Kainate receptor channel opening and gating mechanism.

Kainate receptors, a subclass of ionotropic glutamate receptors, are tetrameric ligand-gated ion channels that mediate excitatory neurotransmission1-4. Kainate receptors modulate neuronal circuits and synaptic plasticity during the development and function of the central nervous system and are implicated in various neurological and psychiatric diseases, including epilepsy, depression, schizophrenia, anxiety and autism5-11. Although structures of kainate receptor domains and subunit assemblies are available12-18, the mechanism of kainate receptor gating remains poorly understood. Here we present cryo-electron microscopy structures of the kainate receptor GluK2 in the presence of the agonist glutamate and the positive allosteric modulators lectin concanavalin A and BPAM344. Concanavalin A and BPAM344 inhibit kainate receptor desensitization and prolong activation by acting as a spacer between the amino-terminal and ligand-binding domains and a stabilizer of the ligand-binding domain dimer interface, respectively. Channel opening involves the kinking of all four pore-forming M3 helices. Our structures reveal the molecular basis of kainate receptor gating, which could guide the development of drugs for treatment of neurological disorders.

Structural mechanism of TRPV3 channel inhibition by the plant-derived coumarin osthole.

TRPV3, a representative of the vanilloid subfamily of TRP channels, is predominantly expressed in skin keratinocytes and has been implicated in cutaneous sensation and associated with numerous skin pathologies and cancers. TRPV3 is inhibited by the natural coumarin derivative osthole, an active ingredient of Cnidium monnieri, which has been used in traditional Chinese medicine for the treatment of a variety of human diseases. However, the structural basis of channel inhibition by osthole has remained elusive. Here we present cryo-EM structures of TRPV3 in complex with osthole, revealing two types of osthole binding sites in the transmembrane region of TRPV3 that coincide with the binding sites of agonist 2-APB. Osthole binding converts the channel pore into a previously unidentified conformation with a widely open selectivity filter and closed intracellular gate. Our structures provide insight into competitive inhibition of TRPV3 by osthole and can serve as a template for the design of osthole chemistry-inspired drugs targeting TRPV3-associated diseases.

Interaction between the transmembrane domains of neurotrophin receptors p75 and TrkA mediates their reciprocal activation.

The neurotrophin receptors p75 and tyrosine protein kinase receptor A (TrkA) play important roles in the development and survival of the nervous system. Biochemical data suggest that p75 and TrkA reciprocally regulate the activities of each other. For instance, p75 is able to regulate the response of TrkA to lower concentrations of nerve growth factor (NGF), and TrkA promotes shedding of the extracellular domain of p75 by α-secretases in a ligand-dependent manner. The current model suggests that p75 and TrkA are regulated by means of a direct physical interaction; however, the nature of such interaction has been elusive thus far. Here, using NMR in micelles, multiscale molecular dynamics, FRET, and functional studies, we identified and characterized the direct interaction between TrkA and p75 through their respective transmembrane domains (TMDs). Molecular dynamics of p75-TMD mutants suggests that although the interaction between TrkA and p75 TMDs is maintained upon mutation, a specific protein interface is required to facilitate TrkA active homodimerization in the presence of NGF. The same mutations in the TMD protein interface of p75 reduced the activation of TrkA by NGF as well as reducing cell differentiation. In summary, we provide a structural model of the p75-TrkA receptor complex necessary for neuronal development stabilized by TMD interactions.

Structure and function of the calcium-selective TRP channel TRPV6.

