Structural Analysis and Activity Correlation of Amphiphilic Cyclic Antimicrobial Peptides Derived from the [W4R4] Scaffold.

In our ongoing quest to design effective antimicrobial peptides (AMPs), this study aimed to elucidate the mechanisms governing cyclic amphiphilic AMPs and their interactions with membranes. The objective was to discern the nature of these interactions and understand how peptide sequence and structure influence antimicrobial activity. We introduced modifications into the established cyclic AMP peptide, [W4R4], incorporating an extra aromatic hydrophobic residue (W), a positively charged residue (R), or the unique 2,5-diketopiperazine (DKP). This study systematically explored the structure-activity relationships (SARs) of a series of cyclic peptides derived from the [W4R4] scaffold, including the first synthesis and evaluation of [W4R4(DKP)]. Structural, dynamic, hydrophobic, and membrane-binding properties of four cyclic peptides ([W4R4], [W5R4], [W4R5], [W4R4(DKP)]) were explored using molecular dynamics simulations within a DOPC/DOPG lipid bilayer that mimics the bacterial membrane. The results revealed distinct SARs linking antimicrobial activity to parameters such as conformational plasticity, immersion depth in the bilayer, and population of the membrane binding mode. Notably, [W4R5] exhibited an optimal "activity/binding to the bacterial membrane" pattern. This multidisciplinary approach efficiently decoded finely regulated SAR profiles, laying a foundation for the rational design of novel antimicrobial peptides.

Molecular Dynamics of DHHC20 Acyltransferase Suggests Principles of Lipid and Protein Substrate Selectivity.

Lipid modification of viral proteins with fatty acids of different lengths (S-acylation) is crucial for virus pathogenesis. The reaction is catalyzed by members of the DHHC family and proceeds in two steps: the autoacylation is followed by the acyl chain transfer onto protein substrates. The crystal structure of human DHHC20 (hDHHC20), an enzyme involved in the acylation of S-protein of SARS-CoV-2, revealed that the acyl chain may be inserted into a hydrophobic cavity formed by four transmembrane (TM) α-helices. To test this model, we used molecular dynamics of membrane-embedded hDHHC20 and its mutants either in the absence or presence of various acyl-CoAs. We found that among a range of acyl chain lengths probed only C16 adopts a conformation suitable for hDHHC20 autoacylation. This specificity is altered if the small or bulky residues at the cavity's ceiling are exchanged, e.g., the V185G mutant obtains strong preferences for binding C18. Surprisingly, an unusual hydrophilic ridge was found in TM helix 4 of hDHHC20, and the responsive hydrophilic patch supposedly involved in association was found in the 3D model of the S-protein TM-domain trimer. Finally, the exchange of critical Thr and Ser residues in the spike led to a significant decrease in its S-acylation. Our data allow further development of peptide/lipid-based inhibitors of hDHHC20 that might impede replication of Corona- and other enveloped viruses.

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.

A Uniquely Stable Trimeric Model of SARS-CoV-2 Spike Transmembrane Domain.

Understanding fusion mechanisms employed by SARS-CoV-2 spike protein entails realistic transmembrane domain (TMD) models, while no reliable approaches towards predicting the 3D structure of transmembrane (TM) trimers exist. Here, we propose a comprehensive computational framework to model the spike TMD only based on its primary structure. We performed amino acid sequence pattern matching and compared the molecular hydrophobicity potential (MHP) distribution on the helix surface against TM homotrimers with known 3D structures and selected an appropriate template for homology modeling. We then iteratively built a model of spike TMD, adjusting "dynamic MHP portraits" and residue variability motifs. The stability of this model, with and without palmitoyl modifications downstream of the TMD, and several alternative configurations (including a recent NMR structure), was tested in all-atom molecular dynamics simulations in a POPC bilayer mimicking the viral envelope. Our model demonstrated unique stability under the conditions applied and conforms to known basic principles of TM helix packing. The original computational framework looks promising and could potentially be employed in the construction of 3D models of TM trimers for a wide range of membrane proteins.

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.

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.

Confined Dynamics of Water in Transmembrane Pore of TRPV1 Ion Channel.

