The elusive role of the SPRY2 domain in RyR1

The second of three SPRY domains (SPRY2, S1085 V1208) located in the skeletal muscle ryanodine receptor (RyR1) is contained within regions of RyR1 that influence EC coupling and bind to imperatoxin A, a toxin probe of RyR1 channel gating. We examined the binding of the F loop (P1107 A1121) in SPRY2 to the ASI/basic region in RyR1 (T3471-G3500, containing both alternatively spliced (ASI) residues and neighboring basic amino acids). We then investigated the possible influence of this interaction on excitation contraction (EC) coupling. A peptide with the F loop sequence and an antibody to the SPRY2 domain each enhanced RyR1 activity at low concentrations and inhibited at higher concentrations. A peptide containing the ASI/basic sequence bound to SPRY2 and binding decreased ~10-fold following mutation or structural disruption of the basic residues. Binding was abolished by mutation of three critical acidic F loop residues. Together these results suggest that the ASI/basic and SPRY2 domains interact in an F loop regulatory module. Although a region that includes the SPRY2 domain influences EC coupling, as does the ASI/basic region, Ca2+ release during ligand- and depolarization-induced RyR1 activation were not altered by mutation of the three critical F loop residues following expression of mutant RyR1 in RyR1-null myotubes. Therefore the electrostatic regulatory interaction between the SPRY2 F loop residues (that bind to imperatoxin A) and the ASI/basic residues of RyR1 does not influence bi-directional DHPR-RyR1 signaling during skeletal EC coupling, possibly because the interaction is interrupted by the influence of factors present in intact muscle cells.     

3D Mapping of the SPRY2 domain of ryanodine receptor 1 by single-particle cryo-EM

The type 1 skeletal muscle ryanodine receptor (RyR1) is principally responsible for Ca(2+) release from the sarcoplasmic reticulum and for the subsequent muscle contraction. The RyR1 contains three SPRY domains. SPRY domains are generally known to mediate protein-protein interactions, however the location of the three SPRY domains in the 3D structure of the RyR1 is not known. Combining immunolabeling and single-particle cryo-electron microscopy we have mapped the SPRY2 domain (S1085-V1208) in the 3D structure of RyR1 using three different antibodies against the SPRY2 domain. Two obstacles for the image processing procedure; limited amount of data and signal dilution introduced by the multiple orientations of the antibody bound in the tetrameric RyR1, were overcome by modifying the 3D reconstruction scheme. This approach enabled us to ascertain that the three antibodies bind to the same region, to obtain a 3D reconstruction of RyR1 with the antibody bound, and to map SPRY2 to the periphery of the cytoplasmic domain of RyR1. We report here the first 3D localization of a SPRY2 domain in any known RyR isoform.     

Expressional changes of RyR1 and RyR2 in PC12 cells after induction of apoptosis

Ca2+ released from endoplasmic reticulum through ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs) can trigger apoptotic or necrotic pathways in cooperation with proapoptotic and/or prosurvival proteins, as those of Bcl-2 family. In such regulatory pathways expressional modulation of these Ca2+ transporters could also be expected. Therefore, our aim was to determine the expressional changes of RyR1 and RyR2 after experimental induction of apoptosis in PC12 cells. Our results showed significant decrease of RyR1 and RyR2 expressions, while caspase-3 and Bax expression significantly increased. We conclude that induction of apoptosis in PC12 cells could result in RyR expression down regulation.     

RyR1/RyR3 chimeras reveal that multiple domains of RyR1 are involved in skeletal-type E-C coupling

Skeletal-type E-C coupling is thought to require a direct interaction between RyR1 and the alpha(1S)-DHPR. Most available evidence suggests that the cytoplasmic II-III loop of the dihydropyridine receptor (DHPR) is the primary source of the orthograde signal. However, identification of the region(s) of RyR1 involved in bidirectional signaling with the alpha(1S)-DHPR remains elusive. To identify these regions we have designed a series of chimeric RyR cDNAs in which different segments of RyR1 were inserted into the corresponding region of RyR3 and expressed in dyspedic 1B5 myotubes. RyR3 provides a preferable background than RyR2 for defining domains essential for E-C coupling because it possesses less sequence homology to RyR1 than the RyR2 backbone used in previous studies. Our data show that two regions of RyR1 (chimera Ch-10 aa 1681-2641 and Ch-9 aa 2642-3770), were independently able to restore skeletal-type E-C coupling to RyR3. These two regions were further mapped and the critical RyR1 residues were 1924-2446 (Ch-21) and 2644-3223 (Ch-19). These results both support and refine the previous hypothesis that multiple domains of RyR1 combine to functionally interact with the DHPR during E-C coupling.     

