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Muscling in on the ryanodine receptor Ivana Y Kuo & Barbara E Ehrlich
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© 2015 Nature America, Inc. All rights reserved.
The ryanodine receptor (RyR), an ion channel regulating intracellular calcium release in excitable cells, has been challenging for structural analysis because of its colossal proportions compared to most other ion channels. Three independent groups have now used recent technological advancements in single-particle cryo-EM to make giant strides in solving the structure of this elusive protein complex. Controlling intracellular calcium is a daunting task for cells, especially in skeletal and cardiac muscle, in which a burst of calcium must be released from intracellular stores for muscle contraction to occur. A rich array of mechanisms to control calcium release in response to cellular demands is therefore required. A major molecular component of the intracellular calcium-release pathway is the RyR, a homo tetrameric protein complex associated with the endoplasmic and/or sarcoplasmic reticulum. The RyR is one of the largest known ion channels, weighing in at a hefty 2.2 MDa. The functional importance of the RyR is highlighted by the debilitating effects arising from mutations to this protein, including heart disease1 (for example, catecholaminergic polymorphic ventricular tachycardia, associated with mutations in cardiac RyR2) and skeletal-muscle dysfunction (for example, malignant hypothermia, associated with mutations in skeletal RyR1 (ref. 2)). The RyR has long been considered to be an ideal target for cryo-EM studies because of its large size and four-fold symmetry3. However, obtaining a reliable structural map of the whole complex has been elusive, partly because the RyR is present only in higher order organisms but mostly because of its large size. As reported in Nature4–6, three groups have now independently solved the structure of RyR1, with resolutions ranging from 5.2 Å (ref. 5) to an unprecedented 3.8 Å (ref. 6), by applying advances in cryo-EM technology7–9. All groups used purified RyR1 protein with its interacting partner FKBP (calstabin) as well as a more sensitive detector and automatic particle-picking software to categorize the particles. Each of the newly described RyR1 structures reveals similar features, thus enabling improved understanding of the RyR1 structure. Prior to these studies, no reliable structure at better than ~10-Å resolution existed10. For Ivana Y. Kuo and Barbara E. Ehrlich are at the Department of Pharmacology and the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut, USA. e-mail: [email protected]
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functional and structural biologists alike, these studies set the stage for an in-depth analysis of the RyR structure and for correlation with normal and pathophysiological studies. The new structures provide a solid footing for prior hypotheses11 and reveal several intriguing features that warrant further investigation. The RyR was previously described as having a mushroom-like shape consisting of an extensive cytoplasmic N terminus on a short transmembrane stalk2,3,12. However, the number of transmembrane segments and the location of several subdomains were uncertain. Importantly, the historic argument regarding the number of transmembrane segments is now resolved, with six in each subunit clearly identified. All three structures confirm that the RyR1 consists of four homomeric units arranged in four-fold symmetry and show new specific interactions among subdomains of the large cytoplasmic N-terminal domain. The relative arrangement of the three SPRY domains, phosphorylation (P1 and P2) domains and the central domain in the cytoplasmic region of the channel are all defined (Fig. 1). One particularly interesting feature is the extended α-solenoid scaffold in the N terminus that connects to key regulatory domains of the pore. The functional importance of this and similar features is certain to be the focus of future studies. The structures provide a high-resolution view of the pore, enabling identification of specific features that regulate ion selectivity and gating13. It is reassuring to see several features common with other cation channels, namely TRP, potassium and sodium channels. As expected, the pore of the RyR1 is formed from stretches of α-helices and a canonical P loop, in which transmembrane segment 6 contains negatively charged residues appropriately situated to confer cation selectivity. The identification of the residues constituting pore-forming segments is a critical finding because the region between transmembrane segment 5 and the P loop is a hotspot for disease-related mutations3. With this new knowledge, functional experiments identifying the effect of certain mutations on ion conductance and structural conformation can be made.
