The scorpion toxin BeKm-1 blocks hERG cardiac potassium channels using an indispensable arginine residue.

BeKm-1 is a peptide toxin from scorpion venom that blocks the pore of the potassium channel hERG (Kv11.1) in the human heart. Although individual protein structures have been resolved, the structure of the complex between hERG and BeKm-1 is unknown. Here, we used molecular dynamics and ensemble docking, guided by previous double-mutant cycle analysis data, to obtain an in silico model of the hERG-BeKm-1 complex. Adding to the previous mutagenesis study of BeKm-1, our model uncovers the key role of residue Arg20, which forms three interactions (a salt bridge and hydrogen bonds) with the channel vestibule simultaneously. Replacement of this residue even by lysine weakens the interactions significantly. In accordance, the recombinantly produced BeKm-1R20K mutant exhibited dramatically decreased activity on hERG. Our model may be useful for future drug design attempts.

Molecular Basis for Mambalgin-2 Interaction with Heterotrimeric α-ENaC/ASIC1a/γ-ENaC Channels in Cancer Cells.

Cancer progression is characterized by microenvironmental acidification. Tumor cells adapt to low environmental pH by activating acid-sensing trimeric ion channels of the DEG/ENaC family. The α-ENaC/ASIC1a/γ-ENaC heterotrimeric channel is a tumor-specific acid-sensing channel, and its targeting can be considered a new strategy for cancer therapy. Mambalgin-2 from the Dendroaspis polylepis venom inhibits the α-ENaC/ASIC1a/γ-ENaC heterotrimer more effectively than the homotrimeric ASIC1a channel, initially proposed as the target of mambalgin-2. Although the molecular basis of such mambalgin selectivity remained unclear. Here, we built the models of the complexes of mambalgin-2 with the α-ENaC/ASIC1a/γ-ENaC and ASIC1a channels, performed MD and predicted the difference in the binding modes. The importance of the 'head' loop region of mambalgin-2 for the interaction with the hetero-, but not with the homotrimeric channel was confirmed by site-directed mutagenesis and electrophysiology. A new mode of allosteric regulation of the ENaC channels by linking the thumb domain of the ASIC1a subunit with the palm domain of the γ-ENaC subunit was proposed. The data obtained provide new insights into the regulation of various types of acid-sensing ion channels and the development of new strategies for cancer treatment.

Fighting Celiac Disease: Improvement of pH Stability of Cathepsin L In Vitro by Computational Design.

Roughly 1% of the global population is susceptible to celiac disease (CD)-inheritable autoimmune inflammation of the small intestine caused by intolerance to gliadin proteins present in wheat, rye, and barley grains, and called gluten in wheat. Classical treatment is a life-long gluten-free diet, which is constraining and costly. An alternative approach is based upon the development and oral reception of effective peptidases that degrade in the stomach immunogenic proline- and glutamine-rich gliadin peptides, which are the cause of the severe reaction in the intestine. In previous research, we have established that the major digestive peptidase of an insect Tribolium castaneum-cathepsin L-hydrolyzes immunogenic prolamins after Gln residues but is unstable in the extremely acidic environment (pH 2-4) of the human stomach and cannot be used as a digestive aid. In this work, using molecular dynamics simulations, we discover the probable cause of the pH instability of cathepsin L-loss of the catalytically competent rotameric state of one of the active site residues, His 275. To "fix" the correct orientation of this residue, we designed a V277A mutant variant, which extends the range of stability of the peptidase in the acidic environment while retaining most of its activity. We suggest this protein as a lead glutenase for the development of oral medical preparation that fights CD and gluten intolerance in susceptible people.

Specific Binding of the α-Component of the Lantibiotic Lichenicidin to the Peptidoglycan Precursor Lipid II Predetermines Its Antimicrobial Activity.

To date, a number of lantibiotics have been shown to use lipid II-a highly conserved peptidoglycan precursor in the cytoplasmic membrane of bacteria-as their molecular target. The α-component (Lchα) of the two-component lantibiotic lichenicidin, previously isolated from the Bacillus licheniformis VK21 strain, seems to contain two putative lipid II binding sites in its N-terminal and C-terminal domains. Using NMR spectroscopy in DPC micelles, we obtained convincing evidence that the C-terminal mersacidin-like site is involved in the interaction with lipid II. These data were confirmed by the MD simulations. The contact area of lipid II includes pyrophosphate and disaccharide residues along with the first isoprene units of bactoprenol. MD also showed the potential for the formation of a stable N-terminal nisin-like complex; however, the conditions necessary for its implementation in vitro remain unknown. Overall, our results clarify the picture of two component lantibiotics mechanism of antimicrobial action.

