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Three-dimensional Localization of Divergent Region 3 of the Ryanodine Receptor to the Clamp-shaped Structures Adjacent to the FKBP Binding Sites

Three-dimensional Localization of Divergent Region 3 of the Ryanodine Receptor to the... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 16, Issue of April 18, pp. 14211–14218, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Three-dimensional Localization of Divergent Region 3 of the Ryanodine Receptor to the Clamp-shaped Structures Adjacent to the FKBP Binding Sites* Received for publication, December 26, 2002, and in revised form, February 5, 2003 Published, JBC Papers in Press, February 7, 2003, DOI 10.1074/jbc.M213164200 Jing Zhang‡, Zheng Liu§, Haruko Masumiya‡ , Ruiwu Wang‡, Dawei Jiang‡ , Fei Li§, Terence Wagenknecht§**, and S. R. Wayne Chen‡ ‡‡ From the ‡Cardiovascular Research Group, Departments of Physiology and Biophysics and of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada, the §Wadsworth Center, New York State Department of Health, Albany, New York 12201, and the **Department of Biomedical Sciences, School of Public Health, State University of New York, Albany, New York 12201 The ryanodine receptor (RyR), located in the sarco(endo) Of the three divergent regions of ryanodine receptors (RyRs), divergent region 3 (DR3) is the best studied and plasmic reticulum of a variety of cells, functions as an intra- is believed to be involved in excitation-contraction cou- cellular Ca release channel and plays an essential role in 2 2 pling as well as in channel regulation by Ca and Mg . various fundamental cellular processes including muscle con- To gain insight into the structural basis of DR3 function, traction and relaxation, fertilization, and apoptosis (1). Three we have determined the location of DR3 in the three- RyR isoforms, RyR1, RyR2, and RyR3, have been identified in dimensional structure of RyR2. We inserted green fluo- mammalian tissues and have been shown to display distinct rescent protein (GFP) into the middle of the DR3 region patterns of expression. RyR1 is predominantly expressed in after Thr-1874 in the sequence. HEK293 cells expressing skeletal muscle, whereas RyR2 is mainly expressed in cardiac this GFP-RyR2 fusion protein, RyR2 were T1874-GFP, muscle and the brain. RyR3 expression is widespread but at readily detected by their green fluorescence, indicating relatively low levels (2–5). The physiological function and proper folding of the inserted GFP. RyR2 was T1874-GFP mechanism of activation are also specific to each RyR isoform. further characterized functionally by assays of Ca re- RyR1 is required for excitation-contraction (EC) coupling in lease and [ H]ryanodine binding. These analyses re- skeletal muscle, in which RyR1 is activated upon depolariza- vealed that RyR2 functions as a caffeine- and T1874-GFP 2 tion by the voltage sensor, the L-type Ca channel (dihydro- ryanodine-sensitive Ca release channel and displays 2 3 pyridine receptor, DHPR), through a direct physical interac- Ca dependence and [ H]ryanodine binding properties tion. RyR2, on the other hand, mediates EC coupling in cardiac similar to those of the wild type RyR2. RyR2 T1874-GFP muscle, and it is activated by Ca influx through the L-type was purified from cell lysates in a single step by affinity 2 2 2 Ca channel via a mechanism known as Ca -induced Ca chromatography using GST-FKBP12.6 as the affinity li- release. Importantly, neither RyR2 nor RyR3 is capable of gand. The three-dimensional structure of the purified substituting for RyR1 in mediating skeletal type EC coupling RyR2 was then reconstructed using cryoelec- T1874-GFP tron microscopy and single particle image analysis. (2, 6 –9). The physiological role of RyR3 is unclear, but RyR3 Comparison of the three-dimensional reconstructions of has been shown to be involved in amplification of the Ca wild type RyR2 and RyR2 revealed the location signal generated by RyR1 in skeletal muscle (3, 10) and in T1874-GFP of the inserted GFP, and hence the DR3 region, in one of regulation of the Ca release activity of RyR1 or RyR2 in the characteristic domains of RyR, domain 9, in the smooth muscle (11). Some of these functional differences clamp-shaped structure adjacent to the FKBP12 and among RyR isoforms are likely to be attributable to differences FKBP12.6 binding sites. COOH-terminal truncation in their primary sequences. One of our goals is to determine analysis demonstrated that a region between 1815 and which differences in the amino acid sequences of the isoforms 1855 near DR3 is essential for GST-FKBP12.6 binding. are responsible for these functional variations. These results provide a structural basis for the role of The amino acid sequences of the three RyR isoforms share a the DR3 region in excitation-contraction coupling and high degree of sequence identity (66 –70%) with their major in channel regulation. variations occurring in three regions, known as divergent re- gions 1, 2, and 3 (DR1, DR2, and DR3) (12). The DR1 region * This work was supported by research grants from the Canadian comprises residues 4254 – 4631 of RyR1 and residues 4210 – Institutes of Health Research and the Heart and Stroke Foundation of 4562 of RyR2. The DR2 region includes residues 1342–1403 of Alberta, N.W.T. and Nunavut (to S. R. W. C.) and by the Muscular RyR1 and residues 1353–1397 of RyR2. Residues 1872–1923 of Dystrophy Association and National Institutes of Health Grant AR40615 (to T. W.). The Resource for Visualization of Biological Com- RyR1 and residues 1852–1890 of RyR2 are denoted as the DR3 plexity was supported by National Institutes of Health Biotechnological region. These divergent regions have been the focus of a num- Resource Grant RR01219. The costs of publication of this article were ber of structural and functional studies. For instance, sequence defrayed in part by the payment of page charges. This article must variations in the DR1 region between RyR1 and RyR2 have therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. been shown to account for the isoforms’ different sensitivities to Supported by the Uehara Memorial Foundation (Japan) and Post- Ca inactivation (13–15), whereas the DR2 region, which is doctoral Fellowships from the Heart and Stroke Foundation of Canada and the Alberta Heritage Foundation for Medical Research (AHFMR). Recipient of the Alex W. Church Graduate Student Award. The abbreviations used are: RyR, ryanodine receptor; CHAPS, 3-[(3- ‡‡ AHFMR Senior Scholar. To whom correspondence should be ad- cholamidopropyl)-dimethylammonio]-1-propane sulfonate; EC, excita- dressed. Tel.: 403-220-4235; Fax: 403-283-4841; E-mail: swchen@ tion-contraction; cryo-EM, cryoelectron microscopy; GFP, green fluores- ucalgary.ca. cent protein; PIPES, 1,4-piperazinediethanesulfonic acid. This paper is available on line at http://www.jbc.org 14211 This is an Open Access article under the CC BY license. 14212 Three-dimensional Localization of the DR3 Region absent in RyR3, was found to be critical for skeletal muscle EC in EC coupling, and they further demonstrate the utility of this coupling, since deletion of DR2 in RyR1 abolished EC coupling approach in correlating linear sequence to three-dimensional (16). structure, thereby allowing an understanding of the structural The DR3 region appears to have multiple roles in RyR chan- basis of RyR function. nel function. In RyR1, DR3 consists of a stretch of 30 nega- EXPERIMENTAL PROCEDURES tively charged amino acid residues that are thought to consti- Materials—Restriction endonucleases and DNA-modifying enzymes tute a low affinity Ca binding site (17). Consistent with this 2 were purchased from New England BioLabs Inc. The anti-RyR and hypothesis, deletion of the DR3 region altered the Ca - and anti-GFP antibodies were obtained from Affinity BioReagents (Golden, Mg -dependent regulation and conduction properties of the CO). The anti-c-Myc antibody was kindly provided by the Immunology RyR1 channel (18 –20). A region of RyR1 between residues 1837 Core Facility at the Wadsworth Center of the New York State Depart- and 2168, encompassing the DR3 region, has also been shown ment of Health. Soybean phosphatidylcholine was obtained from Avanti Polar Lipids, Inc. CHAPS and other reagents were purchased from to interact with the II-III loop of the DHPR and to participate Sigma. in EC coupling (21). Interestingly, the interaction between a Cell Culture and DNA Transfection—HEK293 cells were maintained sequence in the II-III loop of DHPR and RyR1 was enhanced by in Dulbecco’s modified Eagle’s medium as described previously (30). the 12-kDa FK506-binding protein (FKBP12), suggesting that HEK293 cells grown on 100-mm tissue culture dishes for 18 –20 h after FKBP12 may modulate EC coupling in skeletal muscle (22). subculture were transfected with 6 –12 g of wild type (WT) or mutant Recently, we have shown that an NH -terminal fragment con- RyR cDNAs using Ca phosphate precipitation (31). Construction of RyR2 —The cloning and construction of the taining the first 1937 residues of RyR2 is sufficient to enable T1874-GFP 15-kb full-length cDNA encoding the mouse cardiac RyR2 has been GST-FKBP12.6 binding, whereas a shorter NH -terminal frag- described previously (32). The DNA encoding GFP flanked by Gly-rich ment including only the first 1636 residues is unable to bind spacers and an AscI site was obtained by PCR as described previously GST-FKBP12.6, indicating that the region between residues (29). The AscI site was introduced into the DR3 region of RyR2 after 1636 and 1937 of RyR2, encompassing the DR3 region, is re- Thr-1874 in the sequence by overlap-extension using PCR (33). The “outer” primers used were as follows: forward, 5-GATCCTCTGCAGT- quired for FKBP12.6 interaction (23). Whether the correspond- TCATGTCCCTC-3; reverse, 5-TCCATCACTGTCTCATGCATCCC-3. ing region in RyR1 is essential for FKBP12-RyR1 interaction The primers for introducing the AscI site were as follows: forward, has yet to be determined. Taken together, these observations 5-AGTATAGAAGACGGGCGCGCCGAAGGCGAAGAAGAAGCC-3; suggest that residues within or near the DR3 region may be reverse, 5-TTCTTCGCCTTCGGCGCGCCCGTCTTCTATACTGATCT- involved in EC coupling and in channel regulation by Ca , C-3. The Bsu36I (4900)-EcoRV (6439) PCR fragment containing the AscI site was used to replace the Bsu36I (4900)-EcoRV (13,873) frag- Mg , or FKBPs. The molecular mechanisms by which the DR3 ment in the full-length RyR2. The missing EcoRV (6439)-EcoRV region participates in such seemingly diverse functions are, (13,873) fragment was then added back to yield the full-length RyR2 however, unknown. with the inserted AscI site. The AscI-AscI fragment containing GFP and One approach to understanding the roles of individual re- the spacers was then subcloned into the full-length RyR2 at the intro- gions or domains in RyR channel function is to map their duced AscI site. The sequences of all PCR fragments and the orientation three-dimensional locations in the channel structure. The of the inserted GFP cDNA were verified by DNA sequencing analysis. Construction of COOH-terminal Truncation Mutants of RyR2—Con- three-dimensional structures of all three RyR isoforms have struction of the 1–1937 and 1–1636 COOH-terminal truncation mu- been determined to 30 – 40 Å by cryoelectron microscopy tants was described earlier (23). PCR was used to introduce a stop codon (cryo-EM) combined with single particle image processing (24, after residues 1895, 1855, 1815, 1775, and 1735 to generate the COOH- 25). The three RyR isoforms display virtually identical three- terminal truncation mutants, 1–1895, 1–1855, 1–1815, 1–1775, and dimensional structures, as expected based on their high degree 1–1735, respectively. The forward primer used for all truncation mu- tants was 5-GATCCTCTGCAGTTCATGTCCCTC-3. The reverse of sequence identity. The three-dimensional structure of RyR is primers, each containing