wt, and MRP8C42S 1 N-MRP14C3S. The mixtures were MRP8 and MRP14 each contain a single cysteine residue. To made up in a 1:1.2 ratio of labeled to unlabeled protein (0.75–1.0 mM avoid problems associated with the formation of unwanted final concentration). disulfide bonds, the MRP8C42S and MRP14C3S mutant genes All NMR experiments were performed at 27 °C on a Bruker AMX500 1 15 were also constructed and inserted into the pET1120 expres- operating at 499.87 MHz for H and 50.65 MHz for N. Phase-sensitive sion vector. DNA sequencing showed that the gene encoding two-dimensional data were recorded using the method of States et al. (45). H chemical shifts were referenced to the H O peak at 4.75 ppm, MRP14C3S had a single base pair mutation (G to A) at position 15 1 and the N shifts were referenced indirectly using the H frequency of 318. However, both codons (AAG, AAA) code for the amino acid 15 1 the H O resonance (46). The N- H HSQC spectra (47) were recorded lysine, making this a silent mutation. with 16 transients/increment, a total of 128 complex points in t and 512 points in t , and spectral widths of 1500.15 Hz in v and 4000 Hz in v . 2 1 2 Protein Expression All two-dimensional data sets were processed on a Sun SPARCstation LX workstation using FELIX (version 95.0; MSI, San Diego, CA.). The expression vectors containing the cDNA for MRP8wt, Homology Model of MRP8/MRP14 —The model of apo-MRP8/ MRP8C42S, MRP14wt, and MRP14C3S were used to trans- MRP14 was generated using the Homology module of the InsightII form the protease-deficient E. coli strain BL21(DE3). In rich package (MSI, San Diego, CA), based on the low resolution structure media, BL21(DE3) cells transformed with the expression plas- of the apo state of the related S100 protein calcyclin (29). Calcyclin is mids overexpressed proteins that migrated in SDS gels with a symmetric homodimer, so the model was created with MRP8 apparent molecular masses of 8 kDa (MRP8wt, MRP8C42S) or aligned to one subunit and MRP14 aligned to the other. The auto- mated sequence alignment algorithm in the software was used to 14 kDa (MRP14wt, MRP14C3S). Overexpression of MRP8 and define the conserved regions of MRP8 and MRP14. Residues 1– 42 MRP14 proved not to be detrimental to the bacteria, presum- and 51– 88 of MRP8 were assigned to residues 1– 42 and 53–90 of ably because the proteins formed inclusion bodies (Fig. 1). calcyclin, respectively, and residues 4 – 45 and 57–96 of MRP14 were Since these plasmids do not carry the lac repressor gene, high assigned to residues 19–429 and 539–909 of calcyclin, respectively. level expression of these proteins in rich (23 YT) media was After construction of the conserved regions, the loop or “hinge” re- attained without the need for induction. However, in M9 min- gions were generated for residues 43–50 of MRP8 and residues 46 –56 of MRP14, and then the model was minimized using 3000 iterations imal media, protein expression was enhanced considerably af- of steepest descents minimization in the Discover module of InsightII. ter induction with isopropyl-thio-b-D-galactoside. In minimal During minimization, the atomic coordinates for residues 1– 42 and media, cells were grown from mid- to late log phase and in- 51– 88 of MRP8 and residues 4 – 45 and 57–96 of MRP14 were held duced with isopropyl-thio-b-D-galactoside and then grown an fixed. The v angles within the hinge regions of MRP8 (residues additional 24 h before harvesting. 43–50) and MRP14 (residues 46 –56) and the tail region of MRP14 Approximately 100 mg of crude protein could be obtained (residues 97–114) were constrained to 2180° throughout the minimi- zation process. The model was considered sufficiently minimized from 1-liter cultures in 23 YT media, whereas expression lev- when no van der Waals violations were detected. els dropped to nearly half in minimal media. Since tens of milligrams of purified, isotopically enriched protein is neces- RESULTS sary for multidimensional NMR studies, the cost effectiveness Cloning of MRP8 and MRP14 of different carbon sources on expression levels in minimal media were explored. The choice of carbon sources (glycerol or In order to obtain sufficient quantities of protein for biophys- glucose) made little difference to the overall expression levels ical characterization and structure/function studies, the cDNA in minimal media. Excess glucose was shown to increase the containing the entire coding sequence for human MRP8 and expression levels only slightly. This program is available on the World Wide Web at http://chazin.scripps.edu/wisdom. M. Lubienski, J. Glasser, and W. J. Chazin, unpublished results. 