Three-dimensional structure of the type 1 inositol 1,4,5-trisphosphate receptor at 24 A resolution

We report here the first three-dimensional structure of the type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R). From cryo-electron microscopic images of purified receptors embedded in vitreous ice, a three-dimensional structure was determined by use of standard single particle reconstruction techniques. The structure is strikingly different from that of the ryanodine receptor at similar resolution despite molecular similarities between these two calcium release channels. The 24 A resolution structure of the IP(3)R takes the shape of an uneven dumbbell, and is approximately 170 A tall. Its larger end is bulky, with four arms protruding laterally by approximately 50 A and, in comparison with the receptor topology, probably corresponds to the cytoplasmic domain of the receptor. The lateral dimension at the height of the protruding arms is approximately 155 A. The smaller end, whose lateral dimension is approximately 100 A, has structural features indicative of the membrane-spanning domain. A central opening in this domain, which is occluded on the cytoplasmic half, outlines a pathway for calcium flow in the open state of the channel.     

Calcium transport across the sarcoplasmic reticulum: structure and function of Ca2+-ATPase and the ryanodine receptor.

Contraction of striated muscle results from a rise in cytoplasmic calcium concentration in a process termed excitation/contraction coupling. Most of this calcium moves back and forth across the sarcoplasmic-reticulum membrane in cycles of contraction and relaxation. The channel responsible for release from the sarcoplasmic reticulum is the ryanodine receptor, whereas Ca2+-ATPase effects reuptake in an ATP-dependent manner. The structures of these two molecules have been studied by cryoelectron microscopy, with helical crystals in the case of Ca2+-ATPase and as isolated tetramers in the case of ryanodine receptor. Structures of Ca2+-ATPase at 8-A resolution reveal the packing of transmembrane helices and have allowed fitting of a putative ATP-binding domain among the cytoplasmic densities. Comparison of ATPases in different conformations gives hints about the conformational changes that accompany the reaction cycle. Structures of ryanodine receptor at 30-A resolution reveal a multitude of isolated domains in the cytoplasmic portion, as well as a distinct transmembrane assembly. Binding sites for various protein ligands have been determined and conformational changes induced by ATP, calcium and ryanodine have been characterized. Both molecules appear to use large conformational changes to couple interactions in their cytoplasmic domains with calcium transport through their membrane domains, and future studies at higher resolution will focus on the mechanisms for this coupling.     

Three-dimensional structure of ryanodine receptor isoform three in two conformational states as visualized by cryo-electron microscopy

Using cryo-electron microscopy and single particle image processing techniques, we present the first three-dimensional reconstructions of isoform 3 of the ryanodine receptor/calcium release channel (RyR3). Reconstructions were carried out on images obtained from a purified, detergent-solubilized receptor for two different buffer conditions, which were expected to favor open and closed functional states of the channel. As for the heart (RyR2) and skeletal muscle (RyR1) receptor isoforms, RyR3 is a homotetrameric complex comprising two main components, a multidomain cytoplasmic assembly and a smaller ( approximately 20% of the total mass) transmembrane region. Although the isoforms show structural similarities, consistent with the approximately 70% overall sequence identity of the isoforms, detailed comparisons of RyR3 with RyR1 showed one region of highly significant difference between them. This difference indicated additional mass present in RyR1, and it likely corresponds to a region of the RyR1 sequence (residues 1303-1406, known as diversity region 2) that is absent from RyR3. The reconstructions of RyR3 determined under "open" and "closed" conditions were similar to each other in overall architecture. A difference map computed between the two reconstructions reveals subtle changes in conformation at several widely dispersed locations in the receptor, the most prominent of which is a approximately 4 degrees rotation of the transmembrane region with respect to the cytoplasmic assembly.     

