Ryanodine Receptor Structure: Progress and Challenges (2008)

Ryanodine-sensitive Ca2+ release channels, also known as the ryanodine receptors (RyRs), 2The abbreviations used are: RyR, ryanodine receptor; cryo-EM, cryoelectron microscopy; CY, cytoplasmic; TM, transmembrane; aa, amino acids; GFP, green fluorescent protein; IP3, inositol trisphosphate; CaM, calmodulin; FKBP, FK506-binding protein. are homotetramers of an ∼550-kDa subunit (Fig. 1) that are resident proteins of intracellular membranes such as the sarcoplasmic/endoplasmic reticulum. RyRs are responsible for the regulated release of Ca2+ from these lumenal stores. Over the last 10 years, single-particle cryoelectron microscopy (cryo-EM) has produced several low resolution (∼30 Å) structures of RyRs (for review, see Ref. 1Serysheva I.I. Chiu W. Ludtke S.J. Methods Cell Biol. 2007; 79: 407-435Crossref PubMed Scopus (8) Google Scholar). Recently, cryo-EM reconstructions of RyR1 have reached subnanometer resolution (2Ludtke S.J. Serysheva I.I. Hamilton S.L. Chiu W. Structure (Camb.). 2005; 13: 1203-1211Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 3Samso M. Wagenknecht T. Allen P.D. Nat. Struct. Mol. Biol. 2005; 12: 539-544Crossref PubMed Scopus (168) Google Scholar), a breakthrough due to improvements in cryo-specimen preparation, instrumentation, image processing, and three-dimensional reconstruction techniques. RyRs have a mushroom shape with 4-fold symmetry (Fig. 1B) (2Ludtke S.J. Serysheva I.I. Hamilton S.L. Chiu W. Structure (Camb.). 2005; 13: 1203-1211Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Most of the mass of RyR forms a large cytoplasmic (CY) assembly (280 × 280 × 120 Å) that is connected to the transmembrane (TM) region by a stalk-like structure. The CY region is strikingly empty with numerous distinctive structural domains and intervening cavities that appear suitable for interaction with modulators that bind within the N-terminal regions of RyR (Fig. 1). The clamp-shaped regions, located at the corners of the CY assembly, are likely regions for the interdigitation of neighboring RyRs seen in situ (5Block B.A. Franzini-Armstrong C. J. Cell Biol. 1988; 107: 1099-1112Crossref PubMed Scopus (47) Google Scholar) or for interaction with modulators. The clamp-shaped regions are interconnected to form a continuous network between the central rim and the CY stalk-like structure via several bridging densities. The TM region (120 × 120 × 60 Å) is rotated by ∼40° with respect to the CY region. The pore is thought to be composed of six to eight TM segments (RyR1 TM1–8, amino acids (aa) 4277–4323, 4343–4363, 4557–4576, 4637–4662, 4776–4800, 4803–4825, 4834–4854, and 4911–4935 with aa 4854–4911 forming the pore helix and selectivity filter) (Fig. 1A, line 1) (6Du G.G. Sandhu B. Khanna V.K. Guo X.H. MacLennan D.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16725-16730Crossref PubMed Scopus (118) Google Scholar). This arrangement places both the N and C termini of RyR in the cytoplasm. Sequence numbers used in this review refer to rabbit RyR1 (Swiss-Prot accession number P11716) and, where indicated, RyR2 (Swiss-Prot accession number P30957). The best current maps of RyR1 have resolutions of ∼10 Å (2Ludtke S.J. Serysheva I.I. Hamilton S.L. Chiu W. Structure (Camb.). 2005; 13: 1203-1211Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 3Samso M. Wagenknecht T. Allen P.D. Nat. Struct. Mol. Biol. 2005; 12: 539-544Crossref PubMed Scopus (168) Google Scholar), allowing delineation of secondary structure elements in the pore region (Fig. 2A): the helix running across the membrane (helix 1, possibly TM8), the short helix (helix 2, which is likely to be part of the pore helix) that forms the lumenal entrance of the channel, and helix 3 (located parallel to the membrane plane on the CY side of the TM region). Helices 1 and 2 lie near the 4-fold channel axis and resemble the inner (pore-lining) and pore helices in K+ channel structures (7Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Sciences (N. Y.). 1998; 280: 69-77Crossref PubMed Scopus (5768) Google Scholar, 8Jiang Y. Lee A. Chen J. Cadene M. Chait B.T. MacKinnon R. Nature. 2002; 417: 515-522Crossref PubMed Scopus (1209) Google Scholar). The orientation of helix 3 is similar to the slide helix seen in the KirBac1.1 structure (9Kuo A. Gulbis J.M. Antcliff J.F. Rahman T. Lowe E.D. Zimmer J. Cuthbertson J. Ashcroft F.M. Ezaki T. Doyle D.A. Sciences (N. Y.). 