Structure-based prediction of T cell receptor recognition of unseen epitopes using TCRen.

T cell receptor (TCR) recognition of foreign peptides presented by major histocompatibility complex protein is a major event in triggering the adaptive immune response to pathogens or cancer. The prediction of TCR-peptide interactions has great importance for therapy of cancer as well as infectious and autoimmune diseases but remains a major challenge, particularly for novel (unseen) peptide epitopes. Here we present TCRen, a structure-based method for ranking candidate unseen epitopes for a given TCR. The first stage of the TCRen pipeline is modeling of the TCR-peptide-major histocompatibility complex structure. Then a TCR-peptide residue contact map is extracted from this structure and used to rank all candidate epitopes on the basis of an interaction score with the target TCR. Scoring is performed using an energy potential derived from the statistics of TCR-peptide contact preferences in existing crystal structures. We show that TCRen has high performance in discriminating cognate versus unrelated peptides and can facilitate the identification of cancer neoepitopes recognized by tumor-infiltrating lymphocytes.

Inconspicuous Yet Indispensable: The Coronavirus Spike Transmembrane Domain.

Membrane-spanning portions of proteins' polypeptide chains are commonly known as their transmembrane domains (TMDs). The structural organisation and dynamic behaviour of TMDs from proteins of various families, be that receptors, ion channels, enzymes etc., have been under scrutiny on the part of the scientific community for the last few decades. The reason for such attention is that, apart from their obvious role as an "anchor" in ensuring the correct orientation of the protein's extra-membrane domains (in most cases functionally important), TMDs often actively and directly contribute to the operation of "the protein machine". They are capable of transmitting signals across the membrane, interacting with adjacent TMDs and membrane-proximal domains, as well as with various ligands, etc. Structural data on TMD arrangement are still fragmentary at best due to their complex molecular organisation as, most commonly, dynamic oligomers, as well as due to the challenges related to experimental studies thereof. Inter alia, this is especially true for viral fusion proteins, which have been the focus of numerous studies for quite some time, but have provoked unprecedented interest in view of the SARS-CoV-2 pandemic. However, despite numerous structure-centred studies of the spike (S) protein effectuating target cell entry in coronaviruses, structural data on the TMD as part of the entire spike protein are still incomplete, whereas this segment is known to be crucial to the spike's fusogenic activity. Therefore, in attempting to bring together currently available data on the structure and dynamics of spike proteins' TMDs, the present review aims to tackle a highly pertinent task and contribute to a better understanding of the molecular mechanisms underlying virus-mediated fusion, also offering a rationale for the design of novel efficacious methods for the treatment of infectious diseases caused by SARS-CoV-2 and related viruses.

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 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.

On a mechanistic impact of transmembrane tetramerization in the pathological activation of RTKs.

Constitutive activation of receptor tyrosine kinases (RTKs) via different mutations has a strong impact on the development of severe human disorders, including cancer. Here we propose a putative activation scenario of RTKs, whereby transmembrane (TM) mutations can also promote higher-order oligomerization of the receptors that leads to the subsequent ligand-free activation. We illustrate this scenario using a computational modelling framework comprising sequence-based structure prediction and all-atom 1 µs molecular dynamics (MD) simulations in a lipid membrane for a previously characterised oncogenic TM mutation V536E in platelet-derived growth factor receptor alpha (PDGFRA). We show that in the course of MD simulations the mutant TM tetramer retains stable and compact configuration strengthened by tight protein-protein interactions, while the wild type TM tetramer demonstrates looser packing and a tendency to dissociate. Moreover, the mutation affects the characteristic motions of mutated TM helical segments by introducing additional non-covalent crosslinks in the middle of the TM tetramer, which operate as mechanical hinges. This leads to dynamic decoupling of the C-termini from the rigidified N-terminal parts and facilitates more pronounced possible displacement between the C-termini of the mutant TM helical regions that can provide more freedom for mutual rearrangement of the kinase domains located downstream. Our results for the V536E mutation in the context of PDGFRA TM tetramer allow for the possibility that the effect of oncogenic TM mutations can go beyond alternating the structure and dynamics of TM dimeric states and might also promote the formation of higher-order oligomers directly contributing to ligand-independent signalling effectuated by PDGFRA and other RTKs.

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.

Artificial pore blocker acts specifically on voltage-gated potassium channel isoform KV1.6.