a stop codon followed by an EcoRV site were essentially composed of two major components: a cytoplasmic 5-ATGATATCTCACATCTGGAGCAAGCCTTCCTT-3 (1895-stop), 5- assembly and a transmembrane assembly. The cytoplasmic ATGATATCTCAAGCTTCCTTAAACACACTGGG-3 (1855-stop), 5-A- assembly contains at least 10 domains (26). Large conforma- TGATATCTCACGTGGTCCCACCCACAGGATC-3 (1815-stop), 5-GA- tional changes, particularly in the clamp-shaped structure that TATCCTAGTCGTTGCTAATGCTCACGA-3 (1775-stop), and 5-GATA- includes domains 5, 6, 9, and 10, and is located at the corners TCCTACTTTGTCTCCTCTGTCATAG-3 (1735-stop). The Bsu36I (4900)-EcoRV (introduced after the stop codon at various sites) PCR of the cytoplasmic assembly, have been observed when the fragments were used to replace the Bsu36I (4900)-EcoRV (13,873) frag- channel was switched from the closed to the open state (25, 27). ment in the full-length RyR2 cDNA to yield the various COOH-terminal The three-dimensional locations of several ligand binding sites, truncation mutants. All truncation mutants were confirmed by DNA including those for calmodulin, FKBP12, FKBP12.6, and Im- sequencing. peratoxin A, have been identified (24). We have recently Cryoelectron Microscopy and Image Processing—The expression and purification of RyR2 was carried out as described previously mapped the three-dimensional locations of the NH -terminal T1874-GFP (29). The purified RyR2 was diluted 5–10-fold with EM dilution and DR1 regions. The NH T1874-GFP terminus is located at the corners buffer (20 mM Na-PIPES, (pH 7.2), 400 mM KCl, 3 mM EGTA, 0.5% within the clamp structure, whereas the DR1 region maps to CHAPS, 2 mM dithiothreitol, and 2 mg/ml leupeptin). Grids were pre- domain 3, also known as the “handle” domain, adjacent to the pared for cryo-EM according to standard methods (34). Micrographs calmodulin binding site (28, 29). were recorded using low dose protocols on a Philips EM420, equipped In the present study, in order to gain insight into the with low dose kit and a GATAN 626 cryotransfer holder, at a magnifi- cation of 38,600  (2%) as verified by a tobacco mosaic virus standard. structural basis and functions of the DR3 region, we have Each exposure corresponded to an electron dose of 10 e /Å . Micro- constructed and expressed in HEK293 cells a RyR2 fusion graphs were checked for drift, astigmatism, and presence of Thon rings protein in which green fluorescence protein (GFP) was inserted by optical diffraction. Selected electron micrographs were digitized on a into the DR3 region after residue Thr-1874 in the sequence Zeiss/Imaging scanner (Z/I Imaging Corp., Huntsville, AL) with a step (RyR2 ). The RyR2 protein was purified and size of 14 m. Images were processed using the SPIDER/WEB software T1874-GFP T1874-GFP package (35), and three-dimensional reconstructions were obtained then subjected to cryo-EM and single-particle image process- through use of the projection matching procedure (29, 36). The final ing. Three-dimensional reconstruction of RyR2 re- T1874-GFP three-dimensional reconstructions of WT RyR2 and RyR2 were T1874-GFP vealed the localization of GFP, and therefore of DR3, to a region computed, respectively, from 4365 and 4374 particles. 4-Fold symmetry in domain 9 in the clamp-shaped structure, adjacent to the was enforced in both three-dimensional reconstructions. The final res- known FKBP12 and FKBP12.6 binding sites. These results olutions of both reconstructions were estimated to be 34 Å by Fourier suggest a role of the DR3 region in conformational changes and shell correlation using a cut-off value of 0.5 (37). The difference map Three-dimensional Localization of the DR3 Region 14213 FIG.1. Insertion of GFP into RyR2 after residue Thr-1874 in the sequence. The amino acid sequences of the three RyR isoforms differ primarily in three regions known as the divergent regions (DR1, DR2, and DR3) shown by the shaded areas. GFP flanked by two Gly- rich spacers was inserted into the DR3 region after Thr-1874, as indi- cated by a filled box. The linear sequence of RyR2 is denoted by an open rectangle. The hatched area depicts the transmembrane domain (TM). The phosphorylation site (S2808), the calmodulin binding site (CaM; 3614 –3643), the proposed Ca sensor (E3987), and the proposed pore- forming segment (4820 – 4829) are also shown. was calculated by subtracting the three-dimensional volume of the WT RyR2 from the RyR2 volume. T1844-GFP 2 3 Ca Release and [ H]Ryanodine Binding Assays—Measurements of free cytosolic Ca concentrations in the transfected HEK293 cells, 2 3 using fluorescence Ca indicator dye fluo-3, and of equilibrium [ H]ry- anodine binding to cell lysate were done as described previously (38). GST-FKBP12.6 Pull-down, Immunoprecipitation, and Immunoblot- ting Analyses—GST-FKBP12.6 pull-down, immunoprecipitation, and immunoblotting were carried out as described previously (23, 29). RESULTS FIG.2. Expression and functional characterization of RyR2 . HEK293 cells grown on glass coverslips were trans- Insertion of Green Fluorescent Protein into the DR3 Region of T1874-GFP fected with RyR2 cDNA. Transfected cells were fixed with 1% T1874-GFP RyR2—To understand how the DR3 region participates in formaldehyde 24 h after transfection. Fluorescence (a) and phase-con- channel function and regulation, we attempted to map the trast images (b) were recorded under the fluorescence microscope at location of the DR3 region onto the three-dimensional structure 40 magnification (A). B, HEK293 cells were transfected with 12 gof RyR2 cDNA. Fluorescence intensity of the fluo-3-loaded trans- of RyR2. To this end, we constructed a RyR2 fusion protein T1874-GFP fected cells was monitored continuously before and after the addition of (RyR2 ) in which GFP was inserted into the middle of T1874-GFP 2.5 mM caffeine (C)or50 M of ryanodine (R). The sharp decreases in the DR3 region, after residue Thr-1874 in the sequence (Fig. 1). fluorescence intensity immediately after the second and third additions The strategy was to use GFP, a relatively small protein (238 of caffeine were due to fluorescence quenching by caffeine. Similar amino acids, compared with the 5000 amino acids of RyR), as results were obtained from three separate experiments. a structural marker (39, 40). Through comparison of the three- dimensional reconstructions of the WT RyR2 and GFP-inserted pretreated with ryanodine, on the other hand, responded only RyR2, it would then be feasible to map the location of GFP and to the first and not the second or third caffeine stimulation (Fig. thus the region into which GFP was inserted. To minimize 2B, b), suggesting that RyR2 is sensitive to ryanodine T1874-GFP potential perturbation of the channel folding and function of modification (42). The immediate drop in fluorescence after the RyR2, we added two glycine-rich spacers on either side of GFP second and third caffeine stimulations was caused by fluores- (40). cence quenching by caffeine (42). Similar Ca release re- Expression and Functional Characterization of sponses to caffeine and ryanodine were observed in HEK293 RyR2 —Since the fluorescence of GFP depends on cells transfected with WT RyR2 (42). No caffeine- or ryanodine- T1874-GFP the proper folding of its own structure and the structure of induced Ca release was detected in nontransfected HEK293 the inserted region (40, 41), GFP could be used not only as a cells or in cells transfected with pCDNA3 vector DNA (data not structural marker but also as an indicator of proper protein shown). Thus, RyR2 is able to form a caffeine- and T1874-GFP folding. Fig. 2A shows phase-contrast and fluorescent micro- ryanodine-sensitive Ca release channel in HEK293 cells. scopic images of the HEK293 cells transfected with The functional properties of RyR2 were further T1874-GFP RyR2 . Green fluorescent HEK293 cells were readily characterized by [ H]ryanodine binding studies, which have T1874-GFP detected after transfection (Fig. 2A, a), whereas no fluores- been widely used as a functional assay for RyR channel activ- cence was observed in HEK293 cells transfected with the WT ity, since ryanodine has access to its binding site only when the RyR2 (data not shown). The green fluorescence indicates that channel is open (43). Fig. 3A shows [ H]ryanodine binding to a the inserted GFP and its neighboring region are likely to cell lysate prepared from HEK293 cells transfected with have folded properly. A reticular pattern of distribution of RyR2 in the presence of a wide range of Ca concen- T1874-GFP 2 3 RyR2 was observed in most of the fluorescent cells, trations. Analysis of the Ca dependence of [ H]ryanodine T1874-GFP as expected based on its function as an intracellular Ca binding by the Hill equation yielded an EC of 0.20  0.02 M release channel (Fig. 2B). In some cells, however, the fluo- (mean  S.E., n  3). This EC value is similar to that rescence appeared as an amorphous mass due to heavy ex- reported for the WT RyR2 (29). We also performed binding in pression of the RyR2 protein. the presence of various concentrations of [ H]ryanodine (Fig. T1874-GFP To further demonstrate that RyR2 retains proper 3B). Scatchard analysis of these binding data revealed that T1874-GFP folding, we assessed its functional properties. We measured RyR2 exhibited high affinity [ H]ryanodine binding T1874-GFP Ca release from transfected HEK293 cells using the fluores- with a K of 2.75  0.43 nM (n  3), which is also similar to the 2 3 cent Ca indicator dye fluo-3. As shown in Fig. 2B, HEK293 behavior of [ H]ryanodine binding to WT RyR2 reported previ- cells transfected with RyR2 cDNA displayed multiple ously (38). No ryanodine binding activity was detected in ly- T1874-GFP Ca release events in response to repeated stimulation by sates from HEK293 cells transfected with either pCDNA3 vec- caffeine (Fig. 2B, a), an activator of RyRs. Transfected cells tor DNA or no DNA. Taken together, these results suggest that 14214 Three-dimensional Localization of the DR3 Region FIG.4. Purification of RyR2 by GST-FKBP12.6 affinity T1874-GFP chromatography. The RyR2 protein was purified from cell T1874-GFP lysate by affinity chromatography using GST-FKBP12.6 as the affinity ligand. The purified RyR2 and WT RyR2 proteins were solubi- T1874-GFP lized and separated in SDS-PAGE. The gel was then stained with Coomassie Brilliant Blue (CBB)(A). A similar SDS-PAGE gel was transferred to nitrocellulose membrane. The membrane was probed either with the anti-RyR antibody (B) or the anti-GFP antibody (C) for 2 3 Western blotting. Note that the RyR2 protein migrated at a FIG.3. Ca dependence and [ H]ryanodine binding proper- T1874-GFP slightly slower rate in SDS-PAGE than did the WT RyR2, due to the ties of RyR2 . A,[ H]ryanodine binding to cell lysate prepared T1874-GFP addition of GFP. Also note that the anti-GFP antibody reacted with from HEK293 cells transfected with RyR2 was carried out at T1874-GFP 2 3 RyR2 but did not recognize the WT RyR2, indicating that the T1874-GFP various concentrations of Ca in the presence of 5 nM [ H]ryanodine. B, antibody is specific. [ H]ryanodine binding to RyR2 transfected cell lysate was per- T1874-GFP formed at various concentrations of [ H]ryanodine in the presence of 100 M Ca . Data shown are from representative experiments, each major components: a large squarelike cytoplasmic assembly repeated three times. The inset in B shows a Scatchard plot. The (290  290  130 Å) encompassing 10 distinct domains (labeled binding affinity (K ) is 2.75  0.43 (mean  S.E., n  3). The binding density (B ) is 0.25  0.06 pmol/mg protein (mean  S.E., n  3). by numerals in Fig. 6) and a smaller transmembrane assembly max (120  120  70 Å, labeled TA). the insertion of GFP after Thr-1874 does not significantly alter Overall, the three-dimensional reconstruction of the structure or function of RyR2. RyR2 is very similar to that of the recombinant WT T1874-GFP Purification of Recombinant RyR2 Protein by GST- RyR2 obtained previously (29), but close examination reveals T1874-GFP FKBP12.6 Affinity Chromatography—RyR2 retains some small differences. The most noticeable difference was T1874-GFP the ability to interact with GST-FKBP12.6. Taking advantage found on domain 9, one of the domains located within the of this property, we were able to purify the RyR2 “clamp” structures that form each of the corners of the RyR2 T1874-GFP protein from transfected HEK293 cell lysate by affinity chro- square-shaped cytoplasmic assembly (25, 27). Specifically, the matography in a single step using GST-FKBP12.6 as the affin- volume of domain 9 of RyR2 appears to be larger than T1874-GFP ity ligand. The purified protein was analyzed by SDS-PAGE the volume in the corresponding domain in WT RyR2. These and immunoblotting. A single high molecular weight band, differences could result either directly from the GFP insertion which migrated at a slightly slower rate than the WT RyR2 (as or from conformational changes caused by GFP insertion. To expected due to the addition of GFP), was detected in the more precisely determine the differences, we generated a three- purified sample of RyR2 (Fig. 4A). This band was dimensional difference map by subtracting the three-dimen- T1874-GFP recognized by both the anti-RyR and anti-GFP antibodies and sional volume of WT RyR2 from that of RyR2 . The T1844-GFP hence corresponds to RyR2 . On the other hand, the difference regions were displayed in green and superimposed T1874-GFP purified WT RyR2 protein was recognized only by the anti-RyR on the three-dimensional reconstruction of WT RyR2 (shown in antibody and not by the anti-GFP antibody (Fig. 4, B and C). blue) using nearly the same threshold as was used in the RyR2 appears to be expressed in an intact form; no display of RyR2 and WT RyR2 (Fig. 6B). This differ- T1874-GFP T1874-GFP degradation products were detected. ence map clearly indicates four significant differences located Cryo-EM and Three-dimensional Reconstruction of in each domain 9 in the cytoplasmic assembly. This significant RyR2 —The purified RyR2 proteins were pre- difference, repeated four times in the difference map, is not T1874-GFP T1874-GFP served in a thin layer of vitreous ice and imaged by cryo-EM. only due to the 4-fold symmetry enforcement, since RyR2 is a Fig. 5 shows a typical electron micrograph of frozen-hydrated homotetramer composed of four identical monomers. Because RyR2 in which individual RyR2 particles are GFP was inserted into each RyR2 monomer, the difference as a T1874-GFP T1874-GFP readily visualized. The particles displayed an apparently intact result of GFP insertion would be expected to repeat four times structure with multiple orientations similar to those observed in the three-dimensional difference map. in the WT RyR2 (29). We believe that these differences are directly attributable Single particle image processing was then applied to obtain a to the excess mass contributed by the GFP insertion in more detailed structure of RyR2 . Fig. 6A shows a RyR2 , because they are the only significant differences T1874-GFP T1874-GFP surface representation of the three-dimensional reconstruction that appear when the three-dimensional difference map is dis- of RyR2 in three orientations (top, bottom, and side played at a density threshold nearly the same as used to image T1874-GFP views). The overall form of the reconstructed RyR2 the RyR2 and WT RyR2 structures. Furthermore, the T1874-GFP T1874-GFP structure resembles a mushroom in shape and consists of two calculated volume of each of the four difference features in Fig. Three-dimensional Localization of the DR3 Region 14215 FIG.5. Cryoelectron microscopy of RyR2 . A portion of a cryo-EM T1874-GFP micrograph of the purified RyR2 T1874-GFP proteins embedded in a thin layer of vit- reous ice is shown. The tetrameric struc- ture of RyR2 is well preserved. T1874-GFP Several individual RyR2 parti- T1874-GFP cles are marked with white circles. Scale bar, 500 Å. FIG.6. Three-dimensional surface representation of RyR2 and difference map. A, the three-dimensional reconstruction of T1874-GFP RyR2 is shown in green. B, difference map (RyR2  WT RyR2) shown in green is superimposed on the three-dimensional T1874-GFP T1874GFP reconstruction of the WT RyR2 (in blue). The asterisk indicates the equivalent location of the FKBP12 binding site. The three-dimensional reconstructions are shown in three views. Left, top views from the cytoplasmic surface, which in situ would face the transverse-tubule; middle, views toward the bottom of the receptor (i.e. as it would appear if viewed from the lumen of the sarcoplasmic reticulum; right, side views. The numerals on the cytoplasm assembly indicate the distinguishable domains. 6B corresponds to a molecular mass of 28 kDa, assuming a binding sites for FKBP12 and FKBP12.6 are 3 and 2.5 nm, protein density of 1.37 g/cm (26), and this volume agrees well respectively. The close localization of the DR3 region and FKBP with the molecular mass of GFP. Other minor differences are binding sites in the three-dimensional structure of RyR sug- small and insignificant, both in volume and density, and are gests that residues near the DR3 region may be involved in unlikely to correspond to the inserted GFP. Hence, we conclude FKBP12/12.6 interaction. that the DR3 region, as indicated by the inserted GFP, is A Region Upstream of DR3 Is Essential for FKBP12.6 Bind- located in domain 9, one of the characteristic domains that ing—We have previously shown that the first 1937 NH -termi- form the cytoplasmically located clamp structures of RyR2. nal amino acid residues of RyR2, a span that includes the DR3 The FKBP12 binding site was previously mapped to a region region, are sufficient for FKBP12.6 binding (23). To examine at or near the junction of domains 3 and 9 in the three-dimen- the role of the DR3 region in FKBP12.6 interaction, we con- sional structure of RyR1 (indicated by an asterisk in Fig. 6B) structed a series of c-Myc-tagged COOH-terminal truncation (44, 45). Recently, the FKBP12.6 binding site has also been mutants by inserting a stop codon between residues 1636 and mapped to a similar region in the three-dimensional structure 1937 (Fig. 7A). These COOH-terminal truncation mutants of RyR2. Interestingly, the three-dimensional location of the were then expressed in HEK293 cells and their expression was DR3 region, as indicated by the localization of the inserted ascertained by immunoprecipitation using the anti-c-Myc an- GFP, is in close proximity to the FKBP12 and FKBP12.6 bind- tibody, whereas their ability to interact with FKBP12.6 was ing sites. The distances between the DR3 location and the assessed by a GST-FKBP12.6 pull-down assay. As shown in Fig. 7B,NH -terminal fragments containing the first 1815 or fewer residues are unable to bind GST-FKBP12.6, whereas M. Sharma, L. Jeyakumar, S. Fleischer, and T. Wagenknecht, un- published results. NH -terminal fragments including the first 1855 or more res- 2 14216 Three-dimensional Localization of the DR3 Region FIG.8. Co-expression of overlapping NH - and COOH-terminal fragments leads to the synthesis of functional RyR2 channels in HEK293 cells. The NH -terminal fragment containing residues 1–1815 and the COOH-terminal fragment missing the first 1636 NH - terminal residues are shown by filled boxes in A. B, HEK293 cells were transfected with NH -terminal fragment 1–1815 and the COOH-termi- FIG.7. GST-FKBP12.6 binding to COOH-terminal truncation nal fragment D1636 either individually (12 g each) (Ba and Bb)orin mutants of RyR2. A, COOH-terminal truncation mutants are depicted combination (8 g each) (Bc). Fluorescence intensity of the fluo-3-loaded by rectangles (constructs 1–7). The remaining NH -terminal residues of transfected cells was monitored continuously before and after the ad- each COOH-terminal truncation mutant are shown on the left of the dition of 2.5 mM caffeine, indicated by C. A transient increase in fluo- corresponding rectangle. All truncation mutants were tagged with the rescence was detected only in cells co-transfected with mutants 1–1815 c-Myc antibody epitope near the NH terminus, as indicated by small 2 2 and D1636, indicating caffeine-induced Ca release from intracellular open boxes. B, HEK293 cells were transfected with truncation mutants stores. The drops in fluorescence signals immediately after the addition as indicated. The c-Myc-tagged mutant proteins were precipitated from of caffeine in Ba and Bb were due to fluorescence quenching by caffeine. cell lysates by the anti-c-Myc antibody and by GST-FKBP12.6 glutathi- Similar results were obtained from three separate experiments. one-Sepharose. The precipitates were solubilized and separated in SDS- PAGE and stained with Coomassie Brilliant Blue. The expression of and GST-FKBP12.6 binding to mutants 1–1937 and 1–1636 have been level (24). Thus, we conclude that the DR3 region maps to shown previously (23). domain 9 in all three RyR isoforms. Much of the following discussion focuses on the skeletal muscle receptor, RyR1, which idues are sufficient for FKBP12.6 binding. These data indicate has been more intensively characterized than RyR2, with re- that the DR3 region (1852–1890) is unlikely to be critical for spect to the function of DR3. FKBP12.6 binding, whereas a region between residues 1815 Implications for Excitation-Contraction Coupling in Skeletal and 1855 upstream of DR3 is essential for FKBP12.6 interac- Muscle—Comparison of the three-dimensional reconstructions tion. The GST-FKBP12.6 binding-deficient NH -terminal frag- of the closed and open states of RyR1 reveals that several ment, 1–1815, is able to restore the function of an NH -termi- domains, including 5, 6, 9, and 10, in the clamp-shaped struc- nal truncation mutant, D1–1636 (Fig. 8A). HEK293 cells tures at the corners of the cytoplasmic assembly, undergo large transfected with the 1–1815 NH -terminal fragment and the conformational changes and/or movements (25, 27). Based on D1–1636 NH -terminal truncation mutant alone exhibited no these findings and on the observation that the spacing between caffeine-sensitive Ca release activity, whereas caffeine-in- the receptor’s clamp structures is similar to that of a group of duced Ca release was readily observed in cells co-transfected four DHPRs (also known as the tetrad) that abut on RyR1 in with both the 1–1815 and D1–1636 fragments (Fig. 8B). These situ at triad junctions, it has been proposed that the clamp observations indicate that the 1–1815 NH -terminal fragment structures undergo direct physical interaction with DHPRs (25, is functional when co-expressed with an overlapping COOH- 27, 46). The amino acid sequences that make up domains 5, 6, terminal fragment and that the lack of GST-FKBP12.6 binding 9, and 10 are, however, unknown. Several regions in the linear to this fragment is unlikely to result from abnormal expression sequence of RyR1, including the DR2 and DR3 regions (16, 21), or gross structural alterations. and regions between residues 1076 and 1112 (47) and between DISCUSSION residues 2659 and 3720 (48) have been implicated as being By applying cryo-EM and three-dimensional reconstruction important for EC coupling or for interactions between RyR1 to recombinant RyR2 containing a GFP insertion at position and DHPR. Until now, it was not known with a high level of 1874, near the center of the divergent region, DR3, we have confidence which, if any, of these sequences is located in the mapped DR3 to a subregion within domain 9 in the cytoplasmic clamp-shaped structure. This work reveals, for the first time, assembly of the receptor. Also, new evidence has been pre- that the DR3 region forms part of domain 9, one of the domains sented to further implicate a region of the RyR2 sequence that comprising the clamp-shaped structure, and raises the possi- bility that DR3 is involved in the conformational changes in is just upstream of the DR3 region as being required for the binding of the accessory protein, FKBP12.6. Consistent with RyRs that are associated with channel gating. This interpreta- tion is supported by comparative studies of RyR1 and RyR3 in this interpretation, previous mapping studies by cryo-EM of FKBP12-RyR1 and FKBP12.6-RyR2 complexes have placed the closed and open states, which reveal structural differences between the two states in the vicinity of domain 9 (25, 27, 49). FKBP12/FKBP12.6 binding sites adjacent to domain 9 in both receptor isoforms. Although our mapping of the DR3 region Thus, our observations further support the notion that domain 9 is important for EC coupling. was obtained on RyR2, we are confident that essentially the same result would have been obtained for RyR1 and RyR3, DR3 Is Located Near the FKBP12/FKBP12.6 Binding Sites on RyRs—Our results are also suggestive of a structural link because the high degree of sequence identity (70%) among the isoforms means that their tertiary structures should also be between EC coupling and the binding of FKBP12, which is tightly associated with RyR1 (50). FKBP12 has been suggested very similar. Indeed, the three-dimensional reconstructions that have been reported for the three RyR isoforms show strict to be an important regulator of EC coupling, in part because FK506, an immunosuppressant drug that dissociates FKBP12 conservation of the domain architecture of the cytoplasmic region and few significant differences at the 2– 4-nm resolution from RyR1, has been shown to impair EC coupling in skeletal Three-dimensional Localization of the DR3 Region 14217 muscle (51). Moreover, the interaction between RyR1 and the sional locations of residues 1076 –1112 and residues 2659 – II-III loop of DHPR is apparently potentiated by FKBP12 (22). 