2003; 300: 1922-1926Crossref PubMed Scopus (738) Google Scholar). Two recent RyR1 structures (2Ludtke S.J. Serysheva I.I. Hamilton S.L. Chiu W. Structure (Camb.). 2005; 13: 1203-1211Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 3Samso M. Wagenknecht T. Allen P.D. Nat. Struct. Mol. Biol. 2005; 12: 539-544Crossref PubMed Scopus (168) Google Scholar) were determined under conditions favoring the closed channel, the arrangement of the membrane-spanning helix that, in these structures, is quite different. Ludtke et al. (2Ludtke S.J. Serysheva I.I. Hamilton S.L. Chiu W. Structure (Camb.). 2005; 13: 1203-1211Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) found a bent pore-lining helix (Fig. 2A) and suggested that the structure is similar to the open MthK channel (8Jiang Y. Lee A. Chen J. Cadene M. Chait B.T. MacKinnon R. Nature. 2002; 417: 515-522Crossref PubMed Scopus (1209) Google Scholar). This raises questions as to whether “kinking” of the inner helix alone, as proposed for K+ channels, is adequate to open RyRs. However, the conformation of the pore-lining helix in the other high resolution RyR1 cryo-EM structure (3Samso M. Wagenknecht T. Allen P.D. Nat. Struct. Mol. Biol. 2005; 12: 539-544Crossref PubMed Scopus (168) Google Scholar) is straight and resembles the closed KcsA channel structure (7Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Sciences (N. Y.). 1998; 280: 69-77Crossref PubMed Scopus (5768) Google Scholar). These differences may due to resolution or to the conformational state of the channel during the imaging. At the current level of resolution in the three-dimensional structure of RyR, it is not possible to elucidate the number of TM helices. Single-particle cryo-EM has been used to generate structures of RyR1 in different conformational states to explore the structural transitions associated with the RyR gating. RyR1 was frozen under conditions determined from electrophysiological and biochemical studies (10Orlova E.V. Serysheva I.I. van Heel M. Hamilton S.L. Chiu W. Nat. Struct. Biol. 1996; 3: 547-552Crossref PubMed Scopus (146) Google Scholar, 11Serysheva I.I. Schatz M. van Heel M. Chiu W. Hamilton S.L. Biophys. J. 1999; 77: 1936-1944Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 12Sharma M.R. Jeyakumar L.H. Fleischer S. Wagenknecht T. J. Biol. Chem. 2000; 275: 9485-9491Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) to drive the channel into either an opened or a closed state. Although the resolution of these structures was only 25–30 Å, global conformational changes associated with the closed-open transition of the RyR channel were detected in both the CY region (opening of the clamps) and the TM region (mass depletion from the center and a twisting motion of the TM region). Channel opening was proposed to be similar to the opening-closing of the iris in a camera diaphragm. Four approaches are used to identify the locations of specific sequences in the three-dimensional structures: 1) difference mapping with and without bound modulators; 2) difference mapping with and without sequence-specific antibodies; 3) insertion of tags such as green fluorescent protein (GFP) into defined positions in the primary sequence, followed by difference mapping; and 4) docking of molecular models of domains derived from crystal structures into the three-dimensional maps. There are three known RyR isoforms encoded by three distinct genes: RyR1 (expressed primarily in skeletal muscle), RyR2 (expressed in cardiac muscle and brain), and RyR3 (expressed in diaphragm, smooth muscle, and brain). These isoforms share ∼65% sequence identity. Regions of divergence (D1, aa 4254–4631 in RyR1 and aa 4210–4562 in RyR2; D2, aa 1342–1403 in RyR1 and aa 1353–1397 in RyR2; and D3, aa 1872–1923 in RyR1 and aa 1852–1890 in RyR2) are indicated in Fig. 1A (line 2). These divergent regions could be responsible for important isoform-specific functions, or they could represent regions that are not evolutionarily conserved. Three-dimensional structures of all three RyR isoforms determined at ∼30 Å using single-particle cryo-EM are virtually identical (10Orlova E.V. Serysheva I.I. van Heel M. Hamilton S.L. Chiu W. Nat. Struct. Biol. 1996; 3: 547-552Crossref PubMed Scopus (146) Google Scholar, 11Serysheva I.I. Schatz M. van Heel M. Chiu W. Hamilton S.L. Biophys. J. 1999; 77: 1936-1944Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 12Sharma M.R. Jeyakumar L.H. Fleischer S. Wagenknecht T. J. Biol. Chem. 2000; 275: 9485-9491Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Using GFP insertions, D1 