The Mechanism of Selective Recognition of Lipid Substrate by hDHHC20 Enzyme.

S-acylation is a post-translational linkage of long chain fatty acids to cysteines, playing a key role in normal physiology and disease. In human cells, the reaction is catalyzed by a family of 23 membrane DHHC-acyltransferases (carrying an Asp-His-His-Cys catalytic motif) in two stages: (1) acyl-CoA-mediated autoacylation of the enzyme; and (2) further transfer of the acyl chain to a protein substrate. Despite the availability of a 3D-structure of human acyltransferase (hDHHC20), the molecular aspects of lipid selectivity of DHHC-acyltransferases remain unclear. In this paper, using molecular dynamics (MD) simulations, we studied membrane-bound hDHHC20 right before the acylation by C12-, C14-, C16-, C18-, and C20-CoA substrates. We found that: (1) regardless of the chain length, its terminal methyl group always reaches the "ceiling" of the enzyme's cavity; (2) only for C16, an optimal "reactivity" (assessed by a simple geometric criterion) permits the autoacylation; (3) in MD, some key interactions between an acyl-CoA and a protein differ from those in the reference crystal structure of the C16-CoA-hDHHS20 mutant complex (probably, because this structure corresponds to a non-native dimer). These features of specific recognition of full-size acyl-CoA substrates support our previous hypothesis of "geometric and physicochemical selectivity" derived for simplified acyl-CoA analogues.

Membrane-mediated interaction of non-conventional snake three-finger toxins with nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptor of α7 type (α7-nAChR) presented in the nervous and immune systems and epithelium is a promising therapeutic target for cognitive disfunctions and cancer treatment. Weak toxin from Naja kaouthia venom (WTX) is a non-conventional three-finger neurotoxin, targeting α7-nAChR with weak affinity. There are no data on interaction mode of non-conventional neurotoxins with nAChRs. Using α-bungarotoxin (classical three-finger neurotoxin with high affinity to α7-nAChR), we showed applicability of cryo-EM to study complexes of α7-nAChR extracellular ligand-binding domain (α7-ECD) with toxins. Using cryo-EM structure of the α7-ECD/WTX complex, together with NMR data on membrane active site in the WTX molecule and mutagenesis data, we reconstruct the structure of α7-nAChR/WTX complex in the membrane environment. WTX interacts at the entrance to the orthosteric site located at the receptor intersubunit interface and simultaneously forms the contacts with the membrane surface. WTX interaction mode with α7-nAChR significantly differs from α-bungarotoxin's one, which does not contact the membrane. Our study reveals the important role of the membrane for interaction of non-conventional neurotoxins with the nicotinic receptors.

Orientational Preferences of GPI-Anchored Ly6/uPAR Proteins.

Ly6/uPAR proteins regulate many essential functions in the nervous and immune systems and epithelium. Most of these proteins contain single ß-structural LU domains with three protruding loops and are glycosylphosphatidylinositol (GPI)-anchored to a membrane. The GPI-anchor role is currently poorly studied. Here, we investigated the positional and orientational preferences of six GPI-anchored proteins in the receptor-unbound state by molecular dynamics simulations. Regardless of the linker length between the LU domain and GPI-anchor, the proteins interacted with the membrane by polypeptide parts and N-/O-glycans. Lynx1, Lynx2, Lypd6B, and Ly6H contacted the membrane by the loop regions responsible for interactions with nicotinic acetylcholine receptors, while Lypd6 and CD59 demonstrated unique orientations with accessible receptor-binding sites. Thus, GPI-anchoring does not guarantee an optimal 'pre-orientation' of the LU domain for the receptor interaction.

Conserved Structure and Evolution of DPF Domain of PHF10-The Specific Subunit of PBAF Chromatin Remodeling Complex.

Transcription activation factors and multisubunit coactivator complexes get recruited at specific chromatin sites via protein domains that recognize histone modifications. Single PHDs (plant homeodomains) interact with differentially modified H3 histone tails. Double PHD finger (DPF) domains possess a unique structure different from PHD and are found in six proteins: histone acetyltransferases MOZ and MORF; chromatin remodeling complex BAF (DPF1-3); and chromatin remodeling complex PBAF (PHF10). Among them, PHF10 stands out due to the DPF sequence, structure, and functions. PHF10 is ubiquitously expressed in developing and adult organisms as four isoforms differing in structure (the presence or absence of DPF) and transcription regulation functions. Despite the importance of the DPF domain of PHF10 for transcription activation, its structure remains undetermined. We performed homology modeling of the human PHF10 DPF domain and determined common and distinct features in structure and histone modifications recognition capabilities, which can affect PBAF complex chromatin recruitment. We also traced the evolution of DPF1-3 and PHF10 genes from unicellular to vertebrate organisms. The data reviewed suggest that the DPF domain of PHF10 plays an important role in SWI/SNF-dependent chromatin remodeling during transcription activation.