There are many factors that affect RyR1 channel opening, including direct association with the L-type calcium channel. However, in both skeletal and nonskeletal muscle, calcium binding to the EF-hand calciumbinding motif also induces channel opening. In the present structures, the EF hand is well defined and is found as a pair tucked away under the umbrella of the mushroom head of the structure (Fig. 1a). This position is ideally located for regulation of channel opening. Two of the structures4,6 describe an intimate interaction between the EF hands located in the α-solenoid scaffold with a unique linker region inserted between the second and third transmembrane helices. Because binding of calcium is known to change the conformation of the EF hands, a mechanism whereby calcium binding to the EF hand directly communicates to the transmembrane region now comes into focus. To examine the effect of calcium binding to the EF hand on the pore, the structures in the presence and absence of calcium5, albeit at a slightly lower resolution and in very high calcium (10 mM), were compared . In calcium, there is a 1.5-Å shift in the latter helices of the EF hand, which translates to a rotation of the solenoid by 4° (Fig. 1b). This leads to downward rotation of the clamp and handle domains by approximately 5 Å, an outward rotation of transmembrane segments 4 and 5, presumably by the interactions of the linker between transmembrane segments 2 and 3 (refs. 4,6), and expansion by approximately 2 Å of the inner-pore helices formed by transmembrane segments 5 and 6 (Fig. 1b). This expansion would be sufficient for calcium flux, thus showing that binding of calcium to the EF hand can open the pore. Although there is congruency in the general structural features, a closer examination also shows several differences. The structure obtained with a nanodisc preparation5, instead of detergent-solubilized receptor, presents multiple states of the RyR in both calcium-bound and calcium-unbound states. With calcium bound, a class of structures with altered EF-hand conformations was identified along with structural classes presenting no discernible rearrangement
volume 22 number 2 FEbruary 2015 nature structural & molecular biology
news and views a
SPRY domains
P1
Closed
Open
N terminus
P2 Cytosol
Lumen
b
α -so solen lenoid oid α-solenoid C termin terminus EF hand
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© 2015 Nature America, Inc. All rights reserved.
Closed S5 S6
4° 2Å
hotspot mutations, for which X-ray crystallography, NMR and cryo-EM structures exist2,3,12. The new structures confirm the localization of many of these hotspot mutations to critical sites, especially in the pore region, and suggest intriguing interactions among the hotspots, the pore and the regulatory sites. The effects of phosphorylation of RyR2 will be important for understanding heart disease. Also, to understand excitation-contraction coupling more fully in skeletal muscle, it will be essential to determine the structure of the L-type calcium channel in complex with RyR1. Understanding the RyR in combination with its local environment, for example with superresolution microscopy16,17, promises to reveal even more secrets. High-resolution structures of the RyR’s ‘little sister’, a slightly smaller but still huge channel, the inositol trisphosphate receptor (InsP3R), are now anticipated12,18. Because InsP3R is a closely related molecule controlling intracellular calcium release in virtually all cells19,20, we expect an unveiling of its structure in the future. But even now it is possible to conjecture that inositol trisphosphate and calcium bind to the InsP3R under its own mushroom cap in a manner akin to ligand binding at the EF-calcium binding site of the RyR. Clearly it is now time to have the nerve to muscle in on these large proteins. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.
Open
Ca2+
Figure 1 Cartoon representation of RyR1 structure. (a) Cross-sectional arrangement of the RyR1 channel in the closed conformation (left) and in the presence of calcium (right), with purple dots depicting the calcium-binding EF hands. The relative positions of the SPRY domains and P1 and P2 are noted. The arrow represents movement accompanying channel opening. (b) Movements within the transmembrane segments and N terminus that accompany RyR1 channel opening. Top, RyR1 in the closed conformation. Two subunits are shown for simplicity, the one on the right in lighter colors. Only the last N-terminal α-solenoid is shown (red). The EF hand is depicted as dark-purple spheres and the C terminus as light-purple rectangles. Bottom, upon binding of calcium ions (green spheres) to the EF hand, a 4° upward deflection of the α-solenoid translates into a ~2-Å dilation of the pore. S1–S6 denote transmembrane segments 1–6.
