Irina I. Serysheva

Structure of TRPV1 channel revealed by electron cryomicroscopy

The transient receptor potential (TRP) family of ion channels participate in many signaling pathways. TRPV1 functions as a molecular integrator of noxious stimuli, including heat, low pH, and chemical ligands. Here, we report the 3D structure of full-length rat TRPV1 channel expressed in the yeast Saccharomyces cerevisiae and purified by immunoaffinity chromatography. We demonstrate that the recombinant purified TRPV1 channel retains its structural and functional integrity and is suitable for structural analysis. The 19-Å structure of TRPV1 determined by using single-particle electron cryomicroscopy exhibits fourfold symmetry and comprises two distinct regions: a large open basket-like domain, likely corresponding to the cytoplasmic N- and C-terminal portions, and a more compact domain, corresponding to the transmembrane portion. The assignment of transmembrane and cytoplasmic regions was supported by fitting crystal structures of the structurally homologous Kv1.2 channel and isolated TRPV1 ankyrin repeats into the TRPV1 structure.

Determinants for calmodulin binding on voltage-dependent Ca2+ channels

Calmodulin, bound to the α1subunit of the cardiac L-type calcium channel, is required for calcium-dependent inactivation of this channel. Several laboratories have suggested that the site of interaction of calmodulin with the channel is an IQ-like motif in the carboxyl-terminal region of the α1 subunit. Mutations in this IQ motif are linked to L-type Ca2+ current (I Ca) facilitation and inactivation. IQ peptides from L, P/Q, N, and R channels all bind Ca2+calmodulin but not Ca2+-free calmodulin. Another peptide representing a carboxyl-terminal sequence found only in L-type channels (designated the CB domain) binds Ca2+calmodulin and enhances Ca2+-dependent I Cafacilitation in cardiac myocytes, suggesting the CB domain is functionally important. Calmodulin blocks the binding of an antibody specific for the CB sequence to the skeletal muscle L-type Ca2+ channel, suggesting that this is a calmodulin binding site on the intact protein. The binding of the IQ and CB peptides to calmodulin appears to be competitive, signifying that the two sequences represent either independent or alternative binding sites for calmodulin rather than both sequences contributing to a single binding site.

Subnanometer-resolution electron cryomicroscopy-based domain models for the cytoplasmic region of skeletal muscle RyR channel

The skeletal muscle Ca2+ release channel (RyR1), a homotetramer, regulates the release of Ca2+ from the sarcoplasmic reticulum to initiate muscle contraction. In this work, we have delineated the RyR1 monomer boundaries in a subnanometer-resolution electron cryomicroscopy (cryo-EM) density map. In the cytoplasmic region of each RyR1 monomer, 36 α-helices and 7 β-sheets can be resolved. A β-sheet was also identified close to the membrane-spanning region that resembles the cytoplasmic pore structures of inward rectifier K+ channels. Three structural folds, generated for amino acids 12–565 using comparative modeling and cryo-EM density fitting, localize close to regions implicated in communication with the voltage sensor in the transverse tubules. Eleven of the 15 disease-related residues for these domains are mapped to the surface of these models. Four disease-related residues are found in a basin at the interfaces of these regions, creating a pocket in which the immunophilin FKBP12 can fit. Taken together, these results provide a structural context for both channel gating and the consequences of certain malignant hyperthermia and central core disease-associated mutations in RyR1.

Structure of the skeletal muscle calcium release channel activated with Ca2+ and AMP-PCP.

The functional state of the skeletal muscle Ca2+ release channel is modulated by a number of endogenous molecules during excitation-contraction. Using electron cryomicroscopy and angular reconstitution techniques, we determined the three-dimensional (3D) structure of the skeletal muscle Ca2+ release channel activated by a nonhydrolyzable analog of ATP in the presence of Ca2+. These ligands together produce almost maximum activation of the channel and drive the channel population toward a predominately open state. The resulting 30-A 3D reconstruction reveals long-range conformational changes in the cytoplasmic region that might affect the interaction of the Ca2+ release channel with the t-tubule voltage sensor. In addition, a central opening and mass movements, detected in the transmembrane domain of both the Ca(2+)- and the Ca2+/nucleotide-activated channels, suggest a mechanism for channel opening similar to opening-closing of the iris in a camera diaphragm.