Functional interaction of the cytoplasmic domain of triadin with the skeletal ryanodine receptor

Triadin has been shown to co-localize with the ryanodine receptor in the sarcoplasmic reticulum membrane. We show that immunoprecipitation of solubilized sarcoplasmic reticulum membrane with antibodies directed against triadin or ryanodine receptor, leads to the co-immunoprecipitation of ryanodine receptor and triadin. We then investigated the functional importance of the cytoplasmic domain of triadin (residues 1-47) in the control of Ca2+ release from sarcoplasmic reticulum. We show that antibodies directed against a synthetic peptide encompassing residues 2-17, induce a decrease in the rate of Ca2+ release from sarcoplasmic reticulum vesicles as well as a decrease in the open probability of the ryanodine receptor Ca2+ channel incorporated in lipid bilayers. Using surface plasmon resonance spectroscopy, we defined a discrete domain (residues 18-46) of the cytoplasmic part of triadin interacting with the purified ryanodine receptor. This interaction is optimal at low Ca2+ concentration (up to pCa 5) and inhibited by increasing calcium concentration (IC50 of 300 microM). The direct molecular interaction of this triadin domain with the ryanodine receptor was confirmed by overlay assay and shown to induce the inhibition of the Ca2+ channel activity of purified RyR in bilayer. We propose that this interaction plays a critical role in the control, by triadin, of the Ca2+ channel behavior of the ryanodine receptor and therefore may represent an important step in the regulation process of excitation-contraction coupling in skeletal muscle.     

Effects of an alpha-helical ryanodine receptor C-terminal tail peptide on ryanodine receptor activity: modulation by Homer

We have determined the structure of a domain peptide corresponding to the extreme 19 C-terminal residues of the ryanodine receptor Ca2+ release channel. We examined functional interactions between the peptide and the channel, in the absence and in the presence of the regulatory protein Homer. The peptide was partly alpha-helical and structurally homologous to the C-terminal end of the T1 domain of voltage-gated K+ channels. The peptide (0.1-10 microM) inhibited skeletal ryanodine receptor channels when the cytoplasmic Ca2+ concentration was 1 microM; but with 10 microM cytoplasmic Ca2+, skeletal ryanodine receptors were activated by < or = 1.0 microM peptide and inhibited by 10 microM peptide. Cardiac ryanodine receptors on the other hand were inhibited by all peptide concentrations, at both Ca2+ concentrations. When channels did open in the presence of the peptide, they were more likely to open to substate levels. The inhibition and increased fraction of openings to subconductance levels suggested that the domain peptide might destabilise inter-domain interactions that involve the C-terminal tail. We found that Homer 1b not only interacts with the channels, but reduces the inhibitory action of the C-terminal tail peptide, perhaps by stabilizing inter-domain interactions and preventing their disruption.     

Structure of frozen–hydrated triad junctions: A case study in motif searching inside tomograms

We used tomographic reconstructions of frozen–hydrated triad junctions to determine the structure of the macromolecular complex associated with calcium release from the sarcoplasmic reticulum (SR), during excitation–contraction coupling. Using a rapid motif search algorithm with a reference motif of the ryanodine receptor (RyR) provided by single-particle cryo-electron microscopy, 49 receptors were located in five tomograms. Following co-alignment of the receptors and division into quadrants centered on the 4-fold symmetry axis, the receptors were classified using multivariate statistics. Global and class averages reveal that the SR membrane in the vicinity of the receptor is highly curved, creating an open vestibule with a gap of 4 nm between the receptor pore and the calsequestrin layer in the SR lumen. The in-plane densities in the calsequestrin layer have paracrystalline order, consistent with the packing of calsequestrin dimers in the three-dimensional crystal structure. Faint densities (“tethers”) extend to the calsequestrin layer from densities in the SR membrane located 15 nm from the symmetry axis of the RyR. In a class average of RyRs with proximal transverse tubules (TT), a cytoplasmic density is observed near the receptor that could represent the most consistent location of tethers observed in tomograms between the SR and TT membranes.     

Identification of two regions within the cytoplasmic domain of the human interferon-gamma receptor required for function.

Functionally active human interferon-gamma (IFN gamma) receptors require the presence of at least two polypeptides: the IFN gamma receptor and an accessory molecule encoded by a gene on human chromosome 21. Here we have used a murine L cell line that stably contains human chromosome 21 (SCC16-5) to determine whether the receptor's cytoplasmic domain is important for receptor function. SCC16-5 stably transfected with the full-length human IFN gamma receptor cDNA bound, internalized, and responded to human IFN gamma. In contrast, SCC16-5 expressing human IFN gamma receptors lacking a cytoplasmic domain bound human IFN gamma but did not internalize or respond to it. Using a family of IFN gamma receptor deletion mutants, two functionally important regions within the intracellular domain were identified: (a) a membrane proximal region (residues 256-303) required for ligand processing and biologic responsiveness and (b) the carboxyl-terminal 39 amino acids (residues 434-472) needed exclusively for biologic responses.     