3720 and the sequence identity of domains 5 and 10 are The molecular mechanism by which FKBP12 enhances DHPR- unknown. A systematic and comprehensive study by cryo-EM RyR1 interaction and modulates EC coupling is, however, un- and single particle image processing of modified RyR proteins clear. We found that a region including residues 1815–1855, containing GFP insertions into various regions in RyR will upstream of the DR3 region, is essential for GST-FKBP12.6 allow us to further define the structural basis of EC coupling binding and that the three-dimensional location of the DR3 and other functions of the RyR channel. region is very close to the FKBP12 and FKBP12.6 binding sites. Acknowledgments—We thank the Wadsworth Center’s molecular It is thus possible that FKBP12, by binding to a site near the genetics, immunology, and electron microscopy core facilities and the DR3 region may either stabilize a conformation required for Resource for Visualization of Biological Complexity, the Immunology DR3-DHPR interaction, as suggested previously (22), or may Core Facility at the Wadsworth Center of the New York State De- stabilize the DR3-DHPR interaction directly. partment of Health for providing the anti-c-Myc antibody, Dr. Wayne 2 2 R. Giles for continued support, Cindy Brown for excellent technical DR3 and Regulation of RyRs by Ca and Mg —The DR3 assistance, and Jeff Bolstad for a critical reading of the manuscript. region encompasses a highly negatively charged sequence that is thought to form the low affinity Ca binding site. Deletion REFERENCES of the DR3 region affected both Ca -dependent activation and 1. Berridge, M. J., Lipp, P., and Bootman, M. D. (2000) Nat. Rev. Mol. Cell. Biol. inactivation and Mg inhibition of the RyR1 channel (17–20). 1, 11–21 Mg is an essential regulator of EC coupling in skeletal mus- 2. Franzini-Armstrong, C., and Protasi, F. (1997) Physiol. Rev. 77, 699 –729 3. Sutko, J. L., and Airey, J. A. (1996) Physiol. Rev. 76, 1027–1071 cle. It has been proposed that the initial activation of RyR1 by 4. Ogawa, Y., Kurebayashi, N., and Murayama, T. (1999) Adv. Biophys. 36, the voltage sensor during EC coupling involves the removal of 27– 64 the inhibitory effect of cytoplasmic Mg (52). However, the 5. Giannini, G., Conti, A., Mammarella, S., Scrobogna, M., and Sorrentino, V. (1995) J. Cell Biol. 128, 893–904 molecular mechanisms underlying Mg inhibition of RyR 6. Rios, E., and Pizarro, G. (1991) Physiol. Rev. 71, 849 –908 channel activity and EC coupling are unknown. Localization of 7. Fill, M., and Copello, J. A. (2002) Physiol. Rev. 82, 893–922 the DR3 region, and consequently the putative Mg inhibitory 8. Lamb, G. D. (2000) Clin. Exp. Pharmacol. Physiol. 27, 216 –224 9. Bers, D. M. (2002) Nature 415, 198 –205 site, to domain 9 within the clamp structure suggests certain 10. Yang, D., Pan, Z., Takeshima, H., Wu, C., Nagaraj, R. Y., Ma, J., and Cheng, hypotheses as to the role of Mg in conformational changes H. (2001) J. Biol. Chem. 276, 40210 – 40214 11. Lohn, M., Jessner, W., Furstenau, M., Wellner, M., Sorrentino, V., Haller, H., and in EC coupling. For instance, it is possible that Mg binds Luft, F. C., and Gollasch, M. (2001) Circ. Res. 89, 1051–1057 to domain 9, stabilizing RyR1 in an inactive conformation, and 12. Sorrentino, V., and Volpe, P. (1993) Trends Pharmacol. Sci. 14, 98 –103 that the II-III loop of DHPR may bind to the same domain, 13. Du, G. G., and MacLennan, D. H. (1999) J. Biol. Chem. 274, 26120 –26126 14. Du, G. G., Khanna, V. K., and MacLennan, D. H. (2000) J. Biol. Chem. 275, relieving Mg inhibition and resulting in channel openings. 11778 –11783 Binding of Mg to domain 9 may also affect the binding of 15. Nakai, J., Gao, L., Xu, L., Xin, C., Pasek, D. A., and Meissner, G. (1999) FEBS FKBP, which binds adjacent to domain 9 (45). This may explain Lett. 459, 154 –158 16. Yamazawa, T., Takeshima, H., Shimuta, M., and Iino, M. (1997) J. Biol. Chem. the observation that Mg influences the interaction between 272, 8161– 8164 FKBP and RyR (53, 54). It will be of great interest to determine 17. Zorzato, F., Fujii, J., Otsu, K., Phillips, M., Green, N. M., Lai, F. A., Meissner, whether mutations in the DR3 region, particularly among the G., and MacLennan, D. H. (1990) J. Biol. Chem. 265, 2244 –2256 18. Bhat, M. B., Zhao, J., Hayek, S., Freeman, E. C., Takeshima, H., and Ma, J. negatively charged residues, alter Mg inhibition and skeletal (1997) Biophys. J. 73, 1320 –1328 muscle EC coupling. 19. Hayek, S. M., Zhao, J., Bhat, M., Xu, X., Nagaraj, R., Pan, Z., Takeshima, H., DR3 and Channel Conduction—The DR3 region appears to and Ma, J. (1999) FEBS Lett. 461, 157–164 20. Hayek, S. M., Zhu, X., Bhat, M. B., Zhao, J., Takeshima, H., Valdivia, H. H., be involved not only in channel gating but also in channel and Ma, J. (2000) Biochem. J. 351, 57– 65 conduction, since deletion of the DR3 region has been shown to 21. Proenza, C., O’Brien, J., Nakai, J., Mukherjee, S., Allen, P. D., and Beam, K. G. (2002) J. Biol. Chem. 277, 6530 – 6535 alter the conduction properties of RyR1 (20). The DR3 region is 22. O’Reilly, F. M., Robert, M., Jona, I., Szegedi, C., Albrieux, M., Geib, S., De located along the edge of the cytoplasmic assembly, far away Waard, M., Villaz, M., and Ronjat, M. (2002) Biophys. J. 82, 145–155 from the channel pore-forming region located in the transmem- 23. Masumiya, H., Wang, R., Zhang, J., Xiao, B., and Chen, S. R. W. (2003) J. Biol. Chem. 278, 3786 –3792 brane assembly, so how this region influences channel conduc- 24. Wagenknecht, T., and Samso, M. (2002) Front. Biosci. 7, d1464 –74 tion is unclear. It is unlikely that the DR3 region is directly 25. Serysheva, I. I., Schatz, M., van Heel, M., Chiu, W., and Hamilton, S. L. (1999) involved in the formation of the ion conduction pathway. It is, Biophys. J. 77, 1936 –1944 26. Radermacher, M., Rao, V., Grassucci, R., Frank, J., Timerman, A. P., however, possible that conformational changes in the DR3 Fleischer, S., and Wagenknecht, T. (1994) J. Cell Biol. 127, 411– 423 region may have long range effects on both gating and 27. Orlova, E. V., Serysheva, II, van Heel, M., Hamilton, S. L., and Chiu, W. (1996) conduction. Nat. Struct. Biol. 3, 547–552 28. Liu, Z., Zhang, J., Sharma, M. R., Li, P., Chen, S. R., and Wagenknecht, T. In summary, domain 9, which is involved in the structural (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 6104 – 6109 changes that occur upon channel activation and is also in 29. Liu, Z., Zhang, J., Li, P., Chen, S. R., and Wagenknecht, T. (2002) J. Biol. close contact with bound FKBPs, appears to encompass, Chem. 277, 46712– 46719 30. Chen, S. R. W., Li, X., Ebisawa, K., and Zhang, L. (1997) J. Biol. Chem. 272, within its DR3-containing region, a binding site for the II-III 24234 –24246 loop of DHPR and a low affinity cation binding site for Ca 31. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Mg . Accordingly, we propose that domain 9 constitutes 32. Zhao, M., Li, P., Li, X., Zhang, L., Winkfein, R. J., and Chen, S. R. (1999) an essential part of the macromolecular apparatus that car- J. Biol. Chem. 274, 25971–25974 ries out EC coupling. It is probable that multiple regions or 33. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 51–59 domains are involved in EC coupling. The most likely addi- 34. Wagenknecht, T., Frank, J., Boublik, M., Nurse, K., and Ofengand, J. (1988) J. tional candidates for EC coupling are the DR2 region and the Mol. Biol. 203, 753–760 regions between residues 1076 and 1112 and between resi- 35. Frank, J. (1996) Three-Dimensional Electron Microscopy of Macromolecular Assemblies, pp. 54 –125, Academic Press, Inc., San Diego, CA dues 2659 and 3720 or domains 5, 6, and 10, located within 36. Penczek, P. A., Grassucci, R. A., and Frank, J. (1994) Ultramicroscopy 53, the clamp-shaped structure (16, 25, 27, 46 – 48). The DR2 251–270 region is completely missing in RyR3, and comparison of the 37. Malhotra, A., Penczek, P., Agrawal, R. K., Gabashvili, I. S., Grassucci, R. A., Junemann, R., Burkhardt, N., Nierhaus, K. H., and Frank, J. (1998) J. Mol. three-dimensional reconstructions of RyR1 and RyR3 re- Biol. 280, 103–116 vealed a significant difference in domain 6, suggesting that 38. Li, P., and Chen, S. R. (2001) J. Gen. Physiol. 118, 33– 44 domain 6 may include the DR2 region (49). The three-dimen- 39. Doi, N., and Yanagawa, H. (2002) Methods Mol. Biol. 183, 49 –55 14218 Three-dimensional Localization of the DR3 Region 40. Niwa, H., Inouye, S., Hirano, T., Matsuno, T., Kojima, S., Kubota, M., Ohashi, 47. Leong, P., and MacLennan, D. H. (1998) J. Biol. Chem. 273, 7791–7794 M., and Tsuji, F. I. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13617–13622 48. Nakai, J., Sekiguchi, N., Rando, T. A., Allen, P. D., and Beam, K. G. (1998) 41. Waldo, G. S., Standish, B. M., Berendzen, J., and Terwilliger, T. C. (1999) Nat. J. Biol. Chem. 273, 13403–13406 Biotechnol. 17, 691– 695 49. Sharma, M. R., Jeyakumar, L. H., Fleischer, S., and Wagenknecht, T. (2000) 42. Chen, S. R., Li, P., Zhao, M., Li, X., and Zhang, L. (2002) Biophys. J. 82, J. Biol. Chem. 275, 9485–9491 2436 –2447 50. Jayaraman, T., Brillantes, A. M., Timerman, A. P., Fleischer, S., Erdjument- 43. Tanna, B., Welch, W., Ruest, L., Sutko, J. L., and Williams, A. J. (1998) J. Gen. Bromage, H., Tempst, P., and Marks, A. R. (1992) J. Biol. Chem. 267, Physiol. 112, 55– 69 9474 –9477 44. Wagenknecht, T., Grassucci, R., Berkowitz, J., Wiederrecht, G. J., Xin, H. B., 51. Lamb, G. D., and Stephenson, D. G. (1996) J. Physiol. 494, 569 –576 and Fleischer, S. (1996) Biophys. J. 70, 1709 –1715 52. Lamb, G. D. (2002) Front. Biosci. 7, 834 – 842 45. Wagenknecht, T., Radermacher, M., Grassucci, R., Berkowitz, J., Xin, H. B., 53. Bultynck, G., De Smet, P., Rossi, D., Callewaert, G., Missiaen, L., Sorrentino, and Fleischer, S. (1997) J. Biol. Chem. 272, 32463–32471 V., De Smedt, H., and Parys, J. B. (2001) Biochem. J. 354, 413– 422 46. Serysheva, I. I., Ludtke, S. J., Baker, M. R., Chiu, W., and Hamilton, S. L. 54. Bultynck, G., Rossi, D., Callewaert, G., Missiaen, L., Sorrentino, V., Parys, (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 10370 –10375 J. B., and De Smedt, H. (2001) J. Biol. Chem. 276, 47715– 47724 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Three-dimensional Localization of Divergent Region 3 of the Ryanodine Receptor to the Clamp-shaped Structures Adjacent to the FKBP Binding Sites