Epithelial calcium channel TRPV6 is a member of the vanilloid subfamily of TRP channels that is permeable to cations and highly selective to Ca2+ ; it shows constitutive activity regulated negatively by Ca2+ and positively by phosphoinositol and cholesterol lipids. In this review, we describe the molecular structure of TRPV6 and discuss how its structural elements define its unique functional properties. High Ca2+ selectivity of TRPV6 originates from the narrow selectivity filter, where Ca2+ ions are directly coordinated by a ring of anionic aspartate side chains. Divalent cations Ca2+ and Ba2+ permeate TRPV6 pore according to the knock-off mechanism, while tight binding of Gd3+ to the aspartate ring blocks the channel and prevents Na+ from permeating the pore. The iris-like channel opening is accompanied by an α-to-π helical transition in the pore-lining transmembrane helix S6. As a result of this transition, the intracellular halves of the S6 helices bend and rotate by about 100 deg, exposing different residues to the channel pore in the open and closed states. Channel opening is also associated with changes in occupancy of the transmembrane domain lipid binding sites. The inhibitor 2-aminoethoxydiphenyl borate (2-APB) binds to TRPV6 in a pocket formed by the cytoplasmic half of the S1-S4 transmembrane helical bundle and shifts open-closed channel equilibrium towards the closed state by outcompeting lipids critical for activation. Ca2+ inhibits TRPV6 via binding to calmodulin (CaM), which mediates Ca2+ -dependent inactivation. The TRPV6-CaM complex exhibits 1:1 stoichiometry; one TRPV6 tetramer binds both CaM lobes, which adopt a distinct head-to-tail arrangement. The CaM C-terminal lobe plugs the channel through a unique cation-π interaction by inserting the side chain of lysine K115 into a tetra-tryptophan cage at the ion channel pore intracellular entrance. Recent studies of TRPV6 structure and function described in this review advance our understanding of the role of this channel in physiology and pathophysiology and inform new therapeutic design.

Structural basis of the transmembrane domain dimerization and rotation in the activation mechanism of the TRKA receptor by nerve growth factor.

Tropomyosin-receptor kinases (TRKs) are essential for the development of the nervous system. The molecular mechanism of TRKA activation by its ligand nerve growth factor (NGF) is still unsolved. Recent results indicate that at endogenous levels most of TRKA is in a monomer-dimer equilibrium and that the binding of NGF induces an increase of the dimeric and oligomeric forms of this receptor. An unsolved issue is the role of the TRKA transmembrane domain (TMD) in the dimerization of TRKA and the structural details of the TMD in the active dimer receptor. Here, we found that the TRKA-TMD can form dimers, identified the structural determinants of the dimer interface in the active receptor, and validated this interface through site-directed mutagenesis together with functional and cell differentiation studies. Using in vivo cross-linking, we found that the extracellular juxtamembrane region is reordered after ligand binding. Replacement of some residues in the juxtamembrane region with cysteine resulted in ligand-independent active dimers and revealed the preferred dimer interface. Moreover, insertion of leucine residues into the TMD helix induced a ligand-independent TRKA activation, suggesting that a rotation of the TMD dimers underlies NGF-induced TRKA activation. Altogether, our findings indicate that the transmembrane and juxtamembrane regions of TRKA play key roles in its dimerization and activation by NGF.

Purification of native CCL7 and its functional interaction with selected chemokine receptors.

Chemokine receptors form a major sub-family of G protein-coupled receptors (GPCRs) and they are involved in a number of cellular and physiological processes related to our immune response and regulation. A better structural understanding of ligand-binding, activation, signaling and regulation of chemokine receptors is very important to design potentially therapeutic interventions for human disorders arising from aberrant chemokine signaling. One of the key limitations in probing the structural details of chemokine receptors is the availability of large amounts of purified, homogenous and fully functional chemokine ligands, and the commercially available products, are not affordable for in-depth structural studies. Moreover, production of uniformly isotope-labeled chemokines, for example, suitable for NMR-based structural investigation, also remains challenging. Here, we have designed a streamlined approach to express and purify the human chemokine CCL7 as well as its 15N-, 15N/13C-, 2H/15N/13C- isotope-labeled derivatives, at milligram levels using E. coli expression system. Purified CCL7 not only maintains a well-folded three-dimensional structure as analyzed using circular dichroism and 1H/15N NMR but it also induces coupling of heterotrimeric G-proteins and ß-arrestins for selected chemokine receptors in cellular system. We compared cAMP response induced by histidine tagged CCL7 and native CCL7 and found that modification of the N-terminus of CCL7 compromises its functionality. Our strategy presented here may be applicable to other chemokines and therefore, provide a potentially generic and cost-effective approach to produce chemokines in large amounts for functional and structural studies.