12430 Expression and Characterization of MRP8 and MRP14 FIG.2. Chemical cross-linking of MRP8 and MRP14. Purified recombinant MRP8C42S and MRP14C3S were cross-linked using the bis(sulfosuccinimidyl)suberate cross-linking agent. Lane A, solution containing both MRP8C42S and MRP14C3S; lane B, solution contain- ing only MRP8C42S; lane C, solution containing only MRP14C3S. Purification It was apparent early on that overexpression of MRP8 and MRP14 led to formation of inclusion bodies. In fact, no MRP8 (and small amounts of MRP14) was found in the cytosolic or Triton X-100 fractions after cell lysis (Fig. 1). This greatly facilitated the purification process. After treatment with Triton X-100, the inclusion bodies could be pelleted, and the recombi- nant protein could be isolated from the inclusion bodies by dissolving the pellet in 8 M urea. Spectroscopic analysis and dimerization assays (see below) indicated that dialysis of the urea solution resulted in properly folded protein. This is in agreement with experiments, which demonstrated that the protein complex was still recognized by a protein-specific anti- body after unfolding in urea and refolding (24). Purification of recombinant MRP8 and MRP14 involved preparative scale re- verse-phase HPLC followed by Mono-Q ion exchange chroma- tography. All four recombinant proteins eluted from C resin in nearly pure form with only minor amounts of contaminating proteins (data not shown). Ion exchange chromatography pro- duced essentially pure MRP8 and MRP14, with each protein exhibiting a separate and distinct elution profile. Characterization Purified recombinant MRP8 and MRP14 were found to mi- grate in SDS-polyacrylamide gel electrophoresis with apparent molecular masses of 8 and 14 kDa, respectively (Fig. 1, lanes I FIG.3. Circular dichroism spectra of MRP8 and MRP14. Shown are spectra of MRP8C42S (A), MRP14C3S (B), and a 1:1 ratio of and J). The molecular weights of MRP8wt, MRP8C42S, MRP8C42S plus MRP14C3S (C) in the presence of 0.5 mM EDTA (open MRP14wt, and MRP14C3S were confirmed by either electro- circles) and 0.5 mM EDTA, 1.0 mM CaCl (closed circles). spray or MALDI mass spectrometry and were in close agree- ment with calculated molecular weights, determined using a JavaScript program. For MRP14wt and MRP14C3S, the cal- gates (apparently trimer and tetramer). culated molecular masses were ;130 atomic mass units higher The propensity for the formation of homodimers versus het- than those determined by mass spectrometry. This discrepancy erodimers was explored in a second set of experiments. These was attributed to the absence of the initiating methionine in results showed a strong preference for the MRP8/MRP14 het- both proteins, an apparent post-translational modification. An- erodimer in all solutions containing a mixture of both proteins alytical isoelectric focusing was used to measure the isoelectric (Fig. 2, lane A), which is similar to results obtained by points of MRP8C42S (6.7) and MRP14C3S (5.5). Both of these Teigelkamp et al. (21). The formation of heterodimers was values are in excellent agreement with the published values of found to occur both in the absence or presence of Ca (data not 6.7 and 5.6, respectively, for the native MRPs (48). shown). Chemical Cross-linking—To determine the states of associ- Circular Dichroism—The far UV CD spectrum of the wild- ation of recombinant MRP8 and MRP14, chemical cross-linking type and mutant MRP8 and MRP14 proteins indicates a high experiments were employed using bis(sulfosuccinimidyl)suber- degree of a-helical content (Fig. 3), which is characteristic of ate. First, the possibility that the proteins form homodimers the EF-hand family of proteins. The assignment of secondary was explored. The Cys 3 Ser mutants were utilized for these structure and the examination of the calcium-dependent experiments because this obviated the need for stringent meth- changes were made with the Cys 3 Ser mutants, because this ods to keep the proteins reduced. The results from the cross- obviated the need for reducing agents to keep unwanted disul- linking experiments are clear in indicating that both MRP8 fides from forming. The spectra for homogeneous solutions of and MRP14 will form homodimers in homogenous