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 16, Issue of April 18, pp. 14211–14218, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Three-dimensional Localization of Divergent Region 3 of the Ryanodine Receptor to the Clamp-shaped Structures Adjacent to the FKBP Binding Sites* Received for publication, December 26, 2002, and in revised form, February 5, 2003 Published, JBC Papers in Press, February 7, 2003, DOI 10.1074/jbc.M213164200 Jing Zhang‡, Zheng Liu§, Haruko Masumiya‡ , Ruiwu Wang‡, Dawei Jiang‡ , Fei Li§, Terence Wagenknecht§**, and S. R. Wayne Chen‡ ‡‡ From the ‡Cardiovascular Research Group, Departments of Physiology and Biophysics and of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada, the §Wadsworth Center, New York State Department of Health, Albany, New York 12201, and the **Department of Biomedical Sciences, School of Public Health, State University of New York, Albany, New York 12201 The ryanodine receptor (RyR), located in the sarco(endo) Of the three divergent regions of ryanodine receptors (RyRs), divergent region 3 (DR3) is the best studied and plasmic reticulum of a variety of cells, functions as an intra- is believed to be involved in excitation-contraction cou- cellular Ca release channel and plays an essential role in 2 2 pling as well as in channel regulation by Ca and Mg . various fundamental cellular processes including muscle con- To gain insight into the structural basis of DR3 function, traction and relaxation, fertilization, and apoptosis (1). Three we have determined the location of DR3 in the three- RyR isoforms, RyR1, RyR2, and RyR3, have been identified in dimensional structure of RyR2. We inserted green fluo- mammalian tissues and have been shown to display distinct rescent protein (GFP) into the middle of the DR3 region patterns of expression. RyR1 is predominantly expressed in after Thr-1874 in the sequence. HEK293 cells expressing skeletal muscle, whereas RyR2 is mainly expressed in cardiac this GFP-RyR2 fusion protein, RyR2 were T1874-GFP, muscle and the brain. RyR3 expression is widespread but at readily detected by their green fluorescence, indicating relatively low levels (2–5). The physiological function and proper folding of the inserted GFP. RyR2 was T1874-GFP mechanism of activation are also specific to each RyR isoform. further characterized functionally by assays of Ca re- RyR1 is required for excitation-contraction (EC) coupling in lease and [ H]ryanodine binding. These analyses re- skeletal muscle, in which RyR1 is activated upon depolariza- vealed that RyR2 functions as a caffeine- and T1874-GFP 2 tion by the voltage sensor, the L-type Ca channel (dihydro- ryanodine-sensitive Ca release channel and displays 2 3 pyridine receptor, DHPR), through a direct physical interac- Ca dependence and [ H]ryanodine binding properties tion. RyR2, on the other hand, mediates EC coupling in cardiac similar to those of the wild type RyR2. RyR2 T1874-GFP muscle, and it is activated by Ca influx through the L-type was purified from cell lysates in a single step by affinity 2 2 2 Ca channel via a mechanism known as Ca -induced Ca chromatography using GST-FKBP12.6 as the affinity li- release. Importantly, neither RyR2 nor RyR3 is capable of gand. The three-dimensional structure of the purified substituting for RyR1 in mediating skeletal type EC coupling RyR2 was then reconstructed using cryoelec- T1874-GFP tron microscopy and single particle image analysis. (2, 6 –9). The physiological role of RyR3 is unclear, but RyR3 Comparison of the three-dimensional reconstructions of has been shown to be involved in amplification of the Ca wild type RyR2 and RyR2 revealed the location signal generated by RyR1 in skeletal muscle (3, 10) and in T1874-GFP of the inserted GFP, and hence the DR3 region, in one of regulation of the Ca release activity of RyR1 or RyR2 in the characteristic domains of RyR, domain 9, in the smooth muscle (11). Some of these functional differences clamp-shaped structure adjacent to the FKBP12 and among RyR isoforms are likely to be attributable to differences FKBP12.6 binding sites. COOH-terminal truncation in their primary sequences. One of our goals is to determine analysis demonstrated that a region between 1815 and which differences in the amino acid sequences of the isoforms 1855 near DR3 is essential for GST-FKBP12.6 binding. are responsible for these functional variations. These results provide a structural basis for the role of The amino acid sequences of the three RyR isoforms share a the DR3 region in excitation-contraction coupling and high degree of sequence identity (66 –70%) with their major in channel regulation. variations occurring in three regions, known as divergent re- gions 1, 2, and 3 (DR1, DR2, and DR3) (12). The DR1 region * This work was supported by research grants from the Canadian comprises residues 4254 – 4631 of RyR1 and residues 4210 – Institutes of Health Research and the Heart and Stroke Foundation of 4562 of RyR2. The DR2 region includes residues 1342–1403 of Alberta, N.W.T. and Nunavut (to S. R. W. C.) and by the Muscular RyR1 and residues 1353–1397 of RyR2. Residues 1872–1923 of Dystrophy Association and National Institutes of Health Grant AR40615 (to T. W.). The Resource for Visualization of Biological Com- RyR1 and residues 1852–1890 of RyR2 are denoted as the DR3 plexity was supported by National Institutes of Health Biotechnological region. These divergent regions have been the focus of a num- Resource Grant RR01219. The costs of publication of this article were ber of structural and functional studies. For instance, sequence defrayed in part by the payment of page charges. This article must variations in the DR1 region between RyR1 and RyR2 have therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. been shown to account for the isoforms’ different sensitivities to Supported by the Uehara Memorial Foundation (Japan) and Post- Ca inactivation (13–15), whereas the DR2 region, which is doctoral Fellowships from the Heart and Stroke Foundation of Canada and the Alberta Heritage Foundation for Medical Research (AHFMR). Recipient of the Alex W. Church Graduate Student Award. The abbreviations used are: RyR, ryanodine receptor; CHAPS, 3-[(3- ‡‡ AHFMR Senior Scholar. To whom correspondence should be ad- cholamidopropyl)-dimethylammonio]-1-propane sulfonate; EC, excita- dressed. Tel.: 403-220-4235; Fax: 403-283-4841; E-mail: swchen@ tion-contraction; cryo-EM, cryoelectron microscopy; GFP, green fluores- ucalgary.ca. cent protein; PIPES, 1,4-piperazinediethanesulfonic acid. This paper is available on line at http://www.jbc.org 14211 This is an Open Access article under the CC BY license. 14212 Three-dimensional Localization of the DR3 Region absent in RyR3, was found to be critical for skeletal muscle EC in EC coupling, and they further demonstrate the utility of this coupling, since deletion of DR2 in RyR1 abolished EC coupling approach in correlating linear sequence to three-dimensional (16). structure, thereby allowing an understanding of the structural The DR3 region appears to have multiple roles in RyR chan- basis of RyR function. nel function. In RyR1, DR3 consists of a stretch of 30 nega- EXPERIMENTAL PROCEDURES tively charged amino acid residues that are thought to consti- Materials—Restriction endonucleases and DNA-modifying enzymes tute a low affinity Ca binding site (17). Consistent with this 2 were purchased from New England BioLabs Inc. The anti-RyR and hypothesis, deletion of the DR3 region altered the Ca - and anti-GFP antibodies were obtained from Affinity BioReagents (Golden, Mg -dependent regulation and conduction properties of the CO). The anti-c-Myc antibody was kindly provided by the Immunology RyR1 channel (18 –20). A region of RyR1 between residues 1837 Core Facility at the Wadsworth Center of the New York State Depart- and 2168, encompassing the DR3 region, has also been shown ment of Health. Soybean phosphatidylcholine was obtained from Avanti Polar Lipids, Inc. CHAPS and other reagents were purchased from to interact with the II-III loop of the DHPR and to participate Sigma. in EC coupling (21). Interestingly, the interaction between a Cell Culture and DNA Transfection—HEK293 cells were maintained sequence in the II-III loop of DHPR and RyR1 was enhanced by in Dulbecco’s modified Eagle’s medium as described previously (30). the 12-kDa FK506-binding protein (FKBP12), suggesting that HEK293 cells grown on 100-mm tissue culture dishes for 18 –20 h after FKBP12 may modulate EC coupling in skeletal muscle (22). subculture were transfected with 6 –12 g of wild type (WT) or mutant Recently, we have shown that an NH -terminal fragment con- RyR cDNAs using Ca phosphate precipitation (31). Construction of RyR2 —The cloning and construction of the taining the first 1937 residues of RyR2 is sufficient to enable T1874-GFP 15-kb full-length cDNA encoding the mouse cardiac RyR2 has been GST-FKBP12.6 binding, whereas a shorter NH -terminal frag- described previously (32). The DNA encoding GFP flanked by Gly-rich ment including only the first 1636 residues is unable to bind spacers and an AscI site was obtained by PCR as described previously GST-FKBP12.6, indicating that the region between residues (29). The AscI site was introduced into the DR3 region of RyR2 after 1636 and 1937 of RyR2, encompassing the DR3 region, is re- Thr-1874 in the sequence by overlap-extension using PCR (33). The “outer” primers used were as follows: forward, 5-GATCCTCTGCAGT- quired for FKBP12.6 interaction (23). Whether the correspond- TCATGTCCCTC-3; reverse, 5-TCCATCACTGTCTCATGCATCCC-3. ing region in RyR1 is essential for FKBP12-RyR1 interaction The primers for introducing the AscI site were as follows: forward, has yet to be determined. Taken together, these observations 5-AGTATAGAAGACGGGCGCGCCGAAGGCGAAGAAGAAGCC-3; suggest that residues within or near the DR3 region may be reverse, 5-TTCTTCGCCTTCGGCGCGCCCGTCTTCTATACTGATCT- involved in EC coupling and in channel regulation by Ca , C-3. The Bsu36I (4900)-EcoRV (6439) PCR fragment containing the AscI site was used to replace the Bsu36I (4900)-EcoRV (13,873) frag- Mg , or FKBPs. The molecular mechanisms by which the DR3 ment in the full-length RyR2. The missing EcoRV (6439)-EcoRV region participates in such seemingly diverse functions are, (13,873) fragment was then added back to yield the full-length RyR2 however, unknown. with the inserted AscI site. The AscI-AscI fragment containing GFP and One approach to understanding the roles of individual re- the spacers was then subcloned into the full-length RyR2 at the intro- gions or domains in RyR channel function is to map their duced AscI site. The sequences of all PCR fragments and the orientation three-dimensional locations in the channel structure. The of the inserted GFP cDNA were verified by DNA sequencing analysis. Construction of COOH-terminal Truncation Mutants of RyR2—Con- three-dimensional structures of all three RyR isoforms have struction of the 1–1937 and 1–1636 COOH-terminal truncation mu- been determined to 30 – 40 Å by cryoelectron microscopy tants was described earlier (23). PCR was used to introduce a stop codon (cryo-EM) combined with single particle image processing (24, after residues 1895, 1855, 1815, 1775, and 1735 to generate the COOH- 25). The three RyR isoforms display virtually identical three- terminal truncation mutants, 1–1895, 1–1855, 1–1815, 1–1775, and dimensional structures, as expected based on their high degree 1–1735, respectively. The forward primer used for all truncation mu- tants was 5-GATCCTCTGCAGTTCATGTCCCTC-3. The reverse of sequence identity. The three-dimensional structure of RyR is primers, each containing a stop codon followed by an EcoRV site were essentially composed of