NMR structure of a full-length single-pass membrane protein NRADD.

Structural study of any single-pass membrane protein is both an important and challenging task. In this report, we present the structure of a neurotrophin receptor-alike death-domain protein. The structure and dynamics of the protein was investigated by conventional nuclear magnetic resonance techniques in the solution of phospholipid bicelles. The receptor contains two folded regions-α-helical transmembrane domain and globular C-terminal death domain with more than 50% of the rest of backbone being disordered. This is the first structure of a full-length single-pass membrane receptor-alike protein solved by the single method.

Structural Basis of p75 Transmembrane Domain Dimerization.

Dimerization of single span transmembrane receptors underlies their mechanism of activation. p75 neurotrophin receptor plays an important role in the nervous system, but the understanding of p75 activation mechanism is still incomplete. The transmembrane (TM) domain of p75 stabilizes the receptor dimers through a disulfide bond, essential for the NGF signaling. Here we solved by NMR the three-dimensional structure of the p75-TM-WT and the functionally inactive p75-TM-C257A dimers. Upon reconstitution in lipid micelles, p75-TM-WT forms the disulfide-linked dimers spontaneously. Under reducing conditions, p75-TM-WT is in a monomer-dimer equilibrium with the Cys(257) residue located on the dimer interface. In contrast, p75-TM-C257A forms dimers through the AXXXG motif on the opposite face of the α-helix. Biochemical and cross-linking experiments indicate that AXXXG motif is not on the dimer interface of p75-TM-WT, suggesting that the conformation of p75-TM-C257A may be not functionally relevant. However, rather than mediating p75 homodimerization, mutagenesis of the AXXXG motif reveals its functional role in the regulated intramembrane proteolysis of p75 catalyzed by the γ-secretase complex. Our structural data provide an insight into the key role of the Cys(257) in stabilization of the weak transmembrane dimer in a conformation required for the NGF signaling.

Structure of purotoxin-2 from wolf spider: modular design and membrane-assisted mode of action in arachnid toxins.

Traditionally, arachnid venoms are known to contain two particularly important groups of peptide toxins. One is disulfide-rich neurotoxins with a predominance of ß-structure that specifically target protein receptors in neurons or muscle cells. The other is linear cationic cytotoxins that form amphiphilic α-helices and exhibit rather non-specific membrane-damaging activity. In the present paper, we describe the first 3D structure of a modular arachnid toxin, purotoxin-2 (PT2) from the wolf spider Alopecosa marikovskyi (Lycosidae), studied by NMR spectroscopy. PT2 is composed of an N-terminal inhibitor cystine knot (ICK, or knottin) ß-structural domain and a C-terminal linear cationic domain. In aqueous solution, the C-terminal fragment is hyper-flexible, whereas the knottin domain is very rigid. In membrane-mimicking environment, the C-terminal domain assumes a stable amphipathic α-helix. This helix effectively tethers the toxin to membranes and serves as a membrane-access and membrane-anchoring device. Sequence analysis reveals that the knottin + α-helix architecture is quite widespread among arachnid toxins, and PT2 is therefore the founding member of a large family of polypeptides with similar structure motifs. Toxins from this family target different membrane receptors such as P2X in the case of PT2 and calcium channels, but their mechanism of action through membrane access may be strikingly similar.

Cell-free expression of the APP transmembrane fragments with Alzheimer's disease mutations using algal amino acid mixture for structural NMR studies.