Potassium channel blocker crafted by α-hairpinin scaffold engineering.

The α-Hairpinins are a family of plant defense peptides with a common fold presenting two short α-helices stabilized by two invariant S-S-bridges. We have shown previously that substitution of just two amino acid residues in a wheat α-hairpinin Tk-AMP-X2 leads to Tk-hefu-2 that features specific affinity to voltage-gated potassium channels KV1.3. Here, we utilize a combined molecular modeling approach based on molecular dynamics simulations and protein surface topography technique to improve the affinity of Tk-hefu-2 to KV1.3 while preserving its specificity. An important advance of this work compared with our previous studies is transition from the analysis of various physicochemical properties of an isolated toxin molecule to its consideration in complex with its target, a membrane-bound ion channel. As a result, a panel of computationally designed Tk-hefu-2 derivatives was synthesized and tested against KV1.3. The most active mutant Tk-hefu-10 showed a half-maximal inhibitory concentration of ∼150 nM being >10 times more active than Tk-hefu-2 and >200 times more active than the original Tk-hefu. We conclude that α-hairpinins provide an attractive disulfide-stabilized scaffold for the rational design of ion channel inhibitors. Furthermore, the success rate can be considerably increased by the proposed "target-based" iterative strategy of molecular design.

Biochemical Basis of Skin Disease Mal de Meleda: SLURP-1 Mutants Differently Affect Keratinocyte Proliferation and Apoptosis.

Mal de Meleda is an autosomal recessive palmoplantar keratoderma associated with mutations in a gene encoding SLURP-1. SLURP-1 controls growth, differentiation, and apoptosis of keratinocytes by interaction with α7-type nicotinic acetylcholine receptors. SLURP-1 has a three-finger structure with a ß-structural core (head) and three prolonged loops (fingers). To determine the role of SLURP-1 mutations, we produced 22 mutant variants of the protein, including those involved in Mal de Meleda pathogenesis. All mutants except R71H, R71P, T52A, R96P, and L98P were produced in the folded form. SLURP-1 reduces the growth of Het-1A keratinocytes; thus, we studied the influence of the mutations on its antiproliferative activity. Mutations in loops I and III led to the protein inactivation, whereas most mutations in loop II increased SLURP-1 antiproliferative activity. Alanine substitutions of R96 and L98 residues located in the protein head resulted in the appearance of additional pro-apoptotic activity. Our results agree with the diversity of Mal de Meleda phenotypes. Using obtained functional data, the SLURP-1/α7 type nicotinic acetylcholine receptor complex was modeled in silico. Our study provides functional and structural information about the role of the SLURP-1 mutations in Mal de Meleda pathogenesis and predicts SLURP-1 variants, which could drive the disease.

Tuning Scorpion Toxin Selectivity: Switching From KV1.1 to KV1.3.

Voltage-gated potassium channels (KVs) perform vital physiological functions and are targets in different disorders ranging from ataxia and arrhythmia to autoimmune diseases. An important issue is the search for and production of selective ligands of these channels. Peptide toxins found in scorpion venom named KTx excel in both potency and selectivity with respect to some potassium channel isoforms, which may present only minute differences in their structure. Despite several decades of research the molecular determinants of KTx selectivity are still poorly understood. Here we analyze MeKTx13-3 (Kalium ID: α-KTx 3.19) from the lesser Asian scorpion Mesobuthus eupeus, a high-affinity KV1.1 blocker (IC50 ~2 nM); it also affects KV1.2 (IC50 ~100 nM), 1.3 (~10 nM) and 1.6 (~60 nM). By constructing computer models of its complex with KV1.1-1.3 channels we identify specific contacts between the toxin and the three isoforms. We then perform mutagenesis to disturb the identified contacts with KV1.1 and 1.2 and produce recombinant MeKTx13-3_AAAR, which differs by four amino acid residues from the parent toxin. As predicted by the modeling, this derivative shows decreased activity on KV1.1 (IC50 ~550 nM) and 1.2 (~200 nM). It also has diminished activity on KV1.6 (~1500 nM) but preserves KV1.3 affinity as measured using the voltage-clamp technique on mammalian channels expressed in Xenopus oocytes. In effect, we convert a selective KV1.1 ligand into a new specific KV1.3 ligand. MeKTx13-3 and its derivatives are attractive tools to study the structure-function relationship in potassium channel blockers.