Solvation effects play a key role in chemical and biological processes. The microscopic properties of water near molecular surfaces are radically different from those in the bulk. Furthermore, the behavior of water in confined volumes of a nanometer scale, including transmembrane pores of ion channels, is especially nontrivial. Knowledge at the molecular level of structural and dynamic parameters of water in such systems is necessary to understand the mechanisms of ion channels functioning. In this work, the results of molecular dynamics (MD) simulations of water in the pore and selectivity filter domains of TRPV1 (Transient Receptor Potential Vanilloid type 1) membrane channel are considered. These domains represent nanoscale volumes with strongly amphiphilic walls, where physical behavior of water radically differs from that of free hydration (e.g., at protein interfaces) or in the bulk. Inside the pore and filter domains, water reveals a very heterogeneous spatial distribution and unusual dynamics: It forms compact areas localized near polar groups of particular residues. Residence time of water molecules in such areas is at least 1.5 to 3 times larger than that observed for similar groups at the protein surface. Presumably, these water "blobs" play an important role in the functional activity of TRPV1. In particular, they take part in hydration of the hydrophobic TRPV1 pore by localizing up to six waters near the so-called "lower gate" of the channel and reducing by this way the free energy barrier for ion and water transport. Although the channel is formed by four identical protein subunits, which are symmetrically packed in the initial experimental 3D structure, in the course of MD simulations, hydration of the same amino acid residues of individual subunits may differ significantly. This greatly affects the microscopic picture of the distribution of water in the channel and, potentially, the mechanism of its functioning. Therefore, reconstruction of the full picture of TRPV1 channel solvation requires thorough atomistic simulations and analysis. It is important that the naturally occurring porous volumes, like ion-conducting protein domains, reveal much more sophisticated and fine-tuned regulation of solvation than, e.g., artificially designed carbon nanotubes.

Cardiotoxins: Functional Role of Local Conformational Changes.

Cardiotoxins (CTs) from snake venoms are a family of homologous highly basic proteins that have extended hydrophobic patterns on their molecular surfaces. CTs are folded into three ß-structured loops stabilized by four disulfide bridges. Being well-structured in aqueous solution, most of these proteins are membrane-active, although the exact molecular mechanisms of CT-induced cell damage are still poorly understood. To elucidate the structure-function relationships in CTs, a detailed knowledge of their spatial organization and local conformational dynamics is required. Protein domain motions can be either derived from a set of experimental structures or generated via molecular dynamics (MD). At the same time, traditional clustering algorithms in the Cartesian coordinate space often fail to properly take into account the local large-scale dihedral angle transitions that occur in MD simulations. This is because such perturbations are usually offset by changes in the neighboring dihedrals, thus preserving the overall protein fold. States with a "locally perturbed" backbone were found in experimental 3D models of some globular proteins and have been shown to be functionally meaningful. In this work, the possibility of large-scale dihedral angle transitions in the course of long-term MD in explicit water was explored for three CTs with different membrane activities: CT 1, 2 (Naja oxiana) and CT A3 (Naja atra). Analysis of the MD-derived distributions of backbone torsion angles revealed several important common and specific features in the structural/dynamic behavior of these proteins. First, large-amplitude transitions were detected in some residues located in the functionally important loop I region. The K5/L6 pair of residues was found to induce a perturbation of the hydrophobic patterns on the molecular surface of CTs-reversible breaking of a large nonpolar zone ("bottom") into two smaller ones and their subsequent association. Second, the characteristic sizes of these patterns perfectly coincided with the dimensions of the nonpolar zones on the surfaces of model two-component (zwitterionic/anionic) membranes. Taken together with experimental data on the CT-induced leakage of fluorescent dye from such membranes, these results allowed us to formulate a two-stage mechanism of CT-membrane binding. The principal finding of this study is that even local conformational dynamics of CTs can seriously affect their functional activity via a tuning of the membrane binding site - specific "hot spots" (like the K5/L6 pair) in the protein structure.

PREDDIMER: a web server for prediction of transmembrane helical dimers.