Among voltage-gated potassium channel (KV) isoforms, KV1.6 is one of the most widespread in the nervous system. However, there are little data concerning its physiological significance, in part due to the scarcity of specific ligands. The known high-affinity ligands of KV1.6 lack selectivity, and conversely, its selective ligands show low affinity. Here, we present a designer peptide with both high affinity and selectivity to KV1.6. Previously, we have demonstrated that KV isoform-selective peptides can be constructed based on the simplistic α-hairpinin scaffold, and we obtained a number of artificial Tk-hefu peptides showing selective blockage of KV1.3 in the submicromolar range. We have now proposed amino acid substitutions to enhance their activity. As a result, we have been able to produce Tk-hefu-11 that shows an EC50 of ≈70 nM against KV1.3. Quite surprisingly, Tk-hefu-11 turns out to block KV1.6 with even higher potency, presenting an EC50 of ≈10 nM. Furthermore, we have solved the peptide structure and used molecular dynamics to investigate the determinants of selective interactions between artificial α-hairpinins and KV channels to explain the dramatic increase in KV1.6 affinity. Since KV1.3 is not highly expressed in the nervous system, we hope that Tk-hefu-11 will be useful in studies of KV1.6 and its functions.

Variability in the Spatial Structure of the Central Loop in Cobra Cytotoxins Revealed by X-ray Analysis and Molecular Modeling.

Cobra cytotoxins (CTs) belong to the three-fingered protein family and possess membrane activity. Here, we studied cytotoxin 13 from Naja naja cobra venom (CT13Nn). For the first time, a spatial model of CT13Nn with both "water" and "membrane" conformations of the central loop (loop-2) were determined by X-ray crystallography. The "water" conformation of the loop was frequently observed. It was similar to the structure of loop-2 of numerous CTs, determined by either NMR spectroscopy in aqueous solution, or the X-ray method. The "membrane" conformation is rare one and, to date has only been observed by NMR for a single cytotoxin 1 from N. oxiana (CT1No) in detergent micelle. Both CT13Nn and CT1No are S-type CTs. Membrane-binding of these CTs probably involves an additional step-the conformational transformation of the loop-2. To confirm this suggestion, we conducted molecular dynamics simulations of both CT1No and CT13Nn in the Highly Mimetic Membrane Model of palmitoiloleoylphosphatidylglycerol, starting with their "water" NMR models. We found that the both toxins transform their "water" conformation of loop-2 into the "membrane" one during the insertion process. This supports the hypothesis that the S-type CTs, unlike their P-type counterparts, require conformational adaptation of loop-2 during interaction with lipid membranes.

Small Amphiphilic Peptides: Activity Against a Broad Range of Drug-Resistant Bacteria and Structural Insight into Membranolytic Properties.

We report the synthesis and antibacterial activities of a series of amphiphilic membrane-active peptides composed, in part, of various nongenetically coded hydrophobic amino acids. The lead cyclic peptides, 8C and 9C, showed broad-spectrum activity against drug-resistant Gram-positive (minimum inhibitory concentration (MIC) = 1.5-6.2 µg/mL) and Gram-negative (MIC = 12.5-25 µg/mL) bacteria. The cytotoxicity study showed the predominant lethal action of the peptides against bacteria as compared with mammalian cells. A plasma stability study revealed approximately 2-fold higher stability of lead cyclic peptides as compared to their linear counterparts after 24 h of incubation. A calcein dye leakage experiment revealed the membranolytic effect of the cyclic peptides. Nuclear magnetic resonance spectroscopy and molecular dynamics simulation studies of the interaction of the peptides with the phospholipid bilayer provided a solid structural basis to explain the membranolytic action of the peptides with atomistic details. These results highlight the potential of newly designed amphiphilic peptides as the next generation of peptide-based antibiotics.

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.

Stepwise Insertion of Cobra Cardiotoxin CT2 into a Lipid Bilayer Occurs as an Interplay of Protein and Membrane "Dynamic Molecular Portraits".

For many peripheral membrane-binding polypeptides(MBPs), especially ß-structural ones, the precise molecular mechanisms of membrane insertion remain unclear. In most cases, only the terminal water-soluble and membrane-bound states have been elucidated, whereas potential functionally important intermediate stages are still not understood in sufficient detail. In this study, we present one of the first successful attempts to describe step-by-step embedding of the MBP cardiotoxin 2 (CT2) from cobra Naja oxiana venom into a lipid bilayer at the atomistic level. CT2 possesses a highly conservative and rigid ß-structured three-finger fold shared by many other exogenous and endogenous proteins performing a wide variety of functions. The incorporation of CT2 into the lipid bilayer was analyzed via a 2 µs all-atom molecular dynamics (MD) simulation without restraints. This process was shown to occur over a number of distinct steps, while the geometry of initial membrane attachment drastically differs from that of the final equilibrated state. In the latter one, the hydrophobic platform ("bottom") formed by the tips of the three loops is deeply buried into the lipid bilayer. This agrees well with the NMR data obtained earlier for CT2 in detergent micelles. However, the bottom is too bulky to insert itself into the membrane at once. Instead, the gradual immersion of CT2 initiated by the loop-1 was observed. This initial binding stage was also demonstrated in a series of MD runs with varying starting orientations of the toxin with respect to the bilayer surface. Apart from the nonspecific long-range electrostatic attraction and hydrophobic match/mismatch factor, several specific lipid-binding sites were identified in CT2. They were shown to promote membrane insertion by engaging in strong interactions with lipid head groups, fine-tuning the toxin-membrane accommodation. We therefore propose that the toxin insertion relies on the interplay of nonspecific and specific interactions, which are determined by the "dynamic molecular portraits" of the two players, the protein and the membrane. The proposed model does not require protein oligomerization for membrane insertion and can be further employed to design MBPs with predetermined properties with regard to particular membrane targets.