Sevanol and Its Analogues: Chemical Synthesis, Biological Effects and Molecular Docking.

Among acid-sensing ion channels (ASICs), ASIC1a and ASIC3 subunits are the most widespread and prevalent in physiological and pathophysiological conditions. They participate in synaptic plasticity, learning and memory, as well as the perception of inflammatory and neurological pain, making these channels attractive pharmacological targets. Sevanol, a natural lignan isolated from Thymus armeniacus, inhibits the activity of ASIC1a and ASIC3 isoforms, and has a significant analgesic and anti-inflammatory effect. In this work, we described the efficient chemical synthesis scheme of sevanol and its analogues, which allows us to analyze the structure-activity relationships of the different parts of this molecule. We found that the inhibitory activity of sevanol and its analogues on ASIC1a and ASIC3 channels depends on the number and availability of the carboxyl groups of the molecule. At the structural level, we predicted the presence of a sevanol binding site based on the presence of molecular docking in the central vestibule of the ASIC1a channel. We predicted that this site could also be occupied in part by the FRRF-amide peptide, and the competition assay of sevanol with this peptide confirmed this prediction. The intravenous (i.v.), intranasal (i.n.) and, especially, oral (p.o.) administration of synthetic sevanol in animal models produced significant analgesic and anti-inflammatory effects. Both non-invasive methods of sevanol administration (i.n. and p.o.) showed greater efficacy than the invasive (i.v.) method, thus opening new horizons for medicinal uses of sevanol.

Probing temperature and capsaicin-induced activation of TRPV1 channel via computationally guided point mutations in its pore and TRP domains.

In a recent computational study, we revealed some mechanistic aspects of TRPV1 (transient receptor potential channel 1) thermal activation and gating and proposed a set of probable functionally important residues - "hot spots" that have not been characterized experimentally yet. In this work, we analyzed TRPV1 point mutants G643A, I679A + A680G, and K688G/P combining molecular modeling, biochemistry, and electrophysiology. The substitution G643A reduced maximal conductivity that resulted in a normal response to moderate stimuli, but a relatively weak response to more intensive activation. I679A + A680G channel was severely toxic for oocytes most probably due to abnormally increased basal activity of the channel ("always open" gates). The replacement K688G presumably facilitated movements of TRP domain and disturbed its coupling to the pore, thus leading to spontaneous activation and enhanced desensitization of the channel. Finally, mutation K688P was suggested to impair TRP domain directed movement, and the mutated channel showed ~100-fold less sensitivity to the capsaicin, enhanced desensitization and weaker activation by the heat. Our results provide a better understanding of TRPV1 thermal and capsaicin-induced activation and gating. These observations provide a structural basis for understanding some aspects of TRPV1 channel functioning and depict potentially pathogenic mutations.

Impact of external amino acids on fluorescent protein chromophore biosynthesis revealed by molecular dynamics and mutagenesis studies.

The precise positioning of catalytic amino acids against the substrate in an enzyme active site is a crucial factor in biocatalysis. Biosynthesis of the chromophores of fluorescent proteins (FPs) is an autocatalytic process that must conform to these requirements. Here, we show that, in addition to the internal amino acid residues in the proximity of the chromophore, chromophore biosynthesis is influenced by the remote amino acids exposed on the outer surface of the ß-barrel structure of the FP. It has been shown earlier that chromophore biosynthesis of the red FP from Zoanthus sp. (zoan2RFP) proceeds via an immature green state. At the same time, the green state is the final stage of chromophore biosynthesis of green FP (zoanGFP), which is highly homologous to zoan2RFP. It was also shown that a single N66D substitution in the chromophore-forming sequence of zoanGFP might trigger the synthesis of the red chromophore. However, in this case, the synthesis of the red chromophore is incomplete and occurs only at elevated temperatures. Here, we tried to uncover additional structural determinants that govern the biosynthesis of the red chromophore. A comparison of zoanGFP and zoan2RFP revealed intrabarrel amino acid differences at five positions. Exhaustive substitutions of these five positions in zoanGFP-N66D gave rise to zoanGFPmut with the same intrabarrel amino acid composition as zoan2RFP. zoanGFPmut showed only partial green-to-red chromophore transformation at elevated temperatures. To elucidate the extra factors that can affect red chromophore biosynthesis, we performed comparative molecular dynamics simulations of zoan2RFP and zoanGFPmut. The simulations revealed several external amino acids that might influence the arrangement and flexibility of the chromophore-surrounding amino acid residues in these proteins. Mutagenesis experiments confirmed the crucial role of these residues in red chromophore biosynthesis. The obtained zoanGFPmut2 exhibited complete green-to-red transformation, suggesting that the mutated amino acids exposed on the surface of the ß-barrel contribute to red chromophore biosynthesis.