two major components: a cytoplasmic 5-ATGATATCTCACATCTGGAGCAAGCCTTCCTT-3 (1895-stop), 5- assembly and a transmembrane assembly. The cytoplasmic ATGATATCTCAAGCTTCCTTAAACACACTGGG-3 (1855-stop), 5-A- assembly contains at least 10 domains (26). Large conforma- TGATATCTCACGTGGTCCCACCCACAGGATC-3 (1815-stop), 5-GA- tional changes, particularly in the clamp-shaped structure that TATCCTAGTCGTTGCTAATGCTCACGA-3 (1775-stop), and 5-GATA- includes domains 5, 6, 9, and 10, and is located at the corners TCCTACTTTGTCTCCTCTGTCATAG-3 (1735-stop). The Bsu36I (4900)-EcoRV (introduced after the stop codon at various sites) PCR of the cytoplasmic assembly, have been observed when the fragments were used to replace the Bsu36I (4900)-EcoRV (13,873) frag- channel was switched from the closed to the open state (25, 27). ment in the full-length RyR2 cDNA to yield the various COOH-terminal The three-dimensional locations of several ligand binding sites, truncation mutants. All truncation mutants were confirmed by DNA including those for calmodulin, FKBP12, FKBP12.6, and Im- sequencing. peratoxin A, have been identified (24). We have recently Cryoelectron Microscopy and Image Processing—The expression and purification of RyR2 was carried out as described previously mapped the three-dimensional locations of the NH -terminal T1874-GFP (29). The purified RyR2 was diluted 5–10-fold with EM dilution and DR1 regions. The NH T1874-GFP terminus is located at the corners buffer (20 mM Na-PIPES, (pH 7.2), 400 mM KCl, 3 mM EGTA, 0.5% within the clamp structure, whereas the DR1 region maps to CHAPS, 2 mM dithiothreitol, and 2 mg/ml leupeptin). Grids were pre- domain 3, also known as the “handle” domain, adjacent to the pared for cryo-EM according to standard methods (34). Micrographs calmodulin binding site (28, 29). were recorded using low dose protocols on a Philips EM420, equipped In the present study, in order to gain insight into the with low dose kit and a GATAN 626 cryotransfer holder, at a magnifi- cation of 38,600  (2%) as verified by a tobacco mosaic virus standard. structural basis and functions of the DR3 region, we have Each exposure corresponded to an electron dose of 10 e /Å . Micro- constructed and expressed in HEK293 cells a RyR2 fusion graphs were checked for drift, astigmatism, and presence of Thon rings protein in which green fluorescence protein (GFP) was inserted by optical diffraction. Selected electron micrographs were digitized on a into the DR3 region after residue Thr-1874 in the sequence Zeiss/Imaging scanner (Z/I Imaging Corp., Huntsville, AL) with a step (RyR2 ). The RyR2 protein was purified and size of 14 m. Images were processed using the SPIDER/WEB software T1874-GFP T1874-GFP package (35), and three-dimensional reconstructions were obtained then subjected to cryo-EM and single-particle image process- through use of the projection matching procedure (29, 36). The final ing. Three-dimensional reconstruction of RyR2 re- T1874-GFP three-dimensional reconstructions of WT RyR2 and RyR2 were T1874-GFP vealed the localization of GFP, and therefore of DR3, to a region computed, respectively, from 4365 and 4374 particles. 4-Fold symmetry in domain 9 in the clamp-shaped structure, adjacent to the was enforced in both three-dimensional reconstructions. The final res- known FKBP12 and FKBP12.6 binding sites. These results olutions of both reconstructions were estimated to be 34 Å by Fourier suggest a role of the DR3 region in conformational changes and shell correlation using a cut-off value of 0.5 (37). The difference map Three-dimensional Localization of the DR3 Region 14213 FIG.1. Insertion of GFP into RyR2 after residue Thr-1874 in the sequence. The amino acid sequences of the three RyR isoforms differ primarily in three regions known as the divergent regions (DR1, DR2, and DR3) shown by the shaded areas. GFP flanked by two Gly- rich spacers was inserted into the DR3 region after Thr-1874, as indi- cated by a filled box. The linear sequence of RyR2 is denoted by an open rectangle. The hatched area depicts the transmembrane domain (TM). The phosphorylation site (S2808), the calmodulin binding site (CaM; 3614 –3643), the proposed Ca sensor (E3987), and the proposed pore- forming segment (4820 – 4829) are also shown. was calculated by subtracting the three-dimensional volume of the WT RyR2 from the RyR2 volume. T1844-GFP 2 3 Ca Release and [ H]Ryanodine Binding Assays—Measurements of free cytosolic Ca concentrations in the transfected HEK293 cells, 2 3 using fluorescence Ca indicator dye fluo-3, and of equilibrium [ H]ry- anodine binding to cell lysate were done as described previously (38). GST-FKBP12.6 Pull-down, Immunoprecipitation, and Immunoblot- ting Analyses—GST-FKBP12.6 pull-down, immunoprecipitation, and immunoblotting were carried out as described previously (23, 29). RESULTS FIG.2. Expression and functional characterization of RyR2 . HEK293 cells grown on glass coverslips were trans- Insertion of Green Fluorescent Protein into the DR3 Region of T1874-GFP fected with RyR2 cDNA. Transfected cells were fixed with 1% T1874-GFP RyR2—To understand how the DR3 region participates in formaldehyde 24 h after transfection. Fluorescence (a) and phase-con- channel function and regulation, we attempted to map the trast images (b) were recorded under the fluorescence microscope at location of the DR3 region onto the three-dimensional structure 40 magnification (A). B, HEK293 cells were transfected with 12 gof RyR2 cDNA. Fluorescence intensity of the fluo-3-loaded trans- of RyR2. To this end, we constructed a RyR2 fusion protein T1874-GFP fected cells was monitored continuously before and after the addition of (RyR2 ) in which GFP was inserted into the middle of T1874-GFP 2.5 mM caffeine (C)or50 M of ryanodine (R). The sharp decreases in the DR3 region, after residue Thr-1874 in the sequence (Fig. 1). fluorescence intensity immediately after the second and third additions The strategy was to use GFP, a relatively small protein (238 of caffeine were due to fluorescence quenching by caffeine. Similar amino acids, compared with the 5000 amino acids of RyR), as results were obtained from three separate experiments. a structural marker (39, 40). Through comparison of the three- dimensional reconstructions of the WT RyR2 and GFP-inserted pretreated with ryanodine, on the other hand, responded only RyR2, it would then be feasible to map the location of GFP and to the first and not the second or third caffeine stimulation (Fig. thus the region into which GFP was inserted. To minimize 2B, b), suggesting that RyR2 is sensitive to ryanodine T1874-GFP potential perturbation of the channel folding and function of modification (42). The immediate drop in fluorescence after the RyR2, we added two glycine-rich spacers on either side of GFP second and third caffeine stimulations was caused by fluores- (40). cence quenching by caffeine (42). Similar Ca release re- Expression and Functional Characterization of sponses to caffeine and ryanodine were observed in HEK293 RyR2 —Since the fluorescence of GFP depends on cells transfected with WT RyR2 (42). No caffeine- or ryanodine- T1874-GFP the proper folding of its own structure and the structure of induced Ca release was detected in nontransfected HEK293 the inserted region (40, 41), GFP could be used not only as a cells or in cells transfected with pCDNA3 vector DNA (data not structural marker but also as an indicator of proper protein shown). Thus, RyR2 is able to form a caffeine- and T1874-GFP folding. Fig. 2A shows phase-contrast and fluorescent micro- ryanodine-sensitive Ca release channel in HEK293 cells. scopic images of the HEK293 cells transfected with The functional properties of RyR2 were further T1874-GFP RyR2 . Green fluorescent HEK293 cells were readily characterized by [ H]ryanodine binding studies, which have T1874-GFP detected after transfection (Fig. 2A, a), whereas no fluores- been widely used as a functional assay for RyR channel activ- cence was observed in HEK293 cells transfected with the WT ity, since ryanodine has access to its binding site only when the RyR2 (data not shown). The green fluorescence indicates that channel is open (43). Fig. 3A shows [ H]ryanodine binding to a the inserted GFP and its neighboring region are likely to cell lysate prepared from HEK293 cells transfected with have folded properly. A reticular pattern of distribution of RyR2 in the presence of a wide range of Ca concen- T1874-GFP 2 3 RyR2 was observed in most of the fluorescent cells, trations. Analysis of the Ca dependence of [ H]ryanodine T1874-GFP as expected based on its function as an intracellular Ca binding by the Hill equation yielded an EC of 0.20  0.02 M release channel (Fig. 2B). In some cells, however, the fluo- (mean  S.E., n  3). This EC value is similar to that rescence appeared as an amorphous mass due to heavy ex- reported for the WT RyR2 (29). We also performed binding in pression of the RyR2 protein. the presence of various concentrations of [ H]ryanodine (Fig. T1874-GFP To further demonstrate that RyR2 retains proper 3B). Scatchard analysis of these binding data revealed that T1874-GFP folding, we assessed its functional properties. We measured RyR2 exhibited high affinity [ H]ryanodine binding T1874-GFP Ca release from transfected HEK293 cells using the fluores- with a K of 2.75  0.43 nM (n  3), which is also similar to the 2 3 cent Ca indicator dye fluo-3. As shown in Fig. 2B, HEK293 behavior of [ H]ryanodine binding to WT RyR2 reported previ- cells transfected with RyR2 cDNA displayed multiple ously (38). No ryanodine binding activity was detected in ly- T1874-GFP Ca release events in response to repeated stimulation by sates from HEK293 cells transfected with either pCDNA3 vec- caffeine (Fig. 2B, a), an activator of RyRs. Transfected cells tor DNA or no DNA. Taken together, these results suggest that 14214 Three-dimensional Localization of the DR3 Region FIG.4. Purification of RyR2 by GST-FKBP12.6 affinity T1874-GFP chromatography. The RyR2 protein was purified from cell T1874-GFP lysate by affinity chromatography using GST-FKBP12.6 as the affinity ligand. The purified RyR2 and WT RyR2 proteins were solubi- T1874-GFP lized and separated in SDS-PAGE. The gel was then stained with Coomassie Brilliant Blue (CBB)(A). A similar SDS-PAGE gel was transferred to nitrocellulose membrane. The membrane was probed either with the anti-RyR antibody (B) or the anti-GFP antibody (C) for 2 3 Western blotting. Note that the RyR2 protein migrated at a FIG.3. Ca dependence and [ H]ryanodine binding proper- T1874-GFP slightly slower rate in SDS-PAGE than did the WT RyR2, due to the ties of RyR2 . A,[ H]ryanodine binding to cell lysate prepared T1874-GFP addition of GFP. Also note that the anti-GFP antibody reacted with from HEK293 cells transfected with RyR2 was carried out at T1874-GFP 2 3 RyR2 but did not recognize the WT RyR2, indicating that the T1874-GFP various concentrations of Ca in the presence of 5 nM [ H]ryanodine. B, antibody is specific. [ H]ryanodine binding to RyR2 transfected cell lysate was per- T1874-GFP formed at various concentrations of [ H]ryanodine in the presence of 100 M Ca . Data shown are from representative experiments, each major components: a large squarelike cytoplasmic assembly repeated three times. The inset in B shows a Scatchard plot. The (290  290  130 Å) encompassing 10 distinct domains (labeled binding affinity (K ) is 2.75  0.43 (mean  S.E., n  3). The binding density (B ) is 0.25  0.06 pmol/mg protein (mean  S.E., n  3). by numerals in Fig. 6) and a smaller transmembrane assembly max (120  120  70 Å, labeled TA). the insertion of GFP after Thr-1874 does not significantly alter Overall, the three-dimensional reconstruction of the structure or function of RyR2. RyR2 is very similar to that of the recombinant WT T1874-GFP Purification of Recombinant RyR2 Protein by GST- RyR2 obtained previously (29), but close examination reveals T1874-GFP FKBP12.6 Affinity