solutions MRP8 and MRP14 appear quite similar to the spectra obtained (Fig. 2). MRP14 is found to form only the monomer and the for 1:1 mixtures of the two proteins. Using the method of Chen (MRP14) complex. MRP8 is found to form monomer, ho- et al. (44), an estimate of 44, 35, and 40% a-helix was obtained modimer, and a small amount of distinct, higher order aggre- for MRP8C42S alone, MRP14C3S alone, and the mixture of Expression and Characterization of MRP8 and MRP14 12431 FIG.4. Fluorescence emission spec- tra of MRP8 and MRP14. Spectra were measured from 300 to 450 nm using an excitation wavelength of 290 nm. Scans were performed at 1 nm/s with the exci- tation and emission band pass set at 4 nm each. A, MRP8C42S; B, MRP14C3S; C, 1:1 ratio of MRP8C42S plus MRP14C3S in the presence of 0.5 mM EDTA (open circles) and 0.5 mM EDTA, 1.5 mM CaCl (closed circles). Experiments were per- formed on 5 mM protein solutions in 1 mM Tris-HCl at pH 8.5. The dashed line in panel C represents the normalized addi- tions of the spectra of apo-MRP8C42S and apo-MRP14C3S, and the thin solid line represents the normalized addition of the spectra of calcium-loaded MRP8C42S and calcium-loaded MRP14C3S. MRP8C42S plus MRP14C3S, respectively, in the presence of due to the addition of reducing agents to the solution. The EDTA. emission maxima of the spectra (Fig. 4) indicate that the tryp- The spectra in Fig. 3 show that the addition of calcium ions tophan residues of both MRP8 and MRP14 are shielded from to the MRP8/MRP14 heterodimer causes only very small solvent, as the l (emission) for free tryptophan is at 350 nm. max changes in the degree of helicity. While a similar result is It is notable that all tryptophan fluorescence spectra of MRP8 obtained for the homogenous solution of MRP14, there are and MRP14 are asymmetric due to a small shoulder at ;375 surprisingly marked changes in the CD spectrum of MRP8 nm. The signal of free tryptophan is symmetric, and there is no upon the addition of Ca , with a reduction in signal intensity readily apparent explanation for this shoulder. at 222 nm corresponding to an apparent 16% loss in helical In the spectra for the homodimer of MRP8 (Fig. 4A), there is content upon calcium addition. Although similar results have a distinctive 20% drop in fluorescence emission intensity upon been reported for a few other S100 proteins (49, 50), we believe the addition of excess calcium ions. This is accompanied by a that in the case of MRP8, this observation is a consequence of small shift in l (emission) from 338 to 334 nm. In the MRP14 max calcium-induced aggregation of the protein, since higher con- homodimer, a similar 17% reduction in fluorescence emission centrations of calcium ultimately lead to precipitation. intensity is observed upon the addition of excess calcium ions Fluorescence—The presence of a single tryptophan residue in but with only a slight shift in l (emission) from 342 to 341 max both MRP8 and MRP14 provides a ready means to analyze nm (Fig. 4B). These results indicate there are distinct Ca - these proteins by fluorescence spectroscopy. Trp in MRP8 is induced changes in the microenvironment of the respective located in the middle of helix III, and Trp in MRP14 is located tryptophan residues in both homodimers. toward the COOH terminus of helix IV, both in their respective In the spectra for the 1:1 mixture of MRP8 and MRP14 (Fig. COOH-terminal EF hands. An excitation wavelength of 290 nm 4C), a significant but clearly smaller 7% drop in fluorescence was selected to avoid excitation of the four tyrosine residues in emission intensity is observed upon the addition of excess cal- MRP8 and the single tyrosine residue in MRP14. As for other cium ions, accompanied by a small shift in l (emission) from max biophysical experiments, the Cys 3 Ser mutants were utilized 338 to 337 nm. Thus, there appears to be some Ca -induced as opposed to the wild-type protein to minimize adverse affects change in the microenvironments of the two tryptophan probes. 