Protein surface topography as a tool to enhance the selective activity of a potassium channel blocker.

Tk-hefu is an artificial peptide designed based on the α-hairpinin scaffold, which selectively blocks voltage-gated potassium channels Kv1.3. Here we present its spatial structure resolved by NMR spectroscopy and analyze its interaction with channels using computer modeling. We apply protein surface topography to suggest mutations and increase Tk-hefu affinity to the Kv1.3 channel isoform. We redesign the functional surface of Tk-hefu to better match the respective surface of the channel pore vestibule. The resulting peptide Tk-hefu-2 retains Kv1.3 selectivity and displays ∼15 times greater activity compared with Tk-hefu. We verify the mode of Tk-hefu-2 binding to the channel outer vestibule experimentally by site-directed mutagenesis. We argue that scaffold engineering aided by protein surface topography represents a reliable tool for design and optimization of specific ion channel ligands.

Cell-Free Expression of Sodium Channel Domains for Pharmacology Studies. Noncanonical Spider Toxin Binding Site in the Second Voltage-Sensing Domain of Human Nav1.4 Channel.

Voltage-gated sodium (NaV) channels are essential for the normal functioning of cardiovascular, muscular, and nervous systems. These channels have modular organization; the central pore domain allows current flow and provides ion selectivity, whereas four peripherally located voltage-sensing domains (VSDs-I/IV) are needed for voltage-dependent gating. Mutations in the S4 voltage-sensing segments of VSDs in the skeletal muscle channel NaV1.4 trigger leak (gating pore) currents and cause hypokalemic and normokalemic periodic paralyses. Previously, we have shown that the gating modifier toxin Hm-3 from the crab spider Heriaeus melloteei binds to the S3-S4 extracellular loop in VSD-I of NaV1.4 channel and inhibits gating pore currents through the channel with mutations in VSD-I. Here, we report that Hm-3 also inhibits gating pore currents through the same channel with the R675G mutation in VSD-II. To investigate the molecular basis of Hm-3 interaction with VSD-II, we produced the corresponding 554-696 fragment of NaV1.4 in a continuous exchange cell-free expression system based on the Escherichia coli S30 extract. We then performed a combined nuclear magnetic resonance (NMR) and electron paramagnetic resonance spectroscopy study of isolated VSD-II in zwitterionic dodecylphosphocholine/lauryldimethylamine-N-oxide or dodecylphosphocholine micelles. To speed up the assignment of backbone resonances, five selectively 13C,15N-labeled VSD-II samples were produced in accordance with specially calculated combinatorial scheme. This labeling approach provides assignment for ∼50% of the backbone. Obtained NMR and electron paramagnetic resonance data revealed correct secondary structure, quasi-native VSD-II fold, and enhanced ps-ns timescale dynamics in the micelle-solubilized domain. We modeled the structure of the VSD-II/Hm-3 complex by protein-protein docking involving binding surfaces mapped by NMR. Hm-3 binds to VSDs I and II using different modes. In VSD-II, the protruding ß-hairpin of Hm-3 interacts with the S1-S2 extracellular loop, and the complex is stabilized by ionic interactions between the positively charged toxin residue K24 and the negatively charged channel residues E604 or D607. We suggest that Hm-3 binding to these charged groups inhibits voltage sensor transition to the activated state and blocks the depolarization-activated gating pore currents. Our results indicate that spider toxins represent a useful hit for periodic paralyses therapy development and may have multiple structurally different binding sites within one NaV molecule.

KV1.2 channel-specific blocker from Mesobuthus eupeus scorpion venom: Structural basis of selectivity.