Molecular cloning of cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum

We have cloned and sequenced cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum. The cDNA, 16,532 base pairs in length, encodes a protein of 4,969 amino acids with a Mr of 564,711. The deduced amino acid sequence is 66% identical with that of the skeletal muscle ryanodine receptor, but analysis of predicted secondary structures and hydropathy plots suggests that the two isoforms exhibit the same topology in both transmembrane and cytoplasmic domains. A potential ATP binding domain was identified at residues 2619-2652, a potential phosphorylation site at residue 2809, and potential calmodulin binding sites at residues 2775-2807, 2877-2898, and 2998-3016. We suggest that a modulator binding domain in the protein lies between residues 2619 and 3016. Northern blot analysis of mRNA from a variety of tissues demonstrated that the cardiac isoform is expressed in heart and brain, while the skeletal muscle isoform is expressed in both fast- and slow-twitch muscle. No ryanodine receptor mRNA was detected in extracts from smooth muscle or any other non-muscle tissue examined. The two receptors are clearly the products of separate genes, and the gene encoding the cardiac muscle ryanodine receptor was localized to chromosome 1.     

Structural determinants for activation or inhibition of ryanodine receptors by basic residues in the dihydropyridine receptor II-III loop

The structures of peptide A, and six other 7-20 amino acid peptides corresponding to sequences in the A region (Thr671- Leu690) of the skeletal muscle dihydropyridine receptor II-III loop have been examined, and are correlated with the ability of the peptides to activate or inhibit skeletal ryanodine receptor calcium release channels. The peptides adopted either random coil or nascent helix-like structures, which depended upon the polarity of the terminal residues as well as the presence and ionisation state of two glutamate residues. Enhanced activation of Ca2+ release from sarcoplasmic reticulum, and activation of current flow through single ryanodine receptor channels (at -40 mV), was seen with peptides containing the basic residues 681Arg Lys Arg Arg Lys685, and was strongest when the residues were a part of an alpha-helix. Inhibition of channels (at +40 mV) was also seen with peptides containing the five positively charged residues, but was not enhanced in helical peptides. These results confirm the hypothesis that activation of ryanodine receptor channels by the II-III loop peptides requires both the basic residues and their participation in helical structure, and show for the first time that inhibition requires the basic residues, but is not structure-dependent. These findings imply that activation and inhibition result from peptide binding to separate sites on the ryanodine receptor.     

Bradykinin-induced internalization of the human B2 receptor requires phosphorylation of three serine and two threonine residues at its carboxyl tail

The binding of bradykinin (BK) to B2 receptor triggers the internalization of the agonist-receptor complex. To investigate the mechanisms and the receptor structures involved in this fundamental process of receptor regulation, the human B2 receptor was mutated within its cytoplasmic tail by complementary strategies of truncation, deletion, and amino acid substitution. Ligand binding, signal transduction, internalization as well as phosphorylation were studied for the mutated receptors expressed in COS, CHO, and HEK 293 cells. Truncation of 44 out of 55 amino acid residues of the receptor's cytoplasmic tail corresponding to positions 321-364 did not alter the kinetics of BK binding and the receptor coupling to phospholipase C and phospholipase A2. By contrast, truncations after positions 320 and 334, deletions within the segment covering positions 335-351, as well as alanine substitution of serine and threonine residues within segment 335-351 diminished the internalization capacity of the mutant receptors. Mutants with a markedly reduced internalization potential failed to produce BK-induced receptor phosphorylation suggesting that phosphorylation may be involved in receptor internalization. The mutagenesis approaches converged at the conclusion that three serines in positions 339, 346, and 348 and two threonines in positions 342 and 345, contained in a sequence segment that is highly conserved between species, have a critical role in the ligand-dependent internalization and phosphorylation of kinin receptors and can intervene in these processes in an alternative manner. However, mutants lacking these residues were still sensitive to dominant-negative forms of beta-arrestin and dynamin, suggesting the existence of additional receptor structure(s) involved in the receptor sequestration through clathrin-coated vesicles.     