was mapped to subregion 3, whereas both D2 and D3 were mapped to the clamp in subregions 6 and 9, respectively (Fig. 1B). The three divergent regions all appear to be located in the CY domains close to the top of the structure, as would be expected for regions of RyR1 involved in interactions with CaV1.1. However, whether they are involved remains to be determined. Analysis of the primary sequence of RyR1 has revealed several motifs found in other proteins, but the functional roles of these motifs in RyRs are unknown. These motifs include 1) three SPRY domains (aa 582–798, 1014–1209, and 1358–1571) (Fig. 1A, line 3), which are domains found in members of the immunoglobulin superfamily; 2) a sequence that is repeated four times in two tandem domains (aa 842–955, 956–1069, 2726–2845, and 2846–2959) (Fig. 1A, line 4) with two additional truncated repeats located between the two domains (aa 1345–1360 and 1373–1388); 3) five MIR domains (aa 98–153, 160–205, 211–265, 271–334, and 336–394) (Fig. 1A, line 5), which are sequences found in RyRs, inositol trisphosphate receptors, and protein O-mannosyltransferases; and 4) two or more leucine zippers (aa 554–575 and 3039–3071 in RyR1 and aa 565–594, 1604–1632, and 3005–3036 in RyR2) (Fig. 1A, line 6), which are protein-protein interaction motifs and have been suggested to anchor PP1/spinophilin, PP2A/PR130, and protein kinase A/RII (protein kinase A regulatory subunit II)/mAKAP (muscle A-kinase anchoring protein) to RyR2 (13Marks A.R. Marx S.O. Reiken S. Trends Cardiovasc. Med. 2002; 12: 166-170Crossref PubMed Scopus (79) Google Scholar). The MIR domains are all located close to the N terminus, which is part of the clamp domain, suggesting that these domains may contribute to the structure/function of the clamps (4Serysheva I.I. Ludtke S.J. Baker M.L. Cong Y. Topf M. Eramian D. Sali A. Hamilton S.L. Chiu W. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 9610-9615Crossref PubMed Scopus (95) Google Scholar). Computational placement of a homology model based on the crystal structure of the inositol trisphosphate (IP3)-binding and suppressor domains of the IP3 receptor (4Serysheva I.I. Ludtke S.J. Baker M.L. Cong Y. Topf M. Eramian D. Sali A. Hamilton S.L. Chiu W. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 9610-9615Crossref PubMed Scopus (95) Google Scholar) places the first leucine zipper (aa 554–574) in the clamp domain in subregion 7 (Figs. 1B and 2B). If this is indeed the site that anchors kinases or phosphatases to RyR1, the targets of the enzymes may be sequences in close proximity to this site. Stewart et al. (15Stewart R. Zissimopoulos S. Lai F.A. Biochem. J. 2003; 376: 795-799Crossref PubMed Scopus (36) Google Scholar) proposed that the last 15 amino acids in the C-terminal tail are required for tetramer formation. Galvan and Mignery (16Galvan D.L. Mignery G.A. J. Biol. Chem. 2002; 277: 48248-48260Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) also suggested a C-terminal tetramerization domain but suggested that this was between aa 4869 and 5019 in RyR1. These regions are indicated in Fig. 1A (line 7). Because the C termini of RyRs are cytoplasmic, the region of tetramerization must also be in CY domains. The activities of all three RyR isoforms are modulated by a large number of agents, including ATP, Ca2+, Mg2+, calmodulin, FKBP12, junctin, triadin, calsequestrin, S100A, sorcin, and many others. Most of these modulators bind to the CY domains of the RyRs and allosterically regulate the opening of the Ca2+ conduction pathway in the TM region. RyR activities are also modulated by post-translation modifications such as phosphorylation, oxidation, and S-nitrosylation. Prerequisites for the delineation of the mechanisms of channel regulation by individual modulators and modifications are the identity of the sites of modulator interaction or modification in the primary sequence and the location of these sites in the three-dimensional structure. Single-particle cryo-EM has been used to analyze macromolecular interactions of RyR with the modulatory proteins calmodulin and FKBP12 (for original references on these studies, see reviews in Refs. 1Serysheva I.I. Chiu W. Ludtke S.J. Methods Cell Biol. 2007; 79: 407-435Crossref PubMed Scopus (8) Google Scholar and 17Durham W.J. Wehrens X.H.T. Sood S. Hamilton S.L. Carafoli E. Brini M. Calcium Signaling and Disease. Springer-Verlag New New Scholar). Because channel modulators bind in the CY assembly, must be mechanisms changes in the conformation of specific domains in the CY assembly are first into changes in the structure and into changes in the TM helices of the skeletal muscle, the interaction between in the and RyR1 in the has been to an RyR1 to and a which RyR1 the Ca2+ current 2002; PubMed Google Scholar). regions of RyR1 are likely to be involved in the to (Fig. 1A, line in Ref. 2002; PubMed Google Scholar), including aa and and regions between aa 1 and Franzini-Armstrong C. Allen P.