of the EF hand. This heterogeneity shows that careful refinement of the structural arrangement of the RyR domains in the presence of calcium is warranted. It is also worth noting that one structure is presented in the fully dephosphorylated state4, acknowledging the heterogeneity and conformational changes associated with phosphorylation. It is of interest to see how phosphorylation alters structure, because considerable debate surrounds phosphorylation and
RyR2 channel regulation14,15. Finally, the diameter of the closed pore at the constriction point is considerably different in two of the papers4,6. Obtaining structures under different conditions may reveal heterogeneity in the pore. Although there are now three higherresolution structures of RyR1, questions still abound. How do the disease-associated mutations alter the structure to induce changes in function? Prior attention has been placed on
nature structural & molecular biology volume 22 number 2 FEbruary 2015
1. Chen, W. et al. Nat. Med. 20, 184–192 (2014). 2. Lanner, J.T., Georgiou, D.K., Joshi, A.D. & Hamilton, S.L. Cold Spring Harb. Perspect. Biol. 2, a003996 (2010). 3. Van Petegem, F. J. Mol. Biol. 427, 31–55 (2015). 4. Zalk, R. et al. Nature 517, 44–49 (2015). 5. Efremov, R.G., Leitner, A., Aebersold, R. & Raunser, S. Nature 517, 39–43 (2015). 6. Yan, Z. et al. Nature 517, 50–55 (2015). 7. Kuhlbrandt, W. eLife 3, e03678 (2014). 8. Henderson, R. Q. Rev. Biophys. 37, 3–13 (2004). 9. Liao, M., Cao, E., Julius, D. & Cheng, Y. Curr. Opin. Struct. Biol. 27, 1–7 (2014). 10 Ludtke, S.J. & Serysheva, I.I. Curr. Opin. Struct. Biol. 23, 755–762 (2013). 11. Carney, J., Mason, S.A., Viero, C. & Williams, A.J. Curr. Top. Membr. 66, 49–67 (2010). 12. Stathopulos, P.B. et al. Physiology (Bethesda) 27, 331–342 (2012). 13. Gillespie, D., Xu, L. & Meissner, G. Biophys. J. 107, 2263–2273 (2014). 14. Houser, S.R. Circ. Res. 114, 1320–1327 (2014). 15. Dobrev, D. & Wehrens, X.H. Circ. Res. 114, 1311–1319 (2014). 16. Baddeley, D. et al. Proc. Natl. Acad. Sci. USA 106, 22275–22280 (2009). 17. Soeller, C. & Baddeley, D. J. Mol. Cell. Cardiol. 58, 32–40 (2013). 18. Seo, M.D. et al. Nature 483, 108–112 (2012). 19. Foskett, J.K., White, C., Cheung, K.H. & Mak, D.O. Physiol. Rev. 87, 593–658 (2007). 20. Bezprozvanny, I. Cell Calcium 38, 261–272 (2005).
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Muscling in on the ryanodine receptor Ivana Y Kuo & Barbara E Ehrlich
npg
© 2015 Nature America, Inc. All rights reserved.
The ryanodine receptor (RyR), an ion channel regulating intracellular calcium release in excitable cells, has been challenging for structural analysis because of its colossal proportions compared to most other ion channels. Three independent groups have now used recent technological advancements in single-particle cryo-EM to make giant strides in solving the structure of this elusive protein complex. Controlling intracellular calcium is a daunting task for cells, especially in skeletal and cardiac muscle, in which a burst of calcium must be released from intracellular stores for muscle contraction to occur. A rich array of mechanisms to control calcium release in response to cellular demands is therefore required. A major molecular component of the intracellular calcium-release pathway is the RyR, a homo tetrameric protein complex associated with the endoplasmic and/or sarcoplasmic reticulum. The RyR is one of the largest known ion channels, weighing in at a hefty 2.2 MDa. The functional importance of the RyR is highlighted by the debilitating effects arising from mutations to this protein, including heart disease1 (for example, catecholaminergic polymorphic ventricular tachycardia, associated with mutations in cardiac RyR2) and skeletal-muscle dysfunction (for example, malignant hypothermia, associated with mutations in skeletal RyR1 (ref. 2)). The RyR has long been considered to be an ideal target for cryo-EM studies because of its large size and four-fold symmetry3. However, obtaining a reliable structural map of the whole complex has been elusive, partly because the RyR is present only in higher order organisms but mostly because of its large size. As reported in Nature4–6, three groups have now independently solved the structure of RyR1, with resolutions ranging from 5.2 Å (ref. 5) to an unprecedented 3.8 Å (ref. 6), by applying advances in cryo-EM technology7–9. All groups used purified RyR1 protein with its interacting partner FKBP (calstabin) as well as a more sensitive detector and automatic particle-picking software to categorize the particles. Each of the newly described RyR1 structures reveals similar features, thus enabling improved understanding of the RyR1 structure. Prior to these studies, no reliable structure at better than ~10-Å resolution existed10. For Ivana Y. Kuo and Barbara E. Ehrlich are at the Department of Pharmacology and the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut, USA. e-mail: [email protected]
106