Localization of the dantrolene-binding sequence near the FK506-binding protein-binding site in the three-dimensional structure of the ryanodine receptor

Dantrolene is believed to stabilize interdomain interactions between the NH2-terminal and central regions of ryanodine receptors by binding to the NH2-terminal residues 590-609 in skeletal ryanodine receptor (RyR1) and residues 601-620 in cardiac ryanodine receptor (RyR2). To gain further insight into the structural basis of dantrolene action, we have attempted to localize the dantrolene-binding sequence in RyR1/RyR2 by using GFP as a structural marker and three-dimensional cryo-EM. We inserted GFP into RyR2 after residues Arg-626 and Tyr-846 to generate GFP-RyR2 fusion proteins, RyR2Arg-626-GFP and RyR2Tyr-846-GFP. Insertion of GFP after residue Arg-626 abolished the binding of a bulky GST- or cyan fluorescent protein-tagged FKBP12.6 but not the binding of a smaller, nontagged FKBP12.6, suggesting that residue Arg-626 and the dantrolene-binding sequence are located near the FKBP12.6-binding site. Using cryo-EM, we have mapped the three-dimensional location of Tyr-846-GFP to domain 9, which is also adjacent to the FKBP12.6-binding site. To further map the three-dimensional location of the dantrolene-binding sequence, we generated 10 FRET pairs based on four known three-dimensional locations (FKBP12.6, Ser-437-GFP, Tyr-846-GFP, and Ser-2367-GFP). Based on the FRET efficiencies of these FRET pairs and the corresponding distance relationships, we mapped the three-dimensional location of Arg-626-GFP or -cyan fluorescent protein, hence the dantrolene-binding sequence, to domain 9 near the FKBP12.6-binding site but distant to the central region around residue Ser-2367. An allosteric mechanism by which dantrolene stabilizes interdomain interactions between the NH2-terminal and central regions is proposed.     

Native structure and arrangement of inositol-1,4,5-trisphosphate receptor molecules in bovine cerebellar Purkinje cells as studied by quick-freeze deep-etch electron microscopy.

We used quick-freeze deep-etch replica electron microscopy to visualize the native structure of inositol-1,4,5-trisphosphate receptor (IP3R) in the cell. In the dendrites of Purkinje neurons of bovine cerebellum there were many vesicular organelles whose surfaces were covered with a two-dimensional crystalline array of molecules. Detailed examination of the cytoplasmic true surface of such vesicles in replica revealed that the structural unit, identified as IP3R by immunocytochemistry and subsequent Fourier analysis, is a square-shaped assembly and is aligned so that the side of the square is inclined by approximately 20 degrees from the row-line of the lattice. Comparison with the ryanodine receptor (RyaR), another intracellular Ca2+ channel on the endoplasmic reticulum, suggested that IP3R, unlike RyaR, has a very compact structure, potentially reflecting the crucial difference in the function of the cytoplasmic portion of the molecule.     

Mutagenesis of the AT1 receptor reveals different binding modes of angiotensin II and [Sar1]-angiotensin II

Homology modeling of the structure of the AT1 receptor, based on the high resolution rhodopsin crystal structure, indicated that it is unlikely that the binding of AngII to AT1 involves simultaneously all the receptor's residues reported in the literature to participate in this process. Site-directed mutagenesis using Ala substitution of charged residues Lys20, Arg23, Glu91 and Arg93 was performed to evaluate the participation of their side-chains in ligand binding and in triggering the cell's response. A comparative analysis by competition binding and functional assays using angiotensin II and the analog [Sar1]-angiotensin II suggests an important role for Arg23 of AT1 receptor in binding of the natural agonist. It is discussed whether some receptor's residues participate directly in the binding with AngII or whether they are part of a regulatory site.     

Identification and functional characterization of the zeta-chain dimerization motif for TCR surface expression.