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The sites of interaction appear to be a large part of the CY domain of RyR1. on studies C. Franzini-Armstrong C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), the likely regions of RyR1 to be in with include subregions and 6 (Fig. 1B). of the sequences suggested to contribute to have been found within these two of RyR2 in maps to a location between subregions and in RyR2 in maps to a location between subregions and interaction of with these regions of RyR1 could the opening of the clamp and allosterically the channel gating. of RyRs by Ca2+ with the RyR The important modulator of RyR2 in cardiac muscle is Ca2+, where Ca2+ and Ca2+ also RyR1 but on the it either or These that RyR1 has at two RyR1 and RyR2 are at Ca2+ MacLennan D.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar), but RyR1 is more to by of Ca2+ also the of RyRs D. B. D. R. Chen Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). sites for Ca2+ (Fig. 1A, line are located between aa and MacLennan D.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and MacLennan D.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and and S. Biochem. J. PubMed Scopus Google Scholar) and two motifs located at aa and D.A. 1998; PubMed Scopus Google Scholar). in rabbit in RyR2 Ca2+ by Chen J. PubMed Scopus Google Scholar). et al. W. Allen P.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) the motifs and found that these not the to either or RyR in high was in membranes the but the in were by Ca2+ in the These that the contribute to Ca2+ of the channel but are not for Ca2+ regulation of this close to these sites have not been mapped in the three-dimensional structures of RyRs. However, et al. R. Hamilton S.L. Biophys. J. Full Text Full Text PDF PubMed Scopus Google Scholar) suggested an interaction of the regions and with the calmodulin region (aa suggesting that these two are close to 3, where the site is located also bind CaM, which is a protein. RyR1 is the protein of membranes A. Biophys. J. Full Text PDF PubMed Scopus Google Scholar). At Ca2+ both RyR1 and However, at RyR1 but RyR2 D. and A. in Scholar). RyR3 also CaM, and the of on the channel are D.A. Chen 2005; PubMed Scopus Google Scholar). The site on RyR1 aa (Fig. 1A, line Hamilton S.L. 1999; PubMed Scopus Google Scholar, D.A. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) and at an close to aa Hamilton S.L. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). difference the site was found to be located in CY subregion 3 (Fig. 1B). the location of the site is to Å in the of Ca2+ with respect to for M. Wagenknecht T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). This of could be due to a of Ca2+ a of the site RyR1 These that subregion 3 a regulatory for channel and but additional studies are required to elucidate the molecular of regulation of RyR1. FKBP12 and bind to RyRs and regulate activities A.R. 1996; PubMed Scopus Google Scholar). Although FKBP12 and are to bind to all RyR FKBP12 with RyR1, whereas with The bound to RyR3 is not bind to RyR1 and RyR2 on site RyR1 subunit and a closed state of the channel A.R. 1996; PubMed Scopus Google Scholar). The site has been suggested to be between aa and in RyR1 (Fig. 1A, line of this site FKBP12 M. J. Reiken S. Marx S.O. A.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), but this may be due to an regulation of the site S. Lai F.A. Cell Biochem. Biophys. 2005; PubMed Scopus Google Scholar). Using difference mapping of three-dimensional reconstructions of RyR1 with and without FKBP12, the site for FKBP12 is found at the between clamp subregions and and subregion 3, neighboring clamps in the RyR CY region (Fig. 1B). of the location of FKBP12 by difference mapping M. Allen P.D. J. Mol. Biol. PubMed Scopus Google Scholar) with the docking of the IP3 homology model (4Serysheva I.I. Ludtke S.J. Baker M.L. Cong Y. Topf M. Eramian D. Sali A. Hamilton S.L. Chiu W. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 9610-9615Crossref PubMed Scopus (95) Google Scholar) that is a for FKBP12 by and (Fig. 2B). or not FKBP12 at this remains to be protein has been to with RyR1 and to regulate to both and W. J. T. Allen P.D. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus (125) Google Scholar). A possible sequence is found between aa and has been mapped between domains and 6 R. J. Chen Wagenknecht T. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar), suggesting that may also via the clamp domain of RyR1. and S. A. A. C. M. J. J. D. A.R. J. Cell Biol. 2003; PubMed Scopus Google Scholar) have suggested that RyR (aa in RyR1 and aa in RyR2) from RyRs, to Ca2+ A number of other have to these 2007; PubMed Scopus Google Scholar, S. J. R. PubMed Scopus Google Scholar, Chen 2008; PubMed Scopus Google Scholar). site of is aa (aa in sites are indicated in Fig. 1A (line of GFP at in RyR2 in this site to domain to the site B. S. J. R. J. Chen Wagenknecht T. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). The site was mapped by GFP at and was found to be located in to the site B. S. J. Wagenknecht T. Chen Biochem. J. 2008; PubMed Scopus Google Scholar). are in the is the primary of (Fig. 1, line in J. C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). et al. W.J. C. S. Galvan D.L. Baker M.R. R. Hamilton S.L. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar) that RyR1 the found with RyR1 associated with have been by et al. C. Hamilton S.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) under conditions in which the channel was primarily in a open conformation (Fig. 1A, line and by et al. J. M. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) under conditions in which the channel was in a primarily closed Although the differences may be due to differences in it is also possible that different are the channel is in the open closed are the in the three-dimensional be in the docking model (Fig. is in the site and is located in are located in and 6 (Fig. 1B) the region from aa to to these R. J. Chen Wagenknecht T. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). in RyR1 central and of recent review by et al. W.J. Wehrens X.H.T. Sood S. Hamilton S.L. Carafoli E. Brini M. Calcium Signaling and Disease. Springer-Verlag New New Although is for the to in three regions of RyR1, it that the primary sequence (Fig. 1A, line that the channel to be more to whereas central either the channel or the for the location of of the N-terminal be from the docking model (Fig. 2B). Channel opening is associated with structural transitions in the CY clamp subregions and (Fig. 2A) (10Orlova E.V. Serysheva I.I. van Heel M. Hamilton S.L. Chiu W. Nat. Struct. Biol. 1996; 3: 547-552Crossref PubMed Scopus (146) Google Scholar, 11Serysheva I.I. Schatz M. van Heel M. Chiu W. Hamilton S.L. Biophys. J. 1999; 77: 1936-1944Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). the three-dimensional locations of the and sites and other important functional the clamp subregions are likely to be involved in of the channel These a between the conformation of the clamp and channel with many associated with and central in that the channel to open more are located in clamp subregions and (Fig. 2) (4Serysheva I.I. Ludtke S.J. Baker M.L. Cong Y. Topf M. Eramian D. Sali A. Hamilton S.L. Chiu W. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 9610-9615Crossref PubMed Scopus (95) Google Scholar). Although RyR1 was the first structures by single-particle cryo-EM at resolution M. R. J. Fleischer S. Wagenknecht T. J. Cell Biol. PubMed Scopus Google Scholar, I.I. E.V. Chiu W. Hamilton S.L. van Heel M. Nat. Struct. Biol. PubMed Scopus Google Scholar), the RyR assembly remains the for structural due to RyR1 reconstructions at ∼10 Å by two have important into pore structure, and by intracellular (2Ludtke S.J. Serysheva I.I. Hamilton S.L. Chiu W. Structure (Camb.). 2005; 13: 1203-1211Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 3Samso M. Wagenknecht T. Allen P.D. Nat. Struct. Mol. Biol. 2005; 12: 539-544Crossref PubMed Scopus (168) Google Scholar). resolution structures of RyR1, including from cryo-specimen of membrane proteins, of the protein from with and of different conformational are to the channel in a functional state and either to all modulators or to all sites with modulators. New are to the number of has to be and the of are that of these to be are 1) crystal structures of individual or into the cryo-EM and 2) and the models into the cryo-EM the in the resolution of RyR structure are likely to in the with Serysheva