A Ca2+-binding domain in RyR1 that interacts with the calmodulin binding site and modulates channel activity

A fragment of RyR1 (amino acids 4064-4210) is predicted to fold to at least one lobe of calmodulin and to bind Ca(2+). This fragment of RyR1 (R4064-4210) was subcloned, expressed, refolded, and purified. Consistent with the predicted folding pattern, R4064-4210 was found to bind two molecules of Ca(2+) and undergo a structural change upon binding Ca(2+) that exposes hydrophobic amino acids. R4064-4210 also binds to RyR1, the L-type Ca(2+) channel (Cav(1.1)), and several synthetic calmodulin binding peptides. Both R4064-4210 and a peptide representing the calmodulin-binding region of RyR1 (R3614-3643) alter the Ca(2+) dependence of ((3)H)ryanodine binding to RyR1, suggesting that they may both be interfering with an intramolecular interaction between amino acids 4064-4210 and amino acids 3614-3643 in the native RyR1 to alter or regulate the response of the channel to changes in Ca(2+) concentration. The finding that a domain within RyR1 binds Ca(2+) and interacts with calmodulin-binding motifs may provide insights into the mechanism for calcium- and calmodulin-dependent regulation of this channel and perhaps for its regulation by the L-type Ca(2+) channel.     

Expression levels of RyR1 and RyR3 control resting free Ca2+ in skeletal muscle

To better understand the role of the transient expression of ryanodine receptor (RyR) type 3 (RyR3) on Ca²⁺ homeostasis during the development of skeletal muscle, we have analyzed the effect of expression levels of RyR3 and RyR1 on the overall physiology of cultured myotubes and muscle fibers. Dyspedic myotubes were infected with RyR1 or RyR3 containing virions at 0.2, 0.4, 1.0, and 4.0 moieties of infection (MOI), and analysis of their pattern of expression, caffeine sensitivity, and resting free Ca²⁺ concentration ([Ca²⁺]_r) was performed. Although increased MOI resulted in increased expression of each receptor isoform, it did not significantly affect the immunopattern of RyRs or the expression levels of calsequestrin, triadin, or FKBP-12. Interestingly, myotubes expressing RyR3 always had significantly higher [Ca²⁺]_r and lower caffeine EC₅₀ than did cells expressing RyR1. Although some of the increased sensitivity of RyR3 to caffeine could be attributed to the higher [Ca²⁺]_r in RyR3-expressing cells, studies of [³H]ryanodine binding demonstrated intrinsic differences in caffeine sensitivity between RyR1 and RyR3. Tibialis anterior (TA) muscle fibers at different stages of postnatal development exhibited a transient increase in [Ca²⁺]_r coordinately with their level of RyR3 expression. Similarly, adult soleus fibers, which also express RyR3, had higher [Ca²⁺]_r than did adult TA fibers, which exclusively express RyR1. These data show that in skeletal muscle, RyR3 increases [Ca²⁺]_r more than RyR1 does at any expression level. These data suggest that the coexpression of RyR1 and RyR3 at different levels may constitute a novel mechanism by which to regulate [Ca²⁺]_r in skeletal muscle. ryanodine receptor; calcium release; ryanodine binding; muscle fibers     

The pore structure of the closed RyR1 channel

Using single particle electron cryomicroscopy, several helices in the membrane-spanning region of RyR1, including an inner transmembrane helix, a short pore helix, and a helix parallel to the membrane on the cytoplasmic side, have been clearly resolved. Our model places a highly conserved glycine (G4934) at the hinge position of the bent inner helix and two rings of negative charges at the luminal and cytoplasmic mouths of the pore. The kinked inner helix closely resembles the inner helix of the open MthK channel, suggesting that kinking alone does not open RyR1, as proposed for K+ channels.     