We recognized a common dimerization motif between the transmembrane (TM) domain of zeta-chain family members and glycophorin A. We have shown that a glycine within the zeta-dimerization motif is critical for zeta-homodimerization and also for its association with the TCR/CD3 complex. Similarly, two residues within the CD3 delta gamma TM domains have proven to be critical for their interaction with the zeta-homodimer. A three-dimensional homology model of the zeta-chain TM domain highlights potential residues preferentially involved either in the zeta 2-CD3 or zeta 2-TCR alpha beta association, confirming our experimental findings. These results indicate that, for symmetrical reasons, the zeta-homodimer participates in the TCR/CD3 complex assembly by interacting with CD3 gamma delta TM domains, thereby masking their degradation signals located in the cytoplasmic tails.     

Identification of a novel sequence mediating regulated endocytosis of the G protein-coupled alpha-pheromone receptor in yeast.

The Saccharomyces cerevisiae alpha-pheromone receptor, a polytopic, G protein-coupled, membrane protein, is internalized after binding of alpha-factor. Mutational analysis suggested that the first 39 residues of the receptor's cytoplasmic tail carries sufficient information for internalization. A point mutation in one of these 39 residues, K337 to R337, renders the receptor nonfunctional for endocytosis. Other residues, D335 and S338, contribute to the efficiency of internalization. When the sequence DAKSS is added onto a severely truncated receptor, endocytosis of the receptor is restored, showing that this sequence functions to mediate or to signal interaction with the endocytic machinery. Analysis of pheromone response and recovery in strains expressing mutant receptors suggests that receptor internalization is not important for response but contributes to recovery from pheromone.     

Apocalmodulin and Ca2+-calmodulin bind to neighboring locations on the ryanodine receptor.

Calmodulin (CaM) binds to the ryanodine receptor/calcium release channel of skeletal muscle (RyR1), both in the absence and presence of Ca(2+), and regulates the activity of the channel activity by activating and inhibiting it, respectively. Using cryo-electron microscopy and three-dimensional reconstruction, we found that one apoCaM binds per RyR1 subunit along the sides of the cytoplasmic assembly of the receptor. This location is distinct from but close to the location found for Ca(2+)-CaM, providing a structural basis for efficient switching of CaM between these two positions with the oscillating intracellular Ca(2+) concentration that generates muscle relaxation/contraction cycles. The locations of apoCaM and Ca(2+)-CaM at a critical region for RYR1-dihydropyridine receptor interaction are suggestive of a direct role for CaM in the mechanism of excitation-contraction coupling.     

Structure of glutaraldehyde cross-linked ryanodine receptor

The ryanodine receptor (RyR) family of calcium release channels plays a vital role in excitation–contraction coupling (ECC). Along with the dihydropyridine receptor (DHPR), calsequestrin, and several other smaller regulatory and adaptor proteins, RyRs form a large dynamic complex referred to as ECC machinery. Here we describe a simple cross-linking procedure that can be used to stabilize fragile components of the ECC machinery, for the purpose of structural elucidation by single particle cryo-electron microscopy (cryo-EM). As a model system, the complex of the FK506-binding protein (FKBP12) and RyR1 was used to test the cross-linking protocol. Glutaraldehyde fixation led to complete cross-linking of receptor-bound FKBP12 to RyR1, and also to extensive cross-linking of the four subunits comprising RyR to one another without compromising the RyR1 ultrastructure. FKBP12 cross-linked with RyR1 was visualized in 2D averages by single particle cryo-EM. Comparison of control RyR1 and cross-linked RyR1 3D reconstructions revealed minor conformational changes at the transmembrane assembly and at the cytoplasmic region. Intersubunit cross-linking enhanced [³H]ryanodine binding to RyR1. Based on our findings we propose that intersubunit cross-linking of RyR1 by glutaraldehyde induced RyR1 to adopt an open like conformation.     