12432 Expression and Characterization of MRP8 and MRP14 However, since the fluorescence spectra of the MRP8/MRP14 heterodimer reflect the cumulative emission from the two dif- ferent tryptophan probes, the results cannot be interpreted in a straightforward manner because it is not possible to attribute the relative contributions from each. Some insight can be ob- tained from the comparison of the spectrum of the heterodimer versus the two homodimers (Fig. 4C). The curves shown as dashed and thin solid lines were generated by adding the spectra for the MRP8/MRP8 and MRP14/MRP14 homodimers and then dividing by 2 to normalize the protein concentration. There is a very large difference in the calculated and observed fluorescence intensities, which strongly suggests that the mi- croenvironments of one or both of the two probes in the het- erodimer are different from their environments in the respec- tive homodimers. NMR Spectroscopy—The relative integrity of the MRP8 and MRP14 homodimers and the MRP8/MRP14 heterodimer were further examined by NMR spectroscopy. MRP8 has inherent solubility problems in the pH range of ;5.0 – 8.0, where most biomolecular NMR experiments are performed. This, in con- junction with the tendency of both MRPs to aggregate at the millimolar concentrations required for NMR, poses a signifi- cant challenge to NMR analysis. An extensive search of exper- imental conditions was required in order to obtain the spectra shown here. 1 15 1 The one-dimensional H NMR and two-dimensional N- H HSQC experiments on the homogeneous solutions of both MRP8 and MRP14 in the absence of calcium exhibit poor sen- sitivity, broad resonance lines, and limited spectral dispersion. Such observations are typically associated with aggregation 15 1 phenomena, limited structural stability, or conformational ex- FIG.5. 500-MHz N- H HSQC spectra of MRP8 in the het- erodimeric complex in the absence of calcium at pH 8.5 and change. In contrast to the results obtained for the isolated 15 15 27 °C. A, N-enriched MRP8wt and unlabeled MRP14C3S; B, N- proteins, the addition of MRP8 to the solution of MRP14 and of enriched MRP8C42S and unlabeled MRP14C3S. The ratio of N-en- MRP14 to the solution MRP8 result in a radical improvement riched to unlabeled protein is 1:1.2. Details on sample preparation and in resolution and signal dispersion in the spectra. Figs. 5 and 6 acquisition parameters are provided under “Experimental Procedures.” 15 1 show the N- H HSQC spectra of various mixtures of labeled position 14 of the calcium-binding loop in the NH-terminal and unlabeled MRP8 and MRP14. These spectra indicate that pseudo-EF hand (site I). Glutamic acid is highly conserved in MRP8 and MRP14 preferentially form a stable, well packed this position in S100 proteins (Fig. 8) and the EF-hand calcium- heterodimeric complex. binding protein family as a whole (51). This residue is known to The direct addition of calcium to the solutions of the ho- play an important role in calcium binding by providing a bi- modimers and the heterodimer had different effects on each. dentate ligand to the calcium ion and forming an integral part MRP8 immediately forms an insoluble precipitate in direct of a network of hydrogen bonds that serve to stabilize the proportion to the amount of Ca added to the solution. In binding loop (51). Substitution of this Glu residue is known to contrast, MRP14 remains fully soluble throughout a titration drastically reduce calcium affinity (52, 53) and alter the large up to 2 molar equivalents/mol of protein and beyond. This 21 21 Ca -induced conformational changes in the Ca sensors cal- solution gives excellent one-dimensional H and two-dimen- 15 1 modulin and troponin C (54, 55). In analogy to the S100 homo- sional N- H HSQC spectra, as shown in Fig. 7A. The mixture logue p11, which also has an aspartate at this position, we of MRP8 and MRP14 has a similar response to the addition of anticipate that the NH-terminal site of MRP8 is not likely to Ca as MRP8 alone, presumably due to precipitation of MRP8. bind Ca with appreciable affinity. However, we did discover that MRP8 can be titrated into a One of the characteristic properties of S100 proteins is their solution of fully Ca -loaded MRP14 without formation of any tendency to dimerize. A calcium-dependent association of precipitate. NMR spectra obtained on the Ca -loaded het- MRP8 and MRP14 has been reported (21, 48), suggesting the erodimer produced in this manner are clearly distinct from the preferential formation of a 1:1 complex. In fact, the Kyte and spectrum of Ca -loaded MRP14/MRP14 homodimer, strongly Doolittle hydropathy analysis (56) shown in Fig. 9 indicates suggesting that the Ca -loaded MRP8/MRP14 heterodimer is that MRP8 and MRP14 have hydropathic profiles similar to formed and that it has a unique structure (Fig. 7B). S100 proteins known to