RyR1 exhibits lower gain of CICR activity than RyR3 in the SR: evidence for selective stabilization of RyR1 channel

We showed that frog -ryanodine receptor (-RyR) had a lower gain of Ca²⁺-induced Ca²⁺ release (CICR) activity than -RyR in sarcoplasmic reticulum (SR) vesicles, indicating selective "stabilization" of the former isoform (Murayama T and Ogawa Y. J Biol Chem 276: 2953–2960, 2001). To know whether this is also the case with mammalian RyR1, we determined [³H]ryanodine binding of RyR1 and RyR3 in bovine diaphragm SR vesicles. The value of [³H]ryanodine binding (B) was normalized by the number of maximal binding sites (B_max), whereby the specific activity of each isoform was expressed. This B/B_max expression demonstrated that ryanodine binding of individual channels for RyR1 was <15% that for RyR3. Responses to Ca²⁺, Mg²⁺, adenine nucleotides, and caffeine were not substantially different between in situ and purified isoforms. These results suggest that the gain of CICR activity of RyR1 is markedly lower than that of RyR3 in mammalian skeletal muscle, indicating selective stabilization of RyR1 as is true of frog -RyR. The stabilization was partly eliminated by FK506 and partly by solubilization of the vesicles with CHAPS, each of which was additive to the other. In contrast, high salt, which greatly enhances [³H]ryanodine binding, caused only a minor effect on the stabilization of RyR1. None of the T-tubule components, coexisting RyR3, or calmodulin was the cause. The CHAPS-sensitive intra- and intermolecular interactions that are common between mammalian and frog skeletal muscles and the isoform-specific inhibition by FKBP12, which is characteristic of mammals, are likely to be the underlying mechanisms. excitation-contraction coupling; ryanodine binding; ryanodine receptor     

The SPRY domain of SSB-2 adopts a novel fold that presents conserved Par-4-binding residues

The four mammalian SPRY domain-containing SOCS box proteins (SSB-1 to SSB-4) are characterized by a C-terminal SOCS box and a central SPRY domain. We have determined the first SPRY-domain structure, as part of SSB-2, by NMR. This domain adopts a novel fold consisting of a beta-sandwich structure formed by two four-stranded antiparallel beta-sheets with a unique topology. We demonstrate that SSB-1, SSB-2 and SSB-4, but not SSB-3, bind prostate apoptosis response protein-4 (Par-4). Mutational analysis of SSB-2 loop regions identified conserved structural determinants for its interaction with Par-4 and the hepatocyte growth factor receptor, c-Met. Mutations in analogous loop regions of pyrin and midline-1 SPRY domains have been shown to cause Mediterranean fever and Opitz syndrome, respectively. Our findings provide a template for SPRY-domain structure and an insight into the mechanism of SPRY-protein interaction.     

The elusive role of the potential factor X cation-binding exosite-1 in substrate and inhibitor interactions

A number of studies suggest that blood-clotting factor X (FX) uses secondary site(s) to interact (as a substrate) with its activators. Numerous pieces of evidence also imply that, within prothrombinase (as an enzyme), activated FX (FXa) uses exosite(s) for cofactor Va and/or prothrombin recognition. Similarly, FXa exosite(s) seem to govern interaction with inhibitors. An obvious difference between FXa and thrombin resides within a region called exosite-1: positively charged in thrombin and clearly of opposite polarity in FXa. To investigate the role of this potential cation-binding exosite, we prepared a series of mutants within loops 34-40 and 70-80 of FX. Overall, the mutations induced relatively subtle, non-synergistic modulation. The potential exosite was dispensable for FX activation and is unlikely to constitute a critical region for factor Va binding, albeit it is clearly important for prothrombin activation. Our data also implicate loop 34-40 of FXa in the interaction with the tissue factor pathway inhibitor, in prevention of plasminogen activator inhibitor-1 binding, and in tempering inhibition by heparin-activated antithrombin. Compared with FX, mutants with reduced electrostatic potential potentiated thrombin production in FX-depleted plasma, whereas mutants with inverted electrostatic potential impeded clotting. Despite the definite consequences observed, disruption of the potential cation-binding exosite of FX had rather weak effects, far from what would be expected if this region was as crucial as in thrombin.     

Structural and functional insights into the B30.2/SPRY domain

The B30.2/SPRY domain is present in approximately 700 eukaryotic (approximately 150 human) proteins, including medically important proteins such as TRIM5alpha and Pyrin. Nonetheless, the functional role of this modular domain remained unclear. Here, we report the crystal structure of an SPRY-SOCS box family protein GUSTAVUS in complex with Elongins B and C, revealing a highly distorted two-layered beta-sandwich core structure of its B30.2/SPRY domain. Ensuing studies identified one end of the beta-sandwich as the surface interacting with an RNA helicase VASA with a 40 nM dissociation constant. The sequence variation in TRIM5alpha responsible for HIV-1 restriction and most of the mutations in Pyrin causing familial Mediterranean fever map on this surface, implicating the corresponding region in many B30.2/SPRY domains as the ligand-binding site. The amino acids lining the binding surface are highly variable among the B30.2/SPRY domains, suggesting that these domains are protein-interacting modules, which recognize a specific individual partner protein rather than a consensus sequence motif.     