Signaling through sphingolipid microdomains of the plasma membrane: the concept of signaling platform

Transmembrane signaling requires modular interactions between signaling proteins, phosphorylation or dephosphorylation of the interacting protein partners and temporary elaboration of supramolecular structures, to convey the molecular information from the cell surface to the nucleus. Such signaling complexes at the plasma membrane are instrumental in translating the extracellular cues into intracellular signals for gene activation. In the most straightforward case, ligand binding promotes homodimerization of the transmembrane receptor which facilitates modular interactions between the receptor's cytoplasmic domains and intracellular signaling and adaptor proteins. For example, most growth factor receptors contain a cytoplasmic protein tyrosine kinase (PTK) domain and ligand-mediated receptor dimerization leads to cross phosphorylation of tyrosines in the receptor's cytoplasmic domains, an event that initiates the signaling cascade. In other signaling pathways where the receptors have no intrinsic kinase activity, intracellular nonreceptor PTKs (i.e. Src family PTKs, JAKs) are recruited to the cytoplasmic domain of the engaged receptor. Execution of these initial phosphorylations and their translation into efficient cellular stimulation requires concomitant activation of diverse signaling pathways. Availability of stable, preassembled matrices at the plasma membrane would facilitate scaffolding of a large array of receptors, coreceptors, tyrosine kinases and other signaling and adapter proteins, as it is the case in signaling via the T cell antigen receptor. The concept of the signaling platform has gained usage to characterize the membrane structure where many different membrane-bound components need to be assembled in a coordinated manner to carry out signaling. The structural basis of the signaling platform lies in preferential assembly of certain classes of lipids into distinct physical and functional compartments within the plasma membrane. These membrane microdomains or rafts (Figure 1) serve as privileged sites where receptors and proximal signaling molecules optimally interact. In this review, we shall discuss first how signaling platforms are assembled and how receptors and their signaling machinery could be functionally linked in such structures. The second part of our review will deal with selected examples of raft-based signaling pathways in T lymphocytes and NK cells to illustrate the ways in which rafts may facilitate signaling.     

Analysis of the molecular basis for octanal interactions in the expressed rat 17 olfactory receptor

Expression studies have shown that the rat I7 olfactory receptor (OR-I7) responds preferentially to the aldehyde n-octanal. We wished to predict which residues in OR-I7 bind octanal and how the biophysical properties of these residues determine the receptor's odor selectivity. Building on our previous work on aldehyde interactions in olfactory receptors, we constructed a molecular model of OR-I7 based on the 7.5 A resolution three-dimensional map of rhodopsin. Octanal was automatically docked in the model. The results predicted an odor-binding pocket approximately 10 A from the extracellular surface, in a location similar to the epinephrine-binding pocket of the beta-adrenergic receptor and the odor-binding pocket of a previous olfactory receptor model. A lysine on TM4 and an aspartate on TM5 interacted with the aldehyde moiety of octanal. Hydrophobic residues formed Van der Waals contacts with the hydrocarbon portion of octanal. We docked related odor compounds and found that the predicted affinities compared favorably with experimental results. We also tested a number of amino acid substitutions in order to predict their effects on octanal affinity and provide leads for future experimental work.     

Localization of calmodulin binding sites on the ryanodine receptor from skeletal muscle by electron microscopy.

Calmodulin (CaM) is a regulator of the calcium release channel (ryanodine receptor) of the sarcoplasmic reticulum of skeletal and cardiac muscle. The locations where CaM binds on the surface of the skeletal muscle ryanodine receptor were determined by electron microscopy. Wheat germ CaM was labeled specifically at Cys-27 with a maleimide derivative of a 1.4-nm-diameter gold cluster, and the gold-cluster-labeled CaM was bound to the purified ryanodine receptor. The complexes were imaged in the frozen-hydrated state by cryoelectron microscopy with no stains or fixatives present. In the micrographs, gold clusters were frequently observed near the corners of the square-shaped images of the ryanodine receptors. In some images, all four corners of the receptor were occupied by gold clusters. Image averaging allowed the site of CaM binding to be determined in two dimensions with an estimated precision of 4 nm. No changes were apparent in the quaternary structure of the ryanodine receptor upon binding CaM to the resolution attained, about 3 nm. Side views of the ryanodine receptor, in which the receptor is oriented approximately perpendicular to the much more frequent fourfold symmetric views, were occasionally observed, and showed that the CaM binding site is most likely on the surface of the receptor that faces the cytoplasm. We conclude that the CaM binding site is at least 10 nm from the transmembrane channel of the receptor and, consequently, that long-range conformational changes are involved in the modulation of the calcium channel activity of the receptor by CaM.