Structural investigations need ready supply of the isotope labeled proteins with inserted mutations n the quantities sufficient for the heteronuclear NMR. Though cell-free expression system has been widely used in the past years, high startup cost and complex compound composition prevent many researches from the developing this technique, especially for membrane protein production. Here we demonstrate the utility of a robust, cost-optimized cell-free expression technique for production of the physiologically important transmembrane fragment of amyloid precursor protein, APP686-726, containing Alzheimer's disease mutations in the juxtamembrane (E693G, Arctic form) and the transmembrane parts (V717G, London form, or L723P, Australian form). The protein cost was optimized by varying the FM/RM ratio as well as the amino acid concentration. We obtained the wild-type and mutant transmembrane fragments in the pellet mode of continuous exchange cell-free system consuming only commercial algal mixture of the (13)C,(15)N-labeled amino acids. Scaling up analytical tests, we achieved milligram quantity yields of isotope labeled wild-type and mutant APP686-726 for structural studies by high resolution NMR spectroscopy in membrane mimicking environment. The described approach has from 5 to 23-fold cost advantage over the bacterial expression methods described earlier and 1.5 times exceeds our previous result obtained with the longer APP671-726WT fragment.

TRPV3 activation by different agonists accompanied by lipid dissociation from the vanilloid site.

TRPV3 represents both temperature- and ligand-activated transient receptor potential (TRP) channel. Physiologically relevant opening of TRPV3 channels by heat has been captured structurally, while opening by agonists has only been observed in structures of mutant channels. Here, we present cryo-EM structures that illuminate opening and inactivation of wild-type human TRPV3 in response to binding of two types of agonists: either the natural cannabinoid tetrahydrocannabivarin (THCV) or synthetic agonist 2-aminoethoxydiphenylborane (2-APB). We found that THCV binds to the vanilloid site, while 2-APB binds to the S1-S4 base and ARD-TMD linker sites. Despite binding to distally located sites, both agonists induce similar pore opening and cause dissociation of a lipid that occupies the vanilloid site in their absence. Our results uncover different but converging allosteric pathways through which small-molecule agonists activate TRPV3 and provide a framework for drug design and understanding the role of lipids in ion channel function.

Dynamic molecular portraits of ion-conducting pores characterize functional states of TRPV channels.

Structural biology is solving an ever-increasing number of snapshots of ion channel conformational ensembles. Deciphering ion channel mechanisms, however, requires understanding the ensemble dynamics beyond the static structures. Here, we present a molecular modeling-based approach characterizing the ion channel structural intermediates, or their "dynamic molecular portraits", by assessing water and ion conductivity along with the detailed evaluation of pore hydrophobicity and residue packing. We illustrate the power of this approach by analyzing structures of few vanilloid-subfamily transient receptor potential (TRPV) channels. Based on the pore architecture, there are three major states that are common for TRPVs, which we call α-closed, π-closed, and π-open. We show that the pore hydrophobicity and residue packing for the open state is most favorable for the pore conductance. On the contrary, the α-closed state is the most hydrophobic and always non-conducting. Our approach can also be used for structural and functional classification of ion channels.

Structural basis of selective TRPM7 inhibition by the anticancer agent CCT128930.

TRP channels are implicated in various diseases, but high structural similarity between them makes selective pharmacological modulation challenging. Here, we study the molecular mechanism underlying specific inhibition of the TRPM7 channel, which is essential for cancer cell proliferation, by the anticancer agent CCT128930 (CCT). Using cryo-EM, functional analysis, and MD simulations, we show that CCT binds to a vanilloid-like (VL) site, stabilizing TRPM7 in the closed non-conducting state. Similar to other allosteric inhibitors of TRPM7, NS8593 and VER155008, binding of CCT is accompanied by displacement of a lipid that resides in the VL site in the apo condition. Moreover, we demonstrate the principal role of several residues in the VL site enabling CCT to inhibit TRPM7 without impacting the homologous TRPM6 channel. Hence, our results uncover the central role of the VL site for the selective interaction of TRPM7 with small molecules that can be explored in future drug design.