SUMMARY: Here we present PREDDIMER, a web tool for prediction of dimer structure of transmembrane (TM) helices. PREDDIMER allows (i) reconstruction of a number of dimer structures for given sequence(s) of TM protein fragments, (ii) ranking and filtering of predicted structures according to respective values of a scoring function, (iii) visualization of predicted 3D dimer structures and (iv) visualization of surface hydrophobicity of TM helices and their contacting (interface) regions represented as 2D maps. RESULTS: We implemented online the original PREDDIMER algorithm and benchmarked the server on 11 TM sequences, whose 3D dimer conformations were obtained previously by nuclear magnetic resonance spectroscopy. In the most of tested cases backbone root-mean-square deviations of closest predicted conformations from the experimental reference are below 3 Å. A randomization test displays good anticorrelation (-0.82) between values of the scoring function and statistical significance of the prediction 'by chance'. Going beyond a single dimer conformation, our web tool predicts an ensemble of possible conformations, which may be useful for explanation of a functioning of bitopic membrane proteins, e.g. receptor tyrosine kinases. AVAILABILITY AND IMPLEMENTATION: PREDDIMER can be accessed for free on the web at http://model.nmr.ru/preddimer/ CONTACT: newant@gmail.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

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.

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.

Kalium 3.0 is a comprehensive depository of natural, artificial, and labeled polypeptides acting on potassium channels.

Here, we introduce the third release of Kalium database (http://kaliumdb.org/), a manually curated comprehensive depository that accumulates data on polypeptide ligands of potassium channels. The major goal of this amplitudinous update is to summarize findings for natural polypeptide ligands of K+ channels, as well as data for the artificial derivatives of these substances obtained over the decades of exploration. We manually analyzed more than 700 original manuscripts and systematized the information on mutagenesis, production of radio- and fluorescently labeled derivatives, and the molecular pharmacology of K+ channel ligands. As a result, data on more than 1200 substances were processed and added enriching the database content fivefold. We also included the electrophysiological data obtained on the understudied and neglected K+ channels including the heteromeric and concatenated channels. We associated target channels in Kalium with corresponding entries in the official database of the International Union of Basic and Clinical Pharmacology. Kalium was supplemented with an adaptive Statistics page, where users are able to obtain actual data output. Several other improvements were introduced, such as a color code to distinguish the range of ligand activity concentrations and advanced tools for filtration and sorting. Kalium is a fully open-access database, crosslinked to other databases of interest. It can be utilized as a convenient resource containing ample up-to-date information about polypeptide ligands of K+ channels.

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.

libxtc: an efficient library for reading XTC-compressed MD trajectory data.

OBJECTIVE: The purpose of this work is to optimize the processing of molecular dynamics (MD) trajectory data obtained for large biomolecular systems. Two popular software tools were chosen as the reference: the tng and the xdrfile libraries. Current implementation of tng algorithms and library is either fast or storage efficient and xdrfile is storage efficient but slow. Our aim was to combine speed and storage efficiency through the xdrfile's code modification. RESULTS: Here we present libxtc, a ready-to-use library for reading MD trajectory files in xtc format. The effectiveness of libxtc is demonstrated for several biomolecular systems of various sizes (~ 2 × 104 to ~ 2 × 105 atoms). In sequential mode, the performance of libxtc is up to 1.8 times higher and 1.4 times lower than xdrfile and tng, respectively. In parallel mode, libxtc is about 3 and 1.3 times faster than xdrfile and tng. At the same time, MD data stored in the xtc format require about 1.3 times less disk space than those treated with the tng algorithm in the fastest reading mode, which is a noticeable saving especially when the MD trajectory is long and the number of atoms is large-this applies to most biologically relevant systems.

Phospholipase A2 way to hydrolysis: Dint formation, hydrophobic mismatch, and lipid exclusion.