functional and structural biologists alike, these studies set the stage for an in-depth analysis of the RyR structure and for correlation with normal and pathophysiological studies. The new structures provide a solid footing for prior hypotheses11 and reveal several intriguing features that warrant further investigation. The RyR was previously described as having a mushroom-like shape consisting of an extensive cytoplasmic N terminus on a short transmembrane stalk2,3,12. However, the number of transmembrane segments and the location of several subdomains were uncertain. Importantly, the historic argument regarding the number of transmembrane segments is now resolved, with six in each subunit clearly identified. All three structures confirm that the RyR1 consists of four homomeric units arranged in four-fold symmetry and show new specific interactions among subdomains of the large cytoplasmic N-terminal domain. The relative arrangement of the three SPRY domains, phosphorylation (P1 and P2) domains and the central domain in the cytoplasmic region of the channel are all defined (Fig. 1). One particularly interesting feature is the extended α-solenoid scaffold in the N terminus that connects to key regulatory domains of the pore. The functional importance of this and similar features is certain to be the focus of future studies. The structures provide a high-resolution view of the pore, enabling identification of specific features that regulate ion selectivity and gating13. It is reassuring to see several features common with other cation channels, namely TRP, potassium and sodium channels. As expected, the pore of the RyR1 is formed from stretches of α-helices and a canonical P loop, in which transmembrane segment 6 contains negatively charged residues appropriately situated to confer cation selectivity. The identification of the residues constituting pore-forming segments is a critical finding because the region between transmembrane segment 5 and the P loop is a hotspot for disease-related mutations3. With this new knowledge, functional experiments identifying the effect of certain mutations on ion conductance and structural conformation can be made.
There are many factors that affect RyR1 channel opening, including direct association with the L-type calcium channel. However, in both skeletal and nonskeletal muscle, calcium binding to the EF-hand calciumbinding motif also induces channel opening. In the present structures, the EF hand is well defined and is found as a pair tucked away under the umbrella of the mushroom head of the structure (Fig. 1a). This position is ideally located for regulation of channel opening. Two of the structures4,6 describe an intimate interaction between the EF hands located in the α-solenoid scaffold with a unique linker region inserted between the second and third transmembrane helices. Because binding of calcium is known to change the conformation of the EF hands, a mechanism whereby calcium binding to the EF hand directly communicates to the transmembrane region now comes into focus. To examine the effect of calcium binding to the EF hand on the pore, the structures in the presence and absence of calcium5, albeit at a slightly lower resolution and in very high calcium (10 mM), were compared . In calcium, there is a 1.5-Å shift in the latter helices of the EF hand, which translates to a rotation of the solenoid by 4° (Fig. 1b). This leads to downward rotation of the clamp and handle domains by approximately 5 Å, an outward rotation of transmembrane segments 4 and 5, presumably by the interactions of the linker between transmembrane segments 2 and 3 (refs. 4,6), and expansion by approximately 2 Å of the inner-pore helices formed by transmembrane segments 5 and 6 (Fig. 1b). This expansion would be sufficient for calcium flux, thus showing that binding of calcium to the EF hand can open the pore. Although there is congruency in the general structural features, a closer examination also shows several differences. The structure obtained with a nanodisc preparation5, instead of detergent-solubilized receptor, presents multiple states of the RyR in both calcium-bound and calcium-unbound states. With calcium bound, a class of structures with altered EF-hand conformations was identified along with structural classes presenting no discernible rearrangement
volume 22 number 2 FEbruary 2015 nature structural & molecular biology
news and views a
SPRY domains
P1
Closed
Open
N terminus
P2 Cytosol
Lumen
b
α -so solen lenoid oid α-solenoid C termin terminus EF hand
npg
© 2015 Nature America, Inc. All rights reserved.
Closed S5 S6
4° 2Å
hotspot mutations, for which X-ray crystallography, NMR and cryo-EM structures exist2,3,12. The new structures confirm the localization of many of these hotspot mutations to critical sites, especially in the pore region, and suggest intriguing interactions among the hotspots, the pore and the regulatory sites. The effects of phosphorylation of RyR2 will be important for understanding heart disease. Also, to understand excitation-contraction coupling more fully in skeletal muscle, it will be essential to determine the structure of the L-type calcium channel in complex with RyR1. Understanding the RyR in combination with its local environment, for example with superresolution microscopy16,17, promises to reveal even more secrets. High-resolution structures of the RyR’s ‘little sister’, a slightly smaller but still huge channel, the inositol trisphosphate receptor (InsP3R), are now anticipated12,18. Because InsP3R is a closely related molecule controlling intracellular calcium release in virtually all cells19,20, we expect an unveiling of its structure in the future. But even now it is possible to conjecture that inositol trisphosphate and calcium bind to the InsP3R under its own mushroom cap in a manner akin to ligand binding at the EF-calcium binding site of the RyR. Clearly it is now time to have the nerve to muscle in on these large proteins. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.