Transmembrane Peptides as Inhibitors of Protein-Protein Interactions: An Efficient Strategy to Target Cancer Cells?

Cellular functions are regulated by extracellular signals such as hormones, neurotransmitters, matrix ligands, and other chemical or physical stimuli. Ligand binding on its transmembrane receptor induced cell signaling and the recruitment of several interacting partners to the plasma membrane. Nowadays, it is well-established that the transmembrane domain is not only an anchor of these receptors to the membrane, but it also plays a key role in receptor dimerization and activation. Indeed, interactions between transmembrane helices are associated with specific biological activity of the proteins as cell migration, proliferation, or differentiation. Overexpression or constitutive dimerization (due notably to mutations) of these transmembrane receptors are involved in several physiopathological contexts as cancers. The transmembrane domain of tyrosine kinase receptors as ErbB family proteins (implicated in several cancers as HER2 in breast cancer) or other receptors as Neuropilins has been described these last years as a target to inhibit their dimerization/activation using several strategies. In this review, we will focus on the strategy which consists in using peptides to disturb in a specific manner the interactions between transmembrane domains and the signaling pathways (induced by ligand binding) of these receptors involved in cancer. This approach can be extended to inhibit other transmembrane protein dimerization as neuraminidase-1 (the catalytic subunit of elastin receptor complex), Discoidin Domain Receptor 1 (a tyrosine kinase receptor activated by type I collagen) or G-protein coupled receptors (GPCRs) which are involved in cancer processes.

Complex approach for analysis of snake venom α-neurotoxins binding to HAP, the high-affinity peptide.

Snake venom α-neurotoxins, invaluable pharmacological tools, bind with high affinity to distinct subtypes of nicotinic acetylcholine receptor. The combinatorial high-affinity peptide (HAP), homologous to the C-loop of α1 and α7 nAChR subunits, binds biotinylated α-bungarotoxin (αBgt) with nanomolar affinity and might be a protection against snake-bites. Since there are no data on HAP interaction with other toxins, we checked its binding of α-cobratoxin (αCtx), similar to αBgt in action on nAChRs. Using radioiodinated αBgt, we confirmed a high affinity of HAP for αBgt, the complex formation is supported by mass spectrometry and gel chromatography, but only weak binding was registered with αCtx. A combination of protein intrinsic fluorescence measurements with the principal component analysis of the spectra allowed us to measure the HAP-αBgt binding constant directly (29 nM). These methods also confirmed weak HAP interaction with αCtx (>10000 nM). We attempted to enhance it by modification of HAP structure relying on the known structures of α-neurotoxins with various targets and applying molecular dynamics. A series of HAP analogues have been synthesized, HAP[L9E] analogue being considerably more potent than HAP in αCtx binding (7000 nM). The proposed combination of experimental and computational approaches appears promising for analysis of various peptide-protein interactions.

Transmembrane Peptides as a New Strategy to Inhibit Neuraminidase-1 Activation.

Sialidases, or neuraminidases, are involved in several human disorders such as neurodegenerative, infectious and cardiovascular diseases, and cancers. Accumulative data have shown that inhibition of neuraminidases, such as NEU1 sialidase, may be a promising pharmacological target, and selective inhibitors of NEU1 are therefore needed to better understand the biological functions of this sialidase. In the present study, we designed interfering peptides (IntPep) that target a transmembrane dimerization interface previously identified in human NEU1 that controls its membrane dimerization and sialidase activity. Two complementary strategies were used to deliver the IntPep into cells, either flanked to a TAT sequence or non-tagged for solubilization in detergent micelles. Combined with molecular dynamics simulations and heteronuclear nuclear magnetic resonance (NMR) studies in membrane-mimicking environments, our results show that these IntPep are able to interact with the dimerization interface of human NEU1, to disrupt membrane NEU1 dimerization and to strongly decrease its sialidase activity at the plasma membrane. In conclusion, we report here new selective inhibitors of human NEU1 of strong interest to elucidate the biological functions of this sialidase.