Structure-function analyses of human TRPV6 ancestral and derived haplotypes.

TRPV6 is a Ca2+ selective channel that mediates calcium uptake in the gut and contributes to the development and progression of human cancers. TRPV6 is represented by the ancestral and derived haplotypes that differ by three non-synonymous polymorphisms, located in the N-terminal ankyrin repeat domain (C157R), S1-S2 extracellular loop (M378V), and C-terminus (M681T). The ancestral and derived haplotypes were proposed to serve as genomic factors causing a different outcome for cancer patients of African ancestry. We solved cryoelectron microscopy (cryo-EM) structures of ancestral and derived TRPV6 in the open and calmodulin (CaM)-bound inactivated states. Neither state shows substantial structural differences caused by the non-synonymous polymorphisms. Functional properties assessed by electrophysiological recordings and Ca2+ uptake measurements, and water and ion permeation evaluated by molecular modeling also appear similar between the haplotypes. Therefore, ancestral and derived TRPV6 have similar structure and function, implying that other factors are responsible for the differences in susceptibility to cancer.

Structure of human TRPV4 in complex with GTPase RhoA.

Transient receptor potential (TRP) channel TRPV4 is a polymodal cellular sensor that responds to moderate heat, cell swelling, shear stress, and small-molecule ligands. It is involved in thermogenesis, regulation of vascular tone, bone homeostasis, renal and pulmonary functions. TRPV4 is implicated in neuromuscular and skeletal disorders, pulmonary edema, and cancers, and represents an important drug target. The cytoskeletal remodeling GTPase RhoA has been shown to suppress TRPV4 activity. Here, we present a structure of the human TRPV4-RhoA complex that shows RhoA interaction with the membrane-facing surface of the TRPV4 ankyrin repeat domains. The contact interface reveals residues that are mutated in neuropathies, providing an insight into the disease pathogenesis. We also identify the binding sites of the TRPV4 agonist 4α-PDD and the inhibitor HC-067047 at the base of the S1-S4 bundle, and show that agonist binding leads to pore opening, while channel inhibition involves a π-to-α transition in the pore-forming helix S6. Our structures elucidate the interaction interface between hTRPV4 and RhoA, as well as residues at this interface that are involved in TRPV4 disease-causing mutations. They shed light on TRPV4 activation and inhibition and provide a template for the design of future therapeutics for treatment of TRPV4-related diseases.

Human TRPV1 structure and inhibition by the analgesic SB-366791.

Pain therapy has remained conceptually stagnant since the opioid crisis, which highlighted the dangers of treating pain with opioids. An alternative addiction-free strategy to conventional painkiller-based treatment is targeting receptors at the origin of the pain pathway, such as transient receptor potential (TRP) ion channels. Thus, a founding member of the vanilloid subfamily of TRP channels, TRPV1, represents one of the most sought-after pain therapy targets. The need for selective TRPV1 inhibitors extends beyond pain treatment, to other diseases associated with this channel, including psychiatric disorders. Here we report the cryo-electron microscopy structures of human TRPV1 in the apo state and in complex with the TRPV1-specific nanomolar-affinity analgesic antagonist SB-366791. SB-366791 binds to the vanilloid site and acts as an allosteric hTRPV1 inhibitor. SB-366791 binding site is supported by mutagenesis combined with electrophysiological recordings and can be further explored to design new drugs targeting TRPV1 in disease conditions.

Structural mechanisms of TRPM7 activation and inhibition.