Chromatography—RyR2 retains some small differences. The most noticeable difference was T1874-GFP the ability to interact with GST-FKBP12.6. Taking advantage found on domain 9, one of the domains located within the of this property, we were able to purify the RyR2 “clamp” structures that form each of the corners of the RyR2 T1874-GFP protein from transfected HEK293 cell lysate by affinity chro- square-shaped cytoplasmic assembly (25, 27). Specifically, the matography in a single step using GST-FKBP12.6 as the affin- volume of domain 9 of RyR2 appears to be larger than T1874-GFP ity ligand. The purified protein was analyzed by SDS-PAGE the volume in the corresponding domain in WT RyR2. These and immunoblotting. A single high molecular weight band, differences could result either directly from the GFP insertion which migrated at a slightly slower rate than the WT RyR2 (as or from conformational changes caused by GFP insertion. To expected due to the addition of GFP), was detected in the more precisely determine the differences, we generated a three- purified sample of RyR2 (Fig. 4A). This band was dimensional difference map by subtracting the three-dimen- T1874-GFP recognized by both the anti-RyR and anti-GFP antibodies and sional volume of WT RyR2 from that of RyR2 . The T1844-GFP hence corresponds to RyR2 . On the other hand, the difference regions were displayed in green and superimposed T1874-GFP purified WT RyR2 protein was recognized only by the anti-RyR on the three-dimensional reconstruction of WT RyR2 (shown in antibody and not by the anti-GFP antibody (Fig. 4, B and C). blue) using nearly the same threshold as was used in the RyR2 appears to be expressed in an intact form; no display of RyR2 and WT RyR2 (Fig. 6B). This differ- T1874-GFP T1874-GFP degradation products were detected. ence map clearly indicates four significant differences located Cryo-EM and Three-dimensional Reconstruction of in each domain 9 in the cytoplasmic assembly. This significant RyR2 —The purified RyR2 proteins were pre- difference, repeated four times in the difference map, is not T1874-GFP T1874-GFP served in a thin layer of vitreous ice and imaged by cryo-EM. only due to the 4-fold symmetry enforcement, since RyR2 is a Fig. 5 shows a typical electron micrograph of frozen-hydrated homotetramer composed of four identical monomers. Because RyR2 in which individual RyR2 particles are GFP was inserted into each RyR2 monomer, the difference as a T1874-GFP T1874-GFP readily visualized. The particles displayed an apparently intact result of GFP insertion would be expected to repeat four times structure with multiple orientations similar to those observed in the three-dimensional difference map. in the WT RyR2 (29). We believe that these differences are directly attributable Single particle image processing was then applied to obtain a to the excess mass contributed by the GFP insertion in more detailed structure of RyR2 . Fig. 6A shows a RyR2 , because they are the only significant differences T1874-GFP T1874-GFP surface representation of the three-dimensional reconstruction that appear when the three-dimensional difference map is dis- of RyR2 in three orientations (top, bottom, and side played at a density threshold nearly the same as used to image T1874-GFP views). The overall form of the reconstructed RyR2 the RyR2 and WT RyR2 structures. Furthermore, the T1874-GFP T1874-GFP structure resembles a mushroom in shape and consists of two calculated volume of each of the four difference features in Fig. Three-dimensional Localization of the DR3 Region 14215 FIG.5. Cryoelectron microscopy of RyR2 . A portion of a cryo-EM T1874-GFP micrograph of the purified RyR2 T1874-GFP proteins embedded in a thin layer of vit- reous ice is shown. The tetrameric struc- ture of RyR2 is well preserved. T1874-GFP Several individual RyR2 parti- T1874-GFP cles are marked with white circles. Scale bar, 500 Å. FIG.6. Three-dimensional surface representation of RyR2 and difference map. A, the three-dimensional reconstruction of T1874-GFP RyR2 is shown in green. B, difference map (RyR2  WT RyR2) shown in green is superimposed on the three-dimensional T1874-GFP T1874GFP reconstruction of the WT RyR2 (in blue). The asterisk indicates the equivalent location of the FKBP12 binding site. The three-dimensional reconstructions are shown in three views. Left, top views from the cytoplasmic surface, which in situ would face the transverse-tubule; middle, views toward the bottom of the receptor (i.e. as it would appear if viewed from the lumen of the sarcoplasmic reticulum; right, side views. The numerals on the cytoplasm assembly indicate the distinguishable domains. 6B corresponds to a molecular mass of 28 kDa, assuming a binding sites for FKBP12 and FKBP12.6 are 3 and 2.5 nm, protein density of 1.37 g/cm (26), and this volume agrees well respectively. The close localization of the DR3 region and FKBP with the molecular mass of GFP. Other minor differences are binding sites in the three-dimensional structure of RyR sug- small and insignificant, both in volume and density, and are gests that residues near the DR3 region may be involved in unlikely to correspond to the inserted GFP. Hence, we conclude FKBP12/12.6 interaction. that the DR3 region, as indicated by the inserted GFP, is A Region Upstream of DR3 Is Essential for FKBP12.6 Bind- located in domain 9, one of the characteristic domains that ing—We have previously shown that the first 1937 NH -termi- form the cytoplasmically located clamp structures of RyR2. nal amino acid residues of RyR2, a span that includes the DR3 The FKBP12 binding site was previously mapped to a region region, are sufficient for FKBP12.6 binding (23). To examine at or near the junction of domains 3 and 9 in the three-dimen- the role of the DR3 region in FKBP12.6 interaction, we con- sional structure of RyR1 (indicated by an asterisk in Fig. 6B) structed a series of c-Myc-tagged COOH-terminal truncation (44, 45). Recently, the FKBP12.6 binding site has also been mutants by inserting a stop codon between residues 1636 and mapped to a similar region in the three-dimensional structure 1937 (Fig. 7A). These COOH-terminal truncation mutants of RyR2. Interestingly, the three-dimensional location of the were then expressed in HEK293 cells and their expression was DR3 region, as indicated by the localization of the inserted ascertained by immunoprecipitation using the anti-c-Myc an- GFP, is in close proximity to the FKBP12 and FKBP12.6 bind- tibody, whereas their ability to interact with FKBP12.6 was ing sites. The distances between the DR3 location and the assessed by a GST-FKBP12.6 pull-down assay. As shown in Fig. 7B,NH -terminal fragments containing the first 1815 or fewer residues are unable to bind GST-FKBP12.6, whereas M. Sharma, L. Jeyakumar, S. Fleischer, and T. Wagenknecht, un- published results. NH -terminal fragments including the first 1855 or more res- 2 14216 Three-dimensional Localization of the DR3 Region FIG.8. Co-expression of overlapping NH - and COOH-terminal fragments leads to the synthesis of functional RyR2 channels in HEK293 cells. The NH -terminal fragment containing residues 1–1815 and the COOH-terminal fragment missing the first 1636 NH - terminal residues are shown by filled boxes in A. B, HEK293 cells were transfected with NH -terminal fragment 1–1815 and the COOH-termi- FIG.7. GST-FKBP12.6 binding to COOH-terminal truncation nal fragment D1636 either individually (12 g each) (Ba and Bb)orin mutants of RyR2. A, COOH-terminal truncation mutants are depicted combination (8 g each) (Bc). Fluorescence intensity of the fluo-3-loaded by rectangles (constructs 1–7). The remaining NH -terminal residues of transfected cells was monitored continuously before and after the ad- each COOH-terminal truncation mutant are shown on the left of the dition of 2.5 mM caffeine, indicated by C. A transient increase in fluo- corresponding rectangle. All truncation mutants were tagged with the rescence was detected only in cells co-transfected with mutants 1–1815 c-Myc antibody epitope near the NH terminus, as indicated by small 2 2 and D1636, indicating caffeine-induced Ca release from intracellular open boxes. B, HEK293 cells were transfected with truncation mutants stores. The drops in fluorescence signals immediately after the addition as indicated. The c-Myc-tagged mutant proteins were precipitated from of caffeine in Ba and Bb were due to fluorescence quenching by caffeine. cell lysates by the anti-c-Myc antibody and by GST-FKBP12.6 glutathi- Similar results were obtained from three separate experiments. one-Sepharose. The precipitates were solubilized and separated in SDS- PAGE and stained with Coomassie Brilliant Blue. The expression of and GST-FKBP12.6 binding to mutants 1–1937 and 1–1636 have been level (24). Thus, we conclude that the DR3 region maps to shown previously (23). domain 9 in all three RyR isoforms. Much of the following discussion focuses on the skeletal muscle receptor, RyR1, which idues are sufficient for FKBP12.6 binding. These data indicate has been more intensively characterized than RyR2, with re- that the DR3 region (1852–1890) is unlikely to be critical for spect to the function of DR3. FKBP12.6 binding, whereas a region between residues 1815 Implications for Excitation-Contraction Coupling in Skeletal and 1855 upstream of DR3 is essential for FKBP12.6 interac- Muscle—Comparison of the three-dimensional reconstructions tion. The GST-FKBP12.6 binding-deficient NH -terminal frag- of the closed and open states of RyR1 reveals that several ment, 1–1815, is able to restore the function of an NH -termi- domains, including 5, 6, 9, and 10, in the clamp-shaped struc- nal truncation mutant, D1–1636 (Fig. 8A). HEK293 cells tures at the corners of the cytoplasmic assembly, undergo large transfected with the 1–1815 NH -terminal fragment and the conformational changes and/or movements (25, 27). Based on D1–1636 NH -terminal truncation mutant alone exhibited no these findings and on the observation that the spacing between caffeine-sensitive Ca release activity, whereas caffeine-in- the receptor’s clamp structures is similar to that of a group of duced Ca release was readily observed in cells co-transfected four DHPRs (also known as the tetrad) that abut on RyR1 in with both the 1–1815 and D1–1636 fragments (Fig. 8B). These situ at triad junctions, it has been proposed that the clamp observations indicate that the 1–1815 NH -terminal fragment structures undergo direct physical interaction with DHPRs (25, is functional when co-expressed with an overlapping COOH- 27, 46). The amino acid sequences that make up domains 5, 6, terminal fragment and that the lack of GST-FKBP12.6 binding 9, and 10 are, however, unknown. Several regions in the linear to this fragment is unlikely to result from abnormal expression sequence of RyR1, including the DR2 and DR3 regions (16, 21), or gross structural alterations. and regions between residues 1076 and 1112 (47) and between DISCUSSION residues 2659 and 3720 (48) have been implicated as being By applying cryo-EM and three-dimensional reconstruction important for EC coupling or for interactions between RyR1 to recombinant RyR2 containing a GFP insertion at position and DHPR. Until now, it was not known with a high level of 1874, near the center of the divergent region, DR3, we have confidence which, if any, of these sequences is located in the mapped DR3 to a subregion within domain 9 in the cytoplasmic clamp-shaped structure. This work reveals, for the first time, assembly of the receptor. Also, new evidence has been pre- that the DR3 region forms part of domain 9, one of the domains sented to further implicate a region of the RyR2 sequence that comprising the clamp-shaped structure, and raises the possi- bility that DR3 is involved in the conformational changes in is just upstream of the DR3 region as being required for the binding of the accessory protein, FKBP12.6. Consistent with RyRs that are associated with channel gating. This interpreta- tion is supported by comparative studies of RyR1 and RyR3 in this interpretation, previous mapping studies by cryo-EM of FKBP12-RyR1 and FKBP12.6-RyR2 complexes have placed the closed and open states, which reveal structural differences between the two states in the vicinity of domain 9 (25, 27, 49). FKBP12/FKBP12.6 binding sites adjacent to domain 9 in both receptor isoforms. Although our mapping of the DR3 region Thus, our observations further support the notion that domain 9 is important for EC coupling. was obtained on RyR2, we are confident that essentially the same result would have been obtained for RyR1 and RyR3, DR3 Is Located Near the FKBP12/FKBP12.6 Binding Sites on RyRs—Our results are also suggestive of a structural link because the high degree of sequence identity (70%) among the isoforms means that their tertiary structures should also be between EC coupling and the binding of FKBP12, which is tightly associated with RyR1 (50). FKBP12 has been suggested very similar. Indeed, the three-dimensional reconstructions that have been reported for the three RyR isoforms show strict to be an important regulator of EC coupling, in part because FK506, an immunosuppressant drug that dissociates FKBP12 conservation of the domain architecture of the cytoplasmic region and few significant differences at the 2– 4-nm resolution from RyR1, has been shown to impair EC coupling in skeletal Three-dimensional Localization of the DR3 Region 14217 muscle (51). Moreover, the interaction between RyR1 and the sional locations of residues 1076 –1112 and residues 2659 – II-III loop of DHPR is apparently potentiated by FKBP12 (22). 