Open
Ca2+
Figure 1 Cartoon representation of RyR1 structure. (a) Cross-sectional arrangement of the RyR1 channel in the closed conformation (left) and in the presence of calcium (right), with purple dots depicting the calcium-binding EF hands. The relative positions of the SPRY domains and P1 and P2 are noted. The arrow represents movement accompanying channel opening. (b) Movements within the transmembrane segments and N terminus that accompany RyR1 channel opening. Top, RyR1 in the closed conformation. Two subunits are shown for simplicity, the one on the right in lighter colors. Only the last N-terminal α-solenoid is shown (red). The EF hand is depicted as dark-purple spheres and the C terminus as light-purple rectangles. Bottom, upon binding of calcium ions (green spheres) to the EF hand, a 4° upward deflection of the α-solenoid translates into a ~2-Å dilation of the pore. S1–S6 denote transmembrane segments 1–6.
of the EF hand. This heterogeneity shows that careful refinement of the structural arrangement of the RyR domains in the presence of calcium is warranted. It is also worth noting that one structure is presented in the fully dephosphorylated state4, acknowledging the heterogeneity and conformational changes associated with phosphorylation. It is of interest to see how phosphorylation alters structure, because considerable debate surrounds phosphorylation and
RyR2 channel regulation14,15. Finally, the diameter of the closed pore at the constriction point is considerably different in two of the papers4,6. Obtaining structures under different conditions may reveal heterogeneity in the pore. Although there are now three higherresolution structures of RyR1, questions still abound. How do the disease-associated mutations alter the structure to induce changes in function? Prior attention has been placed on
nature structural & molecular biology volume 22 number 2 FEbruary 2015
1. Chen, W. et al. Nat. Med. 20, 184–192 (2014). 2. Lanner, J.T., Georgiou, D.K., Joshi, A.D. & Hamilton, S.L. Cold Spring Harb. Perspect. Biol. 2, a003996 (2010). 3. Van Petegem, F. J. Mol. Biol. 427, 31–55 (2015). 4. Zalk, R. et al. Nature 517, 44–49 (2015). 5. Efremov, R.G., Leitner, A., Aebersold, R. & Raunser, S. Nature 517, 39–43 (2015). 6. Yan, Z. et al. Nature 517, 50–55 (2015). 7. Kuhlbrandt, W. eLife 3, e03678 (2014). 8. Henderson, R. Q. Rev. Biophys. 37, 3–13 (2004). 9. Liao, M., Cao, E., Julius, D. & Cheng, Y. Curr. Opin. Struct. Biol. 27, 1–7 (2014). 10 Ludtke, S.J. & Serysheva, I.I. Curr. Opin. Struct. Biol. 23, 755–762 (2013). 11. Carney, J., Mason, S.A., Viero, C. & Williams, A.J. Curr. Top. Membr. 66, 49–67 (2010). 12. Stathopulos, P.B. et al. Physiology (Bethesda) 27, 331–342 (2012). 13. Gillespie, D., Xu, L. & Meissner, G. Biophys. J. 107, 2263–2273 (2014). 14. Houser, S.R. Circ. Res. 114, 1320–1327 (2014). 15. Dobrev, D. & Wehrens, X.H. Circ. Res. 114, 1311–1319 (2014). 16. Baddeley, D. et al. Proc. Natl. Acad. Sci. USA 106, 22275–22280 (2009). 17. Soeller, C. & Baddeley, D. J. Mol. Cell. Cardiol. 58, 32–40 (2013). 18. Seo, M.D. et al. Nature 483, 108–112 (2012). 19. Foskett, J.K., White, C., Cheung, K.H. & Mak, D.O. Physiol. Rev. 87, 593–658 (2007). 20. Bezprozvanny, I. Cell Calcium 38, 261–272 (2005).
107
