Anton A. Polyansky

Multistate organization of transmembrane helical protein dimers governed by the host membrane.

Association of transmembrane (TM) helices taking place in the cell membrane has an important contribution to the biological function of bitopic proteins, among which receptor tyrosine kinases represent a typical example and a potent target for medical applications. Since this process depends on a complex interplay of different factors (primary structures of TM domains and juxtamembrane regions, composition and phase of the local membrane environment, etc.), it is still far from being fully understood. Here, we present a computational modeling framework, which we have applied to systematically analyze dimerization of 18 TM helical homo- and heterodimers of different bitopic proteins, including the family of epidermal growth factor receptors (ErbBs). For this purpose, we have developed a novel surface-based modeling approach, which not only is able to predict particular conformations of TM dimers in good agreement with experiment but also provides screening of their conformational heterogeneity. Using all-atom molecular dynamics simulations of several of the predicted dimers in different model membranes, we have elucidated a putative role of the environment in selection of particular conformations. Simulation results clearly show that each particular bilayer preferentially stabilizes one of possible dimer conformations, and that the energy gain depends on the interplay between structural properties of the protein and the membrane. Moreover, the character of protein-driven perturbations of the bilayer is reflected in the contribution of a particular membrane to the free energy gain. We have found that the approximated dimerization strength for ErbBs family can be related to their oncogenic ability.

PDGFRA alterations in cancer: characterization of a gain-of-function V536E transmembrane mutant as well as loss-of-function and passenger mutations

Activating mutations in the platelet-derived growth factor (PDGF) receptor alpha (PDGFRA) have been described in patients with gastrointestinal stromal tumors or myeloid malignancies associated with hypereosinophilia. These patients respond well to imatinib mesylate, raising the question as to whether patients with a PDGF receptor mutation in other tumor types should receive a tyrosine kinase inhibitor treatment. We characterized 10 novel somatic point mutations in PDGFRA that have been reported in isolated cases of glioblastoma, melanoma, acute myeloid leukemia, peripheral nerve sheath tumors and neuroendocrine carcinoma. The PDGFRA transmembrane domain mutation V536E stimulated Ba/F3 cell growth and signaling via ERK and STAT5 in the absence of ligand. This mutant, identified in glioblastoma, was strongly inhibited by imatinib. Modeling suggested that the mutation modulates the packing of the transmembrane domain helices in the receptor dimer. By contrast, two mutations in highly conserved residues affected the receptor traffic to the cell surface or kinase activity, thereby preventing the response to PDGF. The other mutations had no significant impact on the receptor activity. This functional analysis matched the predictions of SIFT and PolyPhen for only five mutations and these algorithms do not discriminate gain from loss of function. Finally, an E996K variant that had been identified in a melanoma cell line was not expressed in these cells. Altogether, several newly identified PDGFRA mutations do not activate the receptor and may therefore represent passenger mutations. Our results also underline the importance of characterizing novel kinase alterations in cancer patients.

N-terminal amphipathic helix as a trigger of hemolytic activity in antimicrobial peptides: a case study in latarcins.

In silico structural analyses of sets of α‐helical antimicrobial peptides (AMPs) are performed. Differences between hemolytic and non‐hemolytic AMPs are revealed in organization of their N‐terminal region. A parameter related to hydrophobicity of the N‐terminal part is proposed as a measure of the peptide propensity to exhibit hemolytic and other unwanted cytotoxic activities. Based on the information acquired, a rational approach for selective removal of these properties in AMPs is suggested. A proof of concept is gained through engineering specific mutations that resulted in elimination of the hemolytic activity of AMPs (latarcins) while leaving the beneficial antimicrobial effect intact.

Proteome-wide analysis reveals clues of complementary interactions between mRNAs and their cognate proteins as the physicochemical foundation of the genetic code

Despite more than 50 years of effort, the origin of the genetic code remains enigmatic. Among different theories, the stereochemical hypothesis suggests that the code evolved as a consequence of direct interactions between amino acids and appropriate bases. If indeed true, such physicochemical foundation of the mRNA/protein relationship could also potentially lead to novel principles of protein-mRNA interactions in general. Inspired by this promise, we have recently explored the connection between the physicochemical properties of mRNAs and their cognate proteins at the proteome level. Using experimentally and computationally derived measures of solubility of amino acids in aqueous solutions of pyrimidine analogs together with knowledge-based interaction preferences of amino acids for different nucleobases, we have revealed a statistically significant matching between the composition of mRNA coding sequences and the base-binding preferences of their cognate protein sequences. Our findings provide strong support for the stereochemical hypothesis of genetic code’s origin and suggest the possibility of direct complementary interactions between mRNAs and cognate proteins even in present-day cells.