Scorpion venom is an unmatched source of selective high-affinity ligands of potassium channels. There is a high demand for such compounds to identify and manipulate the activity of particular channel isoforms. The objective of this study was to obtain and characterize a specific ligand of voltage-gated potassium channel KV1.2. As a result, we report the remarkable selectivity of the peptide MeKTx11-1 (α-KTx 1.16) from Mesobuthus eupeus scorpion venom to this channel isoform. MeKTx11-1 is a high-affinity blocker of KV1.2 (IC50 ∼0.2 nM), while its activity against KV1.1, KV1.3, and KV1.6 is 10 000, 330 and 45 000 fold lower, respectively, as measured using the voltage-clamp technique on mammalian channels expressed in Xenopus oocytes. Two substitutions, G9V and P37S, convert MeKTx11-1 to its natural analog MeKTx11-3 (α-KTx 1.17) having 15 times lower activity and reduced selectivity to KV1.2. We produced MeKTx11-1 and MeKTx11-3 as well as their mutants MeKTx11-1(G9V) and MeKTx11-1(P37S) recombinantly and demonstrated that point mutations provide an intermediate effect on selectivity. Key structural elements that explain MeKTx11-1 specificity were identified by molecular modeling of the toxin-channel complexes. Confirming our molecular modeling predictions, site-directed transfer of these elements from the pore region of KV1.2 to KV1.3 resulted in the enhanced sensitivity of mutant KV1.3 channels to MeKTx11-1. We conclude that MeKTx11-1 may be used as a selective tool in neurobiology.

C-Terminal residues in small potassium channel blockers OdK1 and OSK3 from scorpion venom fine-tune the selectivity.

We report isolation, sequencing, and electrophysiological characterization of OSK3 (α-KTx 8.8 in Kalium and Uniprot databases), a potassium channel blocker from the scorpion Orthochirus scrobiculosus venom. Using the voltage clamp technique, OSK3 was tested on a wide panel of 11 voltage-gated potassium channels expressed in Xenopus oocytes, and was found to potently inhibit Kv1.2 and Kv1.3 with IC50 values of ~331nM and ~503nM, respectively. OdK1 produced by the scorpion Odontobuthus doriae differs by just two C-terminal residues from OSK3, but shows marked preference to Kv1.2. Based on the charybdotoxin-potassium channel complex crystal structure, a model was built to explain the role of the variable residues in OdK1 and OSK3 selectivity.

High-Affinity α-Conotoxin PnIA Analogs Designed on the Basis of the Protein Surface Topography Method.

Despite some success for small molecules, elucidating structure-function relationships for biologically active peptides - the ligands for various targets in the organism - remains a great challenge and calls for the development of novel approaches. Some of us recently proposed the Protein Surface Topography (PST) approach, which benefits from a simplified representation of biomolecules' surface as projection maps, which enables the exposure of the structure-function dependencies. Here, we use PST to uncover the "activity pattern" in α-conotoxins - neuroactive peptides that effectively target nicotinic acetylcholine receptors (nAChRs). PST was applied in order to design several variants of the α-conotoxin PnIA, which were synthesized and thoroughly studied. Among the best was PnIA[R9, L10], which exhibits nanomolar affinity for the α7 nAChR, selectivity and a slow wash-out from this target. Importantly, these mutations could hardly be delineated by "standard" structure-based drug design. The proposed combination of PST with a set of experiments proved very efficient for the rational construction of new bioactive molecules.

Temperature-sensitive gating of TRPV1 channel as probed by atomistic simulations of its trans- and juxtamembrane domains.

Heat-activated transient receptor potential channel TRPV1 is one of the most studied eukaryotic proteins involved in temperature sensation. Upon heating, it exhibits rapid reversible pore gating, which depolarizes neurons and generates action potentials. Underlying molecular details of such effects in the pore region of TRPV1 is of a crucial importance to control temperature responses of the organism. Despite the spatial structure of the channel in both open (O) and closed (C) states is known, microscopic nature of channel gating and mechanism of thermal sensitivity are still poorly understood. In this work, we used unrestrained atomistic molecular dynamics simulations of TRPV1 (without N- and C-terminal cytoplasmic domains) embedded into explicit lipid bilayer in its O- and C-states. We found that the pore domain with its neighboring loops undergoes large temperature-dependent conformational transitions in an asymmetric way, when fragments of only one monomer move with large amplitude, freeing the pore upon heating. Such an asymmetrical gating looks rather biologically relevant because it is faster and more reliable than traditionally proposed "iris-like" symmetric scheme of channel opening. Analysis of structural, dynamic, and hydrophobic organization of the pore domain revealed entropy growth upon TRPV1 gating, which is in line with current concepts of thermal sensitivity.