dimerize (57, 58). Both MRP8 and DISCUSSION MRP14 exhibit the same hydrophobic NH and COOH termini The experiments presented here describe the high level ex- and hydrophilic EF-hand regions (32) as calcyclin and S100b. pression and biophysical characterization of recombinant hu- These results imply that both MRP8 and MRP14 should also man MRP8 and MRP14, two members of the S100 calcium- form dimers. binding protein family. Alignment of the calcium-binding loops The absence or presence and the biological significance of of MRP8 and MRP14 with other members of the S100 family disulfide-linked covalent dimers of S100 proteins has been a show that MRP14 contains the conserved sequence determi- topic of some debate (57, 58). It is not known whether MRP8 or nants necessary for calcium-binding in sites I and II that are MRP14 form biologically relevant, disulfide-linked dimers in found in other S100 proteins. MRP8 has a standard binding solution. To examine this possibility, a homology model of the loop in site II but a significant Glu 3 Asp substitution at MRP8/MRP14 complex was assembled based on the three- Expression and Characterization of MRP8 and MRP14 12433 15 1 FIG.6. 500-MHz N- H HSQC spectra of MRP14 in the ho- modimeric and heterodimeric complexes in the absence of cal- cium at pH 8.5 and 27 °C. A, N-enriched MRP14wt and unlabeled 15 1 MRP8C42S. The inset shows the N- H HSQC spectra of the same sample of MRP14wt prior to the addition of unlabeled MRP8C42S. B, 15 1 21 15 15 FIG.7. 500-MHz N- H HSQC spectra of the Ca -loaded N-enriched MRP14C3S and unlabeled MRP8C42S. The ratio of N- MRP8/MRP14 heterodimeric and MRP14/MRP14 homodimeric enriched to unlabeled protein is 1:1.2. Details on sample preparation complexes at pH 8.5 and 27 °C. A, N-enriched MRP14C3s and and acquisition parameters are provided under “Experimental unlabeled MRP8C42S. B, N-enriched MRP14C3S. Each sample con- Procedures.” tained 10 mM Tris-HCl, 1 mM EDTA, and 5 mM CaCl . dimensional solution structure of apo calcyclin determined in this laboratory (29). Inspection of this model suggests that the with the formation of a dimeric (and not higher order) species. 42 3 distance between Cys in MRP8 and Cys in MRP14 would These results suggest a unique complementarity between make it highly unlikely that biologically relevant disulfides MRP8 and MRP14, leading to a well folded stable heterodimer. would form in the heterodimer (Fig. 10). This prompted the The comparison of the fluorescence emission spectra shown production of the two MRP mutants, MRP8C42S and in Fig. 4 strongly suggests distinct differences between the MRP14C3S, which provide a means to avoid problems associ- structures of the homodimers and the heterodimer. The low ated with unwanted disulfide bond formation, particularly dur- fluorescence intensity of Trp in the MRP14/MRP14 ho- ing biophysical characterization at high protein concentrations. modimer (Fig. 4B) suggests that it is more solvent-exposed UV, CD, fluorescence, and especially NMR experiments than is Trp in the MRP8/MRP8 homodimer (Fig. 4A). This showed that the mutant proteins are essentially identical to suggests a rationale for why the co-addition of the normalized wild type. spectra of the homodimers does not correspond to the experi- The propensity of S100 proteins to dimerize and the pairing mentally observed spectrum of the heterodimer. The sequence 88 82 82 of MRP8 and MRP14 in vivo motivated the characterization of homologs of Trp , Met in calcyclin, and Thr in S100b, are the extent to which these two proteins associate with each residues that are known to participate in the formation of a other. Chemical cross-linking experiments showed that ho- stable dimer interface (29, 59). If Trp is partially solvent- modimers (and for MRP8, specific higher order oligomers) are exposed in the homodimer but becomes buried in the well formed in solutions containing only MRP8 or MRP14. In mix- packed heterodimer, then the intensity of Trp fluorescence is tures of the two proteins, the heterodimer is greatly preferred expected to increase in the less polar environment of the het- (in a ratio of at least 10:1; Fig. 2). The uniqueness of the erodimer (47). The more highly sequestered location of Trp in heterodimer is strongly supported by the observation of (i) very the dimer interface is attributed to a greater degree of comple- poor NMR spectral features for the homogenous solutions of mentary in the