Estimation of Conformational Entropy in Protein–Ligand Interactions: A Computational Perspective

Conformational entropy is an important component of the change in free energy upon binding of a ligand to its target protein. As a consequence, development of computational techniques for reliable estimation of conformational entropies is currently receiving an increased level of attention in the context of computational drug design. Here, we review the most commonly used techniques for conformational entropy estimation from classical molecular dynamics simulations. Although by-and-large still not directly used in practical drug design, these techniques provide a golden standard for developing other, computationally less-demanding methods for such applications, in addition to furthering our understanding of protein-ligand interactions in general. In particular, we focus on the quasi-harmonic approximation and discuss different approaches that can be used to go beyond it, most notably, when it comes to treating anharmonic and/or correlated motions. In addition to reviewing basic theoretical formalisms, we provide a concrete set of steps required to successfully calculate conformational entropy from molecular dynamics simulations, as well as discuss a number of practical issues that may arise in such calculations.

Protein Electrostatic Properties Predefining the Level of Surface Hydrophobicity Change upon Phosphorylation.

We use explicit-solvent, molecular dynamics simulations to study the change in polar properties of a solvent-accessible surface for proteins undergoing phosphorylation. We analyze eight different pairs of proteins representing different structural classes in native and phosphorylated states and estimate the polarity of their surface using the molecular hydrophobicity potential approach. Whereas the phosphorylation-induced hydrophobicity change in the vicinity of phosphosites does not vary strongly among the studied proteins, the equivalent change for complete proteins covers a surprisingly wide range of effects including even an increase in the overall hydrophobicity in some cases. Importantly, the observed changes are strongly related to electrostatic properties of proteins, such as the net charge per residue, the distribution of charged side-chain contacts, and the isoelectric point. These features predefine the level of surface hydrophobicity change upon phosphorylation and may thus contribute to the phosphorylation-induced alteration of the interactions between a protein and its environment.

Recent advances in computational modeling of α-helical membrane-active peptides.

Membrane-active peptides (MAPs) represent a broad variety of molecules, and biological functions of most are directly associated with their ability to interact with membranes. Taking into account the effect of MAPs on living cells they can be nominally divided into three major groups - fusion (FPs), antimicrobial/cytolytic (AMPs/CPs) and cell-penetrating (CPPs) peptides. Although spatial structure of different MAPs varies to a great extent, linear α-helical peptides represent the most studied class. These peptides possess relatively simple structural organization and share a set of similar molecular features, which make them very attractive to both experimental and computational studies. Here, we review different molecular modeling methods in prospective of their applications to study of α-helical MAPs. The most sophisticated of them, such as molecular dynamics simulations, give atomistic information about molecular interactions driving peptide binding to the water-lipid interface, cooperative mechanisms of membrane destabilization and thermodynamics of these processes. Significant progress has been achieved in this field during the last few years, resulting in a possibility to observe computationally MAPs action in realistic peptide-to-lipid ratios and over the microsecond timescale. Other relatively simple but powerful approaches allow assessment of important characteristics of MAPs such as α-helical propensity, amphiphilicity, total hydrophobicity, and spatial distribution of charge and hydrophobic/hydrophilic properties, etc. Altogether, computational methods provide efficient basis for rational design of MAPs with predefined properties and a spectrum of biological activities.

Three-dimensional structure/hydrophobicity of latarcins specifies their mode of membrane activity.

Latarcins, linear peptides from the Lachesana tarabaevi spider venom, exhibit a broad-spectrum antimicrobial activity, likely acting on the bacterial cytoplasmic membrane. We study their spatial structures and interaction with model membranes by a combination of experimental and theoretical methods to reveal the structure-activity relationship. In this work, a 26 amino acid peptide, Ltc1, was investigated. Its spatial structure in detergent micelles was determined by (1)H nuclear magnetic resonance (NMR) and refined by Monte Carlo simulations in an implicit water-octanol slab. The Ltc1 molecule was found to form a straight uninterrupted amphiphilic helix comprising 8-23 residues. A dye-leakage fluorescent assay and (31)P NMR spectroscopy established that the peptide does not induce the release of fluorescent marker nor deteriorate the bilayer structure of the membranes. The voltage-clamp technique showed that Ltc1 induces the current fluctuations through planar membranes when the sign of the applied potential coincides with the one across the bacterial inner membrane. This implies that Ltc1 acts on the membranes via a specific mechanism, which is different from the carpet mode demonstrated by another latarcin, Ltc2a, featuring a helix-hinge-helix structure with a hydrophobicity gradient along the peptide chain. In contrast, the hydrophobic surface of the Ltc1 helix is narrow-shaped and extends with no gradient along the axis. We have also disclosed a number of peptides, structurally homologous to Ltc1 and exhibiting similar membrane activity. This indicates that the hydrophobic pattern of the Ltc1 helix and related antimicrobial peptides specifies their activity mechanism. The latter assumes the formation of variable-sized lesions, which depend upon the potential across the membrane.