Phospholipase A2 (PLA2) exerts a wide range of biological effects and attracts a lot of attention of researchers. Two sites are involved in manifestation of PLA2 enzymatic activity: catalytic site responsible for substrate binding and fatty acid cleavage from the sn-2 position of a glycerophospholipid, and interface binding site (IBS) responsible for the protein binding to lipid membrane. IBS is formed by positively charged and hydrophobic amino acids on the outer surface of the protein molecule. Understanding the mechanism of PLA2 interaction with the lipid membrane is the most challenging step in biochemistry of this enzyme. We used a combination of experimental and computer simulation techniques to clarify molecular details of bee venom PLA2 interaction with lipid bilayers formed by palmitoyloleoylphosphatidylcholine or dipalmitoylphosphatidylcholine. We found that after initial enzyme contact with the membrane, a network of hydrogen bonds was formed. This led to deformation of the interacting leaflet and dint formation. The bilayer response to the deformation depended on its phase state. In a gel-phase bilayer, diffusion of lipids is restricted therefore chain melting occurred in both leaflets of the bilayer. In the case of a fluid-phase bilayer, lateral diffusion is possible, and lipid polar head groups were excluded from the contact area. As a result, the bilayer became thinner and a large hydrophobic area was formed. We assume that relative ability of a bilayer to come through lipid redistribution process defines the rate of initial stages of the catalysis.

Structural mechanism of heat-induced opening of a temperature-sensitive TRP channel.

Numerous physiological functions rely on distinguishing temperature through temperature-sensitive transient receptor potential channels (thermo-TRPs). Although the function of thermo-TRPs has been studied extensively, structural determination of their heat- and cold-activated states has remained a challenge. Here, we present cryo-EM structures of the nanodisc-reconstituted wild-type mouse TRPV3 in three distinct conformations: closed, heat-activated sensitized and open states. The heat-induced transformations of TRPV3 are accompanied by changes in the secondary structure of the S2-S3 linker and the N and C termini and represent a conformational wave that links these parts of the protein to a lipid occupying the vanilloid binding site. State-dependent differences in the behavior of bound lipids suggest their active role in thermo-TRP temperature-dependent gating. Our structural data, supported by physiological recordings and molecular dynamics simulations, provide an insight for understanding the molecular mechanism of temperature sensing.

PLATINUM: a web tool for analysis of hydrophobic/hydrophilic organization of biomolecular complexes.

UNLABELLED: The PLATINUM (Protein-Ligand ATtractions Investigation NUMerically) web service is designed for analysis and visualization of hydrophobic/hydrophilic properties of biomolecules supplied as 3D-structures. Furthermore, PLATINUM provides a number of tools for quantitative characterization of the hydrophobic/hydrophilic match in biomolecular complexes e.g. in docking poses. These complement standard scoring functions. The calculations are based on the concept of empirical Molecular Hydrophobicity Potential (MHP). AVAILABILITY: The PLATINUM web tool as well as detailed documentation and tutorial are available free of charge for academic users at http://model.nmr.ru/platinum/. PLATINUM requires Java 5 or higher and Adobe Flash Player 9. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Environmental and dynamic effects explain how nisin captures membrane-bound lipid II.

Antibiotics (AB) resistance is a major threat to global health, thus the development of novel AB classes is urgently needed. Lantibiotics (i.e. nisin) are natural compounds that effectively control bacterial populations, yet their clinical potential is very limited. Nisin targets membrane-embedded cell wall precursor - lipid II - via capturing its pyrophosphate group (PPi), which is unlikely to evolve, and thus represents a promising pharmaceutical target. Understanding of exact molecular mechanism of initial stages of membrane-bound lipid II recognition by water-soluble nisin is indispensable. Here, using molecular simulations, we demonstrate that the structure of lipid II is determined to a large extent by the surrounding water-lipid milieu. In contrast to the bulk solvent, in the bilayer only two conformational states remain capable of nisin binding. In these states PPi manifests a unique arrangement of hydrogen bond acceptors on the bilayer surface. Such a "pyrophosphate pharmacophore" cannot be formed by phospholipids, which explains high selectivity of nisin/lipid II recognition. Similarly, the "recognition module" of nisin, being rather flexible in water, adopts the only stable conformation in the presence of PPi analogue (which mimics the lipid II molecule). We establish the "energy of the pyrophosphate pharmacophore" approach, which effectively distinguishes nisin conformations that can form a complex with PPi. Finally, we propose a molecular model of nisin recognition module/lipid II complex in the bacterial membrane. These results will be employed for further study of lipid II targeting by antimicrobial (poly)cyclic peptides and for design of novel AB prototypes.

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.