A single amino acid change in the SPRY domain of human Trim5alpha leads to HIV-1 restriction

Retroviral restriction factors are cellular proteins that interfere with retrovirus replication at a postpenetration, preintegration stage in the viral life cycle. The first restriction activity described was the mouse Fv1 gene. Three different alleles of Fv1, capable of restricting different murine leukaemia viruses (MLV), have been characterized at the molecular level. Two further activities, Ref1, which acts on MLV, and Lv1, which acts on lentiviruses, have been identified in other mammalian species. Recently, it has become clear that Ref1 and Lv1 are encoded by the same gene, Trim5alpha, which inhibits retrovirus replication in a species-specific manner. A series of chimeras between the human and rhesus monkey Trim5 genes were created to map and identify these specificity determinants. The Trim5alpha SPRY domain was found to be responsible for targeting HIV-1 restriction. By contrast, N-MLV restriction appears dependent on both the coiled-coil domain and the SPRY domain. A single amino acid substitution (R332P) in the human Trim5alpha can confer the ability to restrict HIV-1, suggesting that small changes during evolution may have profound effects on our susceptibility to cross-species infection.     

Identification of ATP-Binding Regions in the RyR1 Ca2+ Release Channel

ATP is an important modulator of gating in type 1 ryanodine receptor (RyR1), also known as a Ca²⁺ release channel in skeletal muscle cells. The activating effect of ATP on this channel is achieved by directly binding to one or more sites on the RyR1 protein. However, the number and location of these sites have yet to be determined. To identify the ATP-binding regions within RyR1 we used 2N₃ATP-2′,3′-Biotin-LC-Hydrazone (BioATP-HDZ), a photo-reactive ATP analog to covalently label the channel. We found that BioATP-HDZ binds RyR1 specifically with an IC₅₀ = 0.6±0.2 mM, comparable with the reported EC50 for activation of RyR1 with ATP. Controlled proteolysis of labeled RyR1 followed by sequence analysis revealed three fragments with apparent molecular masses of 95, 45 and 70 kDa that were crosslinked by BioATP-HDZ and identified as RyR1 sequences. Our analysis identified four glycine-rich consensus motifs that can potentially constitute ATP-binding sites and are located within the N-terminal 95-kDa fragment. These putative nucleotide-binding sequences include amino acids 699–704, 701–706, 1081–1084 and 1195–1200, which are conserved among the three RyR isoforms. Located next to the N-terminal disease hotspot region in RyR1, these sequences may communicate the effects of ATP-binding to channel function by tuning conformational motions within the neighboring cytoplasmic regulatory domains. Two other labeled fragments lack ATP-binding consensus motifs and may form non-canonical ATP-binding sites. Based on domain topology in the 3D structure of RyR1 it is also conceivable that the identified ATP-binding regions, despite their wide separation in the primary sequence, may actually constitute the same non-contiguous ATP-binding pocket within the channel tetramer.     

RyR3 amplifies RyR1-mediated Ca(2+)-induced Ca(2+) release in neonatal mammalian skeletal muscle

The neonatal mammalian skeletal muscle contains both type 1 and type 3 ryanodine receptors (RyR1 and RyR3) located in the sarcoplasmic reticulum membrane. An allosteric interaction between RyR1 and dihydropyridine receptors located in the plasma membrane mediates voltage-induced Ca(2+) release (VICR) from the sarcoplasmic reticulum. RyR3, which disappears in adult muscle, is not involved in VICR, and the role of the transiently expressed RyR3 remains elusive. Here we demonstrate that RyR1 participates in both VICR and Ca(2+)-induced Ca(2+) release (CICR) and that RyR3 amplifies RyR1-mediated CICR in neonatal skeletal muscle. Confocal measurements of intracellular Ca(2+) in primary cultured mouse skeletal myotubes reveal active sites of Ca(2+) release caused by peripheral coupling between dihydropyridine receptors and RyR1. In myotubes lacking RyR3, the peripheral VICR component is unaffected, and RyR1s alone are able to support inward CICR propagation in most cells at an average speed of approximately 190 microm/s. With the co-presence of RyR1 and RyR3 in wild-type cells, unmitigated radial CICR propagates at 2,440 microm/s. Because neonatal skeletal muscle lacks a well developed transverse tubule system, the RyR3 reinforcement of CICR seems to ensure a robust, uniform, and synchronous activation of Ca(2+) release throughout the cell body. Such functional interplay between RyR1 and RyR3 can serve important roles in Ca(2+) signaling of cell differentiation and muscle contraction.     