Modeling Transmembrane Domain Dimers/Trimers of Plexin Receptors: Implications for Mechanisms of Signal Transmission across the Membrane

Single-pass transmembrane (TM) receptors transmit signals across lipid bilayers by helix association or by configurational changes within preformed dimers. The structure determination for such TM regions is challenging and has mostly been accomplished by NMR spectroscopy. Recently, the computational prediction of TM dimer structures is becoming recognized for providing models, including alternate conformational states, which are important for receptor regulation. Here we pursued a strategy to predict helix oligomers that is based on packing considerations (using the PREDDIMER webserver) and is followed by a refinement of structures, utilizing microsecond all-atom molecular dynamics simulations. We applied this method to plexin TM receptors, a family of 9 human proteins, involved in the regulation of cell guidance and motility. The predicted models show that, overall, the preferences identified by PREDDIMER are preserved in the unrestrained simulations and that TM structures are likely to be diverse across the plexin family. Plexin-B1 and –B3 TM helices are regular and tend to associate, whereas plexin-A1, -A2, –A3, -A4, -C1 and –D1 contain sequence elements, such as poly-Glycine or aromatic residues that distort helix conformation and association. Plexin-B2 does not form stable dimers due to the presence of TM prolines. No experimental structural information on the TM region is available for these proteins, except for plexin-C1 dimeric and plexin-B1 – trimeric structures inferred from X-ray crystal structures of the intracellular regions. Plexin-B1 TM trimers utilize Ser and Thr sidechains for interhelical contacts. We also modeled the juxta-membrane (JM) region of plexin-C1 and plexin-B1 and show that it synergizes with the TM structures. The structure and dynamics of the JM region and TM-JM junction provide determinants for the distance and distribution of the intracellular domains, and for their binding partners relative to the membrane. The structures suggest experimental tests and will be useful for the interpretation of future studies.

Modular Organization of α-Toxins from Scorpion Venom Mirrors Domain Structure of Their Targets - Sodium Channels

Background: Scorpion α-toxins affect voltage-gated sodium channels in both mammals and insects. Results: We perform thorough computational analyses of α-toxin molecular architecture and structure-function relationship. Conclusion: Taxon specificity of “orphan” toxins can be predicted from a structural perspective. Significance: The proposed surface mapping technique is a new tool to analyze protein-protein complexes. To gain success in the evolutionary “arms race,” venomous animals such as scorpions produce diverse neurotoxins selected to hit targets in the nervous system of prey. Scorpion α-toxins affect insect and/or mammalian voltage-gated sodium channels (Navs) and thereby modify the excitability of muscle and nerve cells. Although more than 100 α-toxins are known and a number of them have been studied into detail, the molecular mechanism of their interaction with Navs is still poorly understood. Here, we employ extensive molecular dynamics simulations and spatial mapping of hydrophobic/hydrophilic properties distributed over the molecular surface of α-toxins. It is revealed that despite the small size and relatively rigid structure, these toxins possess modular organization from structural, functional, and evolutionary perspectives. The more conserved and rigid “core module” is supplemented with the “specificity module” (SM) that is comparatively flexible and variable and determines the taxon (mammal versus insect) specificity of α-toxin activity. We further show that SMs in mammal toxins are more flexible and hydrophilic than in insect toxins. Concomitant sequence-based analysis of the extracellular loops of Navs suggests that α-toxins recognize the channels using both modules. We propose that the core module binds to the voltage-sensing domain IV, whereas the more versatile SM interacts with the pore domain in repeat I of Navs. These findings corroborate and expand the hypothesis on different functional epitopes of toxins that has been reported previously. In effect, we propose that the modular structure in toxins evolved to match the domain architecture of Navs.