Gating machinery of InsP3R channels revealed by electron cryomicroscopy

Inositol-1,4,5-trisphosphate receptors (InsP3Rs) are ubiquitous ion channels responsible for cytosolic Ca2+ signalling and essential for a broad array of cellular processes ranging from contraction to secretion, and from proliferation to cell death. Despite decades of research on InsP3Rs, a mechanistic understanding of their structure–function relationship is lacking. Here we present the first, to our knowledge, near-atomic (4.7 Å) resolution electron cryomicroscopy structure of the tetrameric mammalian type 1 InsP3R channel in its apo-state. At this resolution, we are able to trace unambiguously ∼85% of the protein backbone, allowing us to identify the structural elements involved in gating and modulation of this 1.3-megadalton channel. Although the central Ca2+-conduction pathway is similar to other ion channels, including the closely related ryanodine receptor, the cytosolic carboxy termini are uniquely arranged in a left-handed α-helical bundle, directly interacting with the amino-terminal domains of adjacent subunits. This configuration suggests a molecular mechanism for allosteric regulation of channel gating by intracellular signals.

Ryanodine receptor structure: progress and challenges.

Ryanodine Receptors as Regulated Guardians of Intracellular Ca Stores Ryanodine-sensitive Ca2 release channels, also known as the ryanodine receptors (RyRs),2 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. 1). Recently, cryo-EM reconstructions of RyR1 have reached subnanometer resolution (2, 3), a breakthrough due to improvements in cryo-specimen preparation, instrumentation, image processing, and three-dimensional reconstruction techniques. RyRs have amushroom shape with 4-fold symmetry (Fig. 1B) (2). 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. TheCY region is strikingly empty with numerous distinctive structural domains and intervening cavities that appear suitable for interactionwithmodulators that bindwithin theN-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 (5) 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 60Å) is rotated by 40°with respect to the CY region.

Structure of the voltage-gated L-type Ca2+ channel by electron cryomicroscopy.

Voltage-dependent L-type Ca2+ channels play important functional roles in many excitable cells. We present a three-dimensional structure of an L-type Ca2+ channel. Electron cryomicroscopy in conjunction with single-particle processing was used to determine a 30-Å resolution structure of the channel protein. The asymmetrical channel structure consists of two major regions: a heart-shaped region connected at its widest end with a handle-shaped region. A molecular model is proposed for the arrangement of this skeletal muscle L-type Ca2+ channel structure with respect to the sarcoplasmic reticulum Ca2+-release channel, the physical partner of the L-type channel for signal transduction during the excitation-contraction coupling in muscle.

Flexible Architecture of IP3R1 by Cryo-EM

Inositol 1,4,5-trisphosphate receptors (IP3Rs) play a fundamental role in generating Ca2+ signals that trigger many cellular processes in virtually all eukaryotic cells. Thus far, the three-dimensional (3D) structure of these channels has remained extremely controversial. Here, we report a subnanometer resolution electron cryomicroscopy (cryo-EM) structure of a fully functional type 1 IP3R from cerebellum in the closed state. The transmembrane region reveals a twisted bundle of four α helices, one from each subunit, that form a funnel shaped structure around the 4-fold symmetry axis, strikingly similar to the ion-conduction pore of K+ channels. The lumenal face of IP3R1 has prominent densities that surround the pore entrance and similar to the highly structured turrets of Kir channels. 3D statistical analysis of the cryo-EM density map identifies high variance in the cytoplasmic region. This structural variation could be attributed to genuine structural flexibility of IP3R1.

The skeletal muscle Ca2+ release channel has an oxidoreductase-like domain

We used a combination of bioinformatics, electron cryomicroscopy, and biochemical techniques to identify an oxidoreductase-like domain in the skeletal muscle Ca2+ release channel protein (RyR1). The initial prediction was derived from sequence-based fold recognition for the N-terminal region (41–420) of RyR1. The putative domain was computationally localized to the clamp domain in the cytoplasmic region of a 22Å structure of RyR1. This localization was subsequently confirmed by difference imaging with a sequence specific antibody. Consistent with the prediction of an oxidoreductase domain, RyR1 binds [3H]NAD+, supporting a model in which RyR1 has a oxidoreductase-like domain that could function as a type of redox sensor.

An allosteric transport mechanism for the AcrAB-TolC multidrug efflux pump.

Bacterial efflux pumps confer multidrug resistance by transporting diverse antibiotics from the cell. In Gram-negative bacteria, some of these pumps form multi-protein assemblies that span the cell envelope. Here, we report the near-atomic resolution cryoEM structures of the Escherichia coli AcrAB-TolC multidrug efflux pump in resting and drug transport states, revealing a quaternary structural switch that allosterically couples and synchronizes initial ligand binding with channel opening. Within the transport-activated state, the channel remains open even though the pump cycles through three distinct conformations. Collectively, our data provide a dynamic mechanism for the assembly and operation of the AcrAB-TolC pump. DOI: http://dx.doi.org/10.7554/eLife.24905.001