The transient receptor potential channel TRPM7 is a master regulator of the organismal balance of divalent cations that plays an essential role in embryonic development, immune responses, cell mobility, proliferation, and differentiation. TRPM7 is implicated in neuronal and cardiovascular disorders, tumor progression and has emerged as a new drug target. Here we use cryo-EM, functional analysis, and molecular dynamics simulations to uncover two distinct structural mechanisms of TRPM7 activation by a gain-of-function mutation and by the agonist naltriben, which show different conformational dynamics and domain involvement. We identify a binding site for highly potent and selective inhibitors and show that they act by stabilizing the TRPM7 closed state. The discovered structural mechanisms provide foundations for understanding the molecular basis of TRPM7 channelopathies and drug development.

Structural mechanism of human oncochannel TRPV6 inhibition by the natural phytoestrogen genistein.

Calcium-selective oncochannel TRPV6 is the major driver of cell proliferation in human cancers. While significant effort has been invested in the development of synthetic TRPV6 inhibitors, natural channel blockers have been largely neglected. Here we report the structure of human TRPV6 in complex with the plant-derived phytoestrogen genistein, extracted from Styphnolobium japonicum, that was shown to inhibit cell invasion and metastasis in cancer clinical trials. Despite the pharmacological value, the molecular mechanism of TRPV6 inhibition by genistein has remained enigmatic. We use cryo-EM combined with electrophysiology, calcium imaging, mutagenesis, and molecular dynamics simulations to show that genistein binds in the intracellular half of the TRPV6 pore and acts as an ion channel blocker and gating modifier. Genistein binding to the open channel causes pore closure and a two-fold symmetrical conformational rearrangement in the S4-S5 and S6-TRP helix regions. The unprecedented mechanism of TRPV6 inhibition by genistein uncovers new possibilities in structure-based drug design.

Molecular pathway and structural mechanism of human oncochannel TRPV6 inhibition by the phytocannabinoid tetrahydrocannabivarin.

The calcium-selective oncochannel TRPV6 is an important driver of cell proliferation in human cancers. Despite increasing interest of pharmacological research in developing synthetic inhibitors of TRPV6, natural compounds acting at this channel have been largely neglected. On the other hand, pharmacokinetics of natural small-molecule antagonists optimized by nature throughout evolution endows these compounds with a medicinal potential to serve as potent and safe next-generation anti-cancer drugs. Here we report the structure of human TRPV6 in complex with tetrahydrocannabivarin (THCV), a natural cannabinoid inhibitor extracted from Cannabis sativa. We use cryo-electron microscopy combined with electrophysiology, calcium imaging, mutagenesis, and molecular dynamics simulations to identify THCV binding sites in the portals that connect the membrane environment surrounding the protein to the central cavity of the channel pore and to characterize the allosteric mechanism of TRPV6 inhibition. We also propose the molecular pathway taken by THCV to reach its binding site. Our study provides a foundation for the development of new TRPV6-targeting drugs.

Structural snapshots of the mechanism of TRPV2 channel activation by small-molecule agonists.

Transient receptor potential (TRP) channels are polymodal sensors that play critical roles in various physiological processes in living organisms. These cation-permeable channels respond to a variety of physical and chemical stimuli, including cold and hot temperatures, acidic pH, and mechanical stress, often determining a sensory frontier of defense against hostile environments. Vanilloid (V) subfamily is the most studied category of TRP channels that includes six closely related members: highly calcium-selective TRPV5-6 and non-selective TRPV1-4. A remarkable feature of TRPV1-4 is their ability to sense heat, which makes them temperature-sensitive TRP channels or thermo-TRPs. TRPV channels are associated with a multitude of human diseases, including cancers, chronic pain, cardiovascular, neurological and nociceptive disorders. Despite the great clinical interest, pharmacology of TRPV channels remains largely undeveloped because of insufficient knowledge about the mechanisms of their regulation. For instance, activation of TRPV channels by small molecules or heat remains poorly understood. Numerous identified TRPV channel agonists, while effective in physiological experiments, appear limited in their ability to act in the conditions of structural biology experiments. In this regard, the recent study by Pumroy et al. [1] makes a significant contribution towards our understanding of TRPV2 structural dynamics that leads to opening of this channel in physiological conditions.