3720 and the sequence identity of domains 5 and 10 are The molecular mechanism by which FKBP12 enhances DHPR- unknown. A systematic and comprehensive study by cryo-EM RyR1 interaction and modulates EC coupling is, however, un- and single particle image processing of modified RyR proteins clear. We found that a region including residues 1815–1855, containing GFP insertions into various regions in RyR will upstream of the DR3 region, is essential for GST-FKBP12.6 allow us to further define the structural basis of EC coupling binding and that the three-dimensional location of the DR3 and other functions of the RyR channel. region is very close to the FKBP12 and FKBP12.6 binding sites. Acknowledgments—We thank the Wadsworth Center’s molecular It is thus possible that FKBP12, by binding to a site near the genetics, immunology, and electron microscopy core facilities and the DR3 region may either stabilize a conformation required for Resource for Visualization of Biological Complexity, the Immunology DR3-DHPR interaction, as suggested previously (22), or may Core Facility at the Wadsworth Center of the New York State De- stabilize the DR3-DHPR interaction directly. partment of Health for providing the anti-c-Myc antibody, Dr. Wayne 2 2 R. Giles for continued support, Cindy Brown for excellent technical DR3 and Regulation of RyRs by Ca and Mg —The DR3 assistance, and Jeff Bolstad for a critical reading of the manuscript. region encompasses a highly negatively charged sequence that is thought to form the low affinity Ca binding site. Deletion REFERENCES of the DR3 region affected both Ca -dependent activation and 1. Berridge, M. J., Lipp, P., and Bootman, M. D. (2000) Nat. Rev. Mol. Cell. Biol. inactivation and Mg inhibition of the RyR1 channel (17–20). 1, 11–21 Mg is an essential regulator of EC coupling in skeletal mus- 2. Franzini-Armstrong, C., and Protasi, F. (1997) Physiol. Rev. 77, 699 –729 3. Sutko, J. L., and Airey, J. A. (1996) Physiol. Rev. 76, 1027–1071 cle. It has been proposed that the initial activation of RyR1 by 4. Ogawa, Y., Kurebayashi, N., and Murayama, T. (1999) Adv. Biophys. 36, the voltage sensor during EC coupling involves the removal of 27– 64 the inhibitory effect of cytoplasmic Mg (52). However, the 5. Giannini, G., Conti, A., Mammarella, S., Scrobogna, M., and Sorrentino, V. (1995) J. Cell Biol. 128, 893–904 molecular mechanisms underlying Mg inhibition of RyR 6. Rios, E., and Pizarro, G. (1991) Physiol. Rev. 71, 849 –908 channel activity and EC coupling are unknown. Localization of 7. Fill, M., and Copello, J. A. (2002) Physiol. Rev. 82, 893–922 the DR3 region, and consequently the putative Mg inhibitory 8. Lamb, G. D. (2000) Clin. Exp. Pharmacol. Physiol. 27, 216 –224 9. Bers, D. M. (2002) Nature 415, 198 –205 site, to domain 9 within the clamp structure suggests certain 10. Yang, D., Pan, Z., Takeshima, H., Wu, C., Nagaraj, R. Y., Ma, J., and Cheng, hypotheses as to the role of Mg in conformational changes H. (2001) J. Biol. Chem. 276, 40210 – 40214 11. Lohn, M., Jessner, W., Furstenau, M., Wellner, M., Sorrentino, V., Haller, H., and in EC coupling. For instance, it is possible that Mg binds Luft, F. C., and Gollasch, M. (2001) Circ. Res. 89, 1051–1057 to domain 9, stabilizing RyR1 in an inactive conformation, and 12. Sorrentino, V., and Volpe, P. (1993) Trends Pharmacol. Sci. 14, 98 –103 that the II-III loop of DHPR may bind to the same domain, 13. Du, G. G., and MacLennan, D. H. (1999) J. Biol. Chem. 274, 26120 –26126 14. Du, G. G., Khanna, V. K., and MacLennan, D. H. (2000) J. Biol. Chem. 275, relieving Mg inhibition and resulting in channel openings. 11778 –11783 Binding of Mg to domain 9 may also affect the binding of 15. Nakai, J., Gao, L., Xu, L., Xin, C., Pasek, D. A., and Meissner, G. (1999) FEBS FKBP, which binds adjacent to domain 9 (45). This may explain Lett. 459, 154 –158 16. Yamazawa, T., Takeshima, H., Shimuta, M., and Iino, M. (1997) J. Biol. Chem. the observation that Mg influences the interaction between 272, 8161– 8164 FKBP and RyR (53, 54). It will be of great interest to determine 17. Zorzato, F., Fujii, J., Otsu, K., Phillips, M., Green, N. M., Lai, F. A., Meissner, whether mutations in the DR3 region, particularly among the G., and MacLennan, D. H. (1990) J. Biol. Chem. 265, 2244 –2256 18. Bhat, M. B., Zhao, J., Hayek, S., Freeman, E. C., Takeshima, H., and Ma, J. negatively charged residues, alter Mg inhibition and skeletal (1997) Biophys. J. 73, 1320 –1328 muscle EC coupling. 19. Hayek, S. M., Zhao, J., Bhat, M., Xu, X., Nagaraj, R., Pan, Z., Takeshima, H., DR3 and Channel Conduction—The DR3 region appears to and Ma, J. (1999) FEBS Lett. 461, 157–164 20. Hayek, S. M., Zhu, X., Bhat, M. B., Zhao, J., Takeshima, H., Valdivia, H. H., be involved not only in channel gating but also in channel and Ma, J. (2000) Biochem. J. 351, 57– 65 conduction, since deletion of the DR3 region has been shown to 21. Proenza, C., O’Brien, J., Nakai, J., Mukherjee, S., Allen, P. D., and Beam, K. G. (2002) J. Biol. Chem. 277, 6530 – 6535 alter the conduction properties of RyR1 (20). The DR3 region is 22. O’Reilly, F. M., Robert, M., Jona, I., Szegedi, C., Albrieux, M., Geib, S., De located along the edge of the cytoplasmic assembly, far away Waard, M., Villaz, M., and Ronjat, M. (2002) Biophys. J. 82, 145–155 from the channel pore-forming region located in the transmem- 23. Masumiya, H., Wang, R., Zhang, J., Xiao, B., and Chen, S. R. W. (2003) J. Biol. Chem. 278, 3786 –3792 brane assembly, so how this region influences channel conduc- 24. Wagenknecht, T., and Samso, M. (2002) Front. Biosci. 7, d1464 –74 tion is unclear. It is unlikely that the DR3 region is directly 25. Serysheva, I. I., Schatz, M., van Heel, M., Chiu, W., and Hamilton, S. L. (1999) involved in the formation of the ion conduction pathway. It is, Biophys. J. 77, 1936 –1944 26. Radermacher, M., Rao, V., Grassucci, R., Frank, J., Timerman, A. P., however, possible that conformational changes in the DR3 Fleischer, S., and Wagenknecht, T. (1994) J. Cell Biol. 127, 411– 423 region may have long range effects on both gating and 27. Orlova, E. V., Serysheva, II, van Heel, M., Hamilton, S. L., and Chiu, W. (1996) conduction. Nat. Struct. Biol. 3, 547–552 28. Liu, Z., Zhang, J., Sharma, M. R., Li, P., Chen, S. R., and Wagenknecht, T. In summary, domain 9, which is involved in the structural (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 6104 – 6109 changes that occur upon channel activation and is also in 29. Liu, Z., Zhang, J., Li, P., Chen, S. R., and Wagenknecht, T. (2002) J. Biol. close contact with bound FKBPs, appears to encompass, Chem. 277, 46712– 46719 30. Chen, S. R. W., Li, X., Ebisawa, K., and Zhang, L. (1997) J. Biol. Chem. 272, within its DR3-containing region, a binding site for the II-III 24234 –24246 loop of DHPR and a low affinity cation binding site for Ca 31. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Mg . Accordingly, we propose that domain 9 constitutes 32. Zhao, M., Li, P., Li, X., Zhang, L., Winkfein, R. J., and Chen, S. R. (1999) an essential part of the macromolecular apparatus that car- J. Biol. Chem. 274, 25971–25974 ries out EC coupling. It is probable that multiple regions or 33. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 51–59 domains are involved in EC coupling. The most likely addi- 34. Wagenknecht, T., Frank, J., Boublik, M., Nurse, K., and Ofengand, J. (1988) J. tional candidates for EC coupling are the DR2 region and the Mol. Biol. 203, 753–760 regions between residues 1076 and 1112 and between resi- 35. Frank, J. (1996) Three-Dimensional Electron Microscopy of Macromolecular Assemblies, pp. 54 –125, Academic Press, Inc., San Diego, CA dues 2659 and 3720 or domains 5, 6, and 10, located within 36. Penczek, P. A., Grassucci, R. A., and Frank, J. (1994) Ultramicroscopy 53, the clamp-shaped structure (16, 25, 27, 46 – 48). The DR2 251–270 region is completely missing in RyR3, and comparison of the 37. Malhotra, A., Penczek, P., Agrawal, R. K., Gabashvili, I. S., Grassucci, R. A., Junemann, R., Burkhardt, N., Nierhaus, K. H., and Frank, J. (1998) J. Mol. three-dimensional reconstructions of RyR1 and RyR3 re- Biol. 280, 103–116 vealed a significant difference in domain 6, suggesting that 38. Li, P., and Chen, S. R. (2001) J. Gen. Physiol. 118, 33– 44 domain 6 may include the DR2 region (49). The three-dimen- 39. Doi, N., and Yanagawa, H. (2002) Methods Mol. Biol. 183, 49 –55 14218 Three-dimensional Localization of the DR3 Region 40. Niwa, H., Inouye, S., Hirano, T., Matsuno, T., Kojima, S., Kubota, M., Ohashi, 47. Leong, P., and MacLennan, D. H. (1998) J. Biol. Chem. 273, 7791–7794 M., and Tsuji, F. I. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13617–13622 48. Nakai, J., Sekiguchi, N., Rando, T. A., Allen, P. D., and Beam, K. G. (1998) 41. Waldo, G. S., Standish, B. M., Berendzen, J., and Terwilliger, T. C. (1999) Nat. J. Biol. Chem. 273, 13403–13406 Biotechnol. 17, 691– 695 49. Sharma, M. R., Jeyakumar, L. H., Fleischer, S., and Wagenknecht, T. (2000) 42. Chen, S. R., Li, P., Zhao, M., Li, X., and Zhang, L. (2002) Biophys. J. 82, J. Biol. Chem. 275, 9485–9491 2436 –2447 50. Jayaraman, T., Brillantes, A. M., Timerman, A. P., Fleischer, S., Erdjument- 43. Tanna, B., Welch, W., Ruest, L., Sutko, J. L., and Williams, A. J. (1998) J. Gen. Bromage, H., Tempst, P., and Marks, A. R. (1992) J. Biol. Chem. 267, Physiol. 112, 55– 69 9474 –9477 44. Wagenknecht, T., Grassucci, R., Berkowitz, J., Wiederrecht, G. J., Xin, H. B., 51. Lamb, G. D., and Stephenson, D. G. (1996) J. Physiol. 494, 569 –576 and Fleischer, S. (1996) Biophys. J. 70, 1709 –1715 52. Lamb, G. D. (2002) Front. Biosci. 7, 834 – 842 45. Wagenknecht, T., Radermacher, M., Grassucci, R., Berkowitz, J., Xin, H. B., 53. Bultynck, G., De Smet, P., Rossi, D., Callewaert, G., Missiaen, L., Sorrentino, and Fleischer, S. (1997) J. Biol. Chem. 272, 32463–32471 V., De Smedt, H., and Parys, J. B. (2001) Biochem. J. 354, 413– 422 46. Serysheva, I. I., Ludtke, S. J., Baker, M. R., Chiu, W., and Hamilton, S. L. 54. Bultynck, G., Rossi, D., Callewaert, G., Missiaen, L., Sorrentino, V., Parys, (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 10370 –10375 J. B., and De Smedt, H. (2001) J. Biol. Chem. 276, 47715– 47724

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