Antibacterial activity of cardiotoxin-like basic polypeptide from cobra venom.

Antibacterial activity of the three-finger toxins from cobra venom, including cytotoxin 3 from N. kaouthia, cardiotoxin-like basic polypeptide A5 from N. naja (CLBP), and alpha-neurotoxin from N. oxiana venom, was investigated. All toxins failed to influence Gram-negative bacteria. The most pronounced activity against Bacillus subtilis was demonstrated by CLBP. The latter is ascribed to the presence of additional Lys-residues within the membrane-binding motif of this toxin.

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.

Kalium 2.0, a comprehensive database of polypeptide ligands of potassium channels.

Potassium channels are the most diverse group of ion channels in humans. They take vital parts in numerous physiological processes and their malfunction gives rise to a range of pathologies. In addition to small molecules, there is a wide selection of several hundred polypeptide ligands binding to potassium channels, the majority of which have been isolated from animal venoms. Until recently, only scorpion toxins received focused attention being systematically assembled in the manually curated Kalium database, but there is a diversity of well-characterized potassium channel ligands originating from other sources. To address this issue, here we present the updated and improved Kalium 2.0 that covers virtually all known polypeptide ligands of potassium channels and reviews all available pharmacological data. In addition to an expansion, we have introduced several new features to the database including posttranslational modification annotation, indication of ligand mode of action, BLAST search, and possibility of data export.

Atomistic mechanism of the constitutive activation of PDGFRA via its transmembrane domain.

Single-point mutations in the transmembrane (TM) region of receptor tyrosine kinases (RTKs) can lead to abnormal ligand-independent activation. We use a combination of computational modeling, NMR spectroscopy and cell experiments to analyze in detail the mechanism of how TM domains contribute to the activation of wild-type (WT) PDGFRA and its oncogenic V536E mutant. Using a computational framework, we scan all positions in PDGFRA TM helix for identification of potential functional mutations for the WT and the mutant and reveal the relationship between the receptor activity and TM dimerization via different interfaces. This strategy also allows us design a novel activating mutation in the WT (I537D) and a compensatory mutation in the V536E background eliminating its constitutive activity (S541G). We show both computationally and experimentally that single-point mutations in the TM region reshape the TM dimer ensemble and delineate the structural and dynamic determinants of spontaneous activation of PDGFRA via its TM domain. Our atomistic picture of the coupling between TM dimerization and PDGFRA activation corroborates the data obtained for other RTKs and provides a foundation for developing novel modulators of the pathological activity of PDGFRA.

The role of hydrophobic /hydrophilic balance in the activity of structurally flexible vs. rigid cytolytic polypeptides and analogs developed on their basis.

INTRODUCTION: Being important representatives of various proteomes, membrane-active cationic peptides (CPs) are attractive objects as lead compounds in the design of new antibacterial, anticancer, antifungal, and antiviral molecules. Numerous CPs are found in insect and snake venoms, where many of them reveal cytolytic properties. Due to advances in omics technologies, the number of such peptides is growing dramatically. Areas covered: To understand structure-function relationships for CPs in a living cell, detailed analysis of their hydrophobic/hydrophilic properties is indispensable. We consider two structural classes of membrane-active CPs: latarcins (Ltc) from spider and cardiotoxins (CTXs) from snake venoms. While the former are void off disulfide bonds and conformationally flexible, the latter are structurally rigid and cross-linked with disulfide bonds. In order to elucidate structure-activity relationships behind their antibacterial, anticancer, and hemolytic effects, the properties of these polypeptides are considered on a side-by-side basis. Expert commentary: An ever-increasing number of venom-derived membrane-active polypeptides require new methods for identification of their functional propensities and sequence-based design of novel pharmacological substances. We address these issues considering a number of the designed peptides, based either on Ltc or CTX sequences. Experimental and computer modeling techniques required for these purposes are delineated.

Improving therapeutic potential of antibacterial spider venom peptides: coarse-grain molecular dynamics guided approach.

AIM: Spider venom is a rich source of antibacterial peptides, whose hemolytic activity is often excessive. METHODOLOGY: How to get rid of it? Using latarcins from Lachesana tarabaevi and oxyopinin Oxt 4a from Oxyopes takobius spider venoms we performed coarse-grained molecular dynamics simulations of these peptides in the presence of lipid bilayers, mimicking erythrocyte membranes. This identified hemolytically active fragments within Oxt 4a and latarcins. Then, we synthesized five 20-residue peptides, containing different parts of the Oxt 4a and latarcin-1 sequence, carrying mutations within the identified regions. CONCLUSION: The antibacterial and hemolytic tests suggested that the three of the synthesized peptides demonstrated substantial decrease in hemolytic activity, retaining, or even exceeding antibacterial potential of the parent peptides.