RyR2 disease mutations at the C-terminal domain intersubunit interface alter closed-state stability and channel activation

Ryanodine receptors (RyRs) are ion channels that mediate the release of Ca²⁺ from the sarcoplasmic reticulum/endoplasmic reticulum, mutations of which are implicated in a number of human diseases. The adjacent C-terminal domains (CTDs) of cardiac RyR (RyR2) interact with each other to form a ring-like tetrameric structure with the intersubunit interface undergoing dynamic changes during channel gating. This mobile CTD intersubunit interface harbors many disease-associated mutations. However, the mechanisms of action of these mutations and the role of CTD in channel function are not well understood. Here, we assessed the impact of CTD disease-associated mutations P4902S, P4902L, E4950K, and G4955E on Ca²⁺− and caffeine-mediated activation of RyR2. The G4955E mutation dramatically increased both the Ca²⁺-independent basal activity and Ca²⁺-dependent activation of [³H]ryanodine binding to RyR2. The P4902S and E4950K mutations also increased Ca²⁺ activation but had no effect on the basal activity of RyR2. All four disease mutations increased caffeine-mediated activation of RyR2 and reduced the threshold for activation and termination of spontaneous Ca²⁺ release. G4955D dramatically increased the basal activity of RyR2, whereas G4955K mutation markedly suppressed channel activity. Similarly, substitution of P4902 with a negatively charged residue (P4902D), but not a positively charged residue (P4902K), also dramatically increased the basal activity of RyR2. These data suggest that electrostatic interactions are involved in stabilizing the CTD intersubunit interface and that the G4955E disease mutation disrupts this interface, and thus the stability of the closed state. Our studies shed new insights into the mechanisms of action of RyR2 CTD disease mutations.     

The elusive activity of the Yersinia protein kinase A kinase domain is revealed

Yersinia spp. pathogens use their type III secretion system to translocate effectors that manipulate host signaling pathways during infection. Although molecular targets for five of the six known Yersinia effectors are known, the target for the serine/threonine kinase domain of Yersinia protein kinase A (YpkA) has remained elusive. Recently, Navarro et al. (2007) demonstrated that YpkA phosphorylates Galphaq, and inhibits Galphaq-mediated signaling. Inhibition by YpkA could contribute to one of the most documented symptoms of Yersinia pestis infection, extensive bleeding.     

Ca2+ influx through alpha1S DHPR may play a role in regulating Ca2+ release from RyR1 in skeletal muscle

Skeletal muscle fiber types differ in their contents of total phosphate, which includes inorganic phosphate (P_i) and high-energy organic pools of ATP and phosphocreatine (PCr). At steady state, uptake of P_i into the cell must equal the rate of efflux, which is expected to be a function of intracellular P_i concentration. We measured ³²P-labeled P_i uptake rates in different muscle fiber types to determine whether they are proportional to cellular P_i content. P_i uptake rates in isolated, perfused rat hindlimb muscles were linear over time and highest in soleus (2.42 ± 0.17 µmol·g^–1·h^–1), lower in red gastrocnemius (1.31 ± 0.11 µmol·g^–1·h^–1), and lowest in white gastrocnemius (0.49 ± 0.06 µmol·g^–1·h^–1). Reasonably similar rates were obtained in vivo. P_i uptake rates at plasma P_i concentrations of 0.3–1.7 mM confirm that the P_i uptake process is nearly saturated at normal plasma P_i levels. P_i uptake rate correlated with cellular P_i content (r = 0.99) but varied inversely with total phosphate content. Sodium-phosphate cotransporter (PiT-1) protein expression in soleus and red gastrocnemius were similar to each other and seven- to eightfold greater than PiT-1 expression in white gastrocnemius. That the PiT-1 expression pattern did not match the pattern of P_i uptake across fiber types implies that other factors are involved in regulating P_i uptake in skeletal muscle. Furthermore, fractional turnover of the cellular P_i pool (0.67, 0.57, and 0.33 h^–1 in soleus, red gastrocnemius, and white gastrocnemius, respectively) varies among fiber types, indicating differential management of intracellular P_i, likely due to differences in resistance to P_i efflux from the fiber. inorganic phosphate; sodium-inorganic phosphate transporters; PiT-2; inorganic phosphate efflux