packing of the hydrophobic side chains in the either MRP8 or MRP14 but far superior results for the 1:1 heterodimer versus the homodimer. The significance of this mixture (Figs. 5 and 6); (ii) no evidence for a second set of hypothesis must now be explored by the combination of site- signals in any of the NMR spectra of the heterodimer, which directed mutagenesis and more detailed structural analysis. suggests that the equilibrium concentration of homooligomers We have characterized the Ca dependence of the struc- must be less than 1:50; (iii) NMR line widths and sensitivity in tural properties of the MRP8, MRP14, and MRP8/MRP14 15 1 the two-dimensional N- H HSQC spectra that are consistent dimers. The CD spectra show that calcium addition leads to 12434 Expression and Characterization of MRP8 and MRP14 FIG.8. Sequences of the calcium-binding loops of some S100 proteins. Data are adapted from Potts et al. (29). FIG. 10. Proximity of cysteine residues in the homology model of the MRP8/MRP14 heterodimer. The structure is represented as a backbone ribbon with the MRP8 chain lighter than the MRP14 chain. 42 3 The side chains of Cys in MRP8 and Cys in MRP14 are shown in a space-filling representation. For greater clarity, residues 96 –114 of MRP14 are not displayed. in intensity is consistent with that seen for calgranulin C and FIG.9. Hydropathic character of calcyclin, S100b, MRP8, and S100b (50, 64), suggesting that there are distinct calcium- MRP14. The numbering refers to structurally conserved regions of each induced structural changes for the MRP8/MRP8, MRP14/ protein as determined from sequence analysis (29). Hydrophilicity val- MRP14, and MRP8/MRP14 dimers. The presence of Ca -in- ues were calculated using a seven-residue window size according to 15 1 2 6 42 duced changes is most clearly exemplified in the N- H HSQC Kyte and Doolittle (56) using in house software. Residues Asp –Leu 50 87 and Asp –Ala of human calcyclin (solid line) overlaid with residues spectra of the Ca -loaded MRP14/MRP14 homodimer and the 4 40 50 88 Glu –Leu and Gln –Phe of bovine S100b (hatched line). B, residues MRP8/MRP14 heterodimer (Fig. 7, A and B, respectively). Each 4 42 48 87 Glu –Cys and Lys –Ala of human MRP8 (solid line) overlaid with spectrum is distinctly different from their respective apo coun- 7 45 56 107 residues Gln –Leu and Glu –Pro of human MRP14 (hatched line). terparts (Fig. 6), which directly indicates that changes are brought on by Ca binding. The ability to obtain spectra only slight changes in helicity for the MRP14 homodimer and indicative of a stable well packed homodimer of MRP14, but not the MRP8/MRP14 heterodimer. However, there is a distinct of MRP8, is an intriguing result in light of the hypothesis that reduction in molar ellipticity in the spectrum of MRP8 upon MRP14 alone, but not MRP8 alone, has biologically relevant calcium addition, similar to that seen for other homodimeric activity in specific inflammatory events. S100 proteins (e.g. S100A3 (50) and S100b (49)). However, in In summary, the chemical cross-linking, CD, fluorescence, the case of MRP8, there are potential complications due to the and NMR analysis of MRP8 and MRP14 show that a stable well limited solubility in the presence of calcium, such that the packed heterodimeric complex is preferentially formed both in change in CD intensity could also arise from aggregation. In the absence and presence of calcium ions. These results indi- addition, changes in secondary structure upon calcium binding cate that the functionally relevant form of the MRP8 and are not expected, since there is no change in the distribution of MRP14 complex is a heterodimer. Furthermore, the corre- secondary structure seen upon Ca -loading in the three-di- sponding studies of isolated MRP14 strongly suggest that its mensional structures of calcyclin (60) and S100b (61, 62) or expression and functional roles independent of MRP8 are as- other EF-hand CaBPs (e.g. Ref. 63). sociated with a stable dimeric state. The determination of the The fluorescence data show distinct changes in emission three-dimensional solution structures of MRP8/MRP14 and intensity upon calcium addition for all three dimers. This loss MRP14/MRP14 is currently in progress in this laboratory. Expression and Characterization of MRP8 and MRP14 12435 28. Zimmer, D. B., Chessher, J., and Song, W. (1996) Biochim. Biophys. 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