Pavel E. Volynsky

Spatial Structure of the Dimeric Transmembrane Domain of the Growth Factor Receptor ErbB2 Presumably Corresponding to the Receptor Active State

Proper lateral dimerization of the transmembrane domains of receptor tyrosine kinases is required for biochemical signal transduction across the plasma membrane. The spatial structure of the dimeric transmembrane domain of the growth factor receptor ErbB2 embedded into lipid bicelles was obtained by solution NMR, followed by molecular dynamics relaxation in an explicit lipid bilayer. ErbB2 transmembrane segments associate in a right-handed α-helical bundle through the N-terminal tandem GG4-like motif Thr652-X3-Ser656-X3-Gly660, providing an explanation for the pathogenic power of some oncogenic mutations.

Unique dimeric structure of BNip3 transmembrane domain suggests membrane permeabilization as a cell death trigger.

BNip3 is a prominent representative of apoptotic Bcl-2 proteins with rather unique properties initiating an atypical programmed cell death pathway resembling both necrosis and apoptosis. Many Bcl-2 family proteins modulate the permeability state of the outer mitochondrial membrane by forming homo- and hetero-oligomers. The structure and dynamics of the homodimeric transmembrane domain of BNip3 were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics energy relaxation in an explicit lipid bilayer. The right-handed parallel helix-helix structure of the domain with a hydrogen bond-rich His-Ser node in the middle of the membrane, accessibility of the node for water, and continuous hydrophilic track across the membrane suggest that the domain can provide an ion-conducting pathway through the membrane. Incorporation of the BNip3 transmembrane domain into an artificial lipid bilayer resulted in pH-dependent conductivity increase. A possible biological implication of the findings in relation to triggering necrosis-like cell death by BNip3 is discussed.

Spatial Structure and pH-dependent Conformational Diversity of Dimeric Transmembrane Domain of the Receptor Tyrosine Kinase EphA1

Eph receptors are found in a wide variety of cells in developing and mature tissues and represent the largest family of receptor tyrosine kinases, regulating cell shape, movements, and attachment. The receptor tyrosine kinases conduct biochemical signals across plasma membrane via lateral dimerization in which their transmembrane domains play an important role. Structural-dynamic properties of the homodimeric transmembrane domain of the EphA1 receptor were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics in explicit lipid bilayer. EphA1 transmembrane segments associate in a right-handed parallel α-helical bundle, region (544-569)2, through the N-terminal glycine zipper motif A550X3G554X3G558. Under acidic conditions, the N terminus of the transmembrane helix is stabilized by an N-capping box formed by the uncharged carboxyl group of Glu547, whereas its deprotonation results in a rearrangement of hydrogen bonds, fractional unfolding of the helix, and a realignment of the helix-helix packing with appearance of additional minor dimer conformation utilizing seemingly the C-terminal GG4-like dimerization motif A560X3G564. This can be interpreted as the ability of the EphA1 receptor to adjust its response to ligand binding according to extracellular pH. The dependence of the pKa value of Glu547 and the dimer conformational equilibrium on the lipid head charge suggests that both local environment and membrane surface potential can modulate dimerization and activation of the receptor. This makes the EphA1 receptor unique among the Eph family, implying its possible physiological role as an “extracellular pH sensor,” and can have relevant physiological implications.

Left-Handed Dimer of EphA2 Transmembrane Domain: Helix Packing Diversity among Receptor Tyrosine Kinases

The Eph receptor tyrosine kinases and their membrane-bound ephrin ligands control a diverse array of cell-cell interactions in the developing and adult organisms. During signal transduction across plasma membrane, Eph receptors, like other receptor tyrosine kinases, are involved in lateral dimerization and subsequent oligomerization presumably with proper assembly of their single-span transmembrane domains. Spatial structure of dimeric transmembrane domain of EphA2 receptor embedded into lipid bicelle was obtained by solution NMR, showing a left-handed parallel packing of the transmembrane helices (535-559)(2). The helices interact through the extended heptad repeat motif L(535)X(3)G(539)X(2)A(542)X(3)V(546)X(2)L(549) assisted by intermolecular stacking interactions of aromatic rings of (FF(557))(2), whereas the characteristic tandem GG4-like motif A(536)X(3)G(540)X(3)G(544) is not used, enabling another mode of helix-helix association. Importantly, a similar motif AX(3)GX(3)G as was found is responsible for right-handed dimerization of transmembrane domain of the EphA1 receptor. These findings serve as an instructive example of the diversity of transmembrane domain formation within the same family of protein kinases and seem to favor the assumption that the so-called rotation-coupled activation mechanism may take place during the Eph receptor signaling. A possible role of membrane lipid rafts in relation to Eph transmembrane domain oligomerization and Eph signal transduction was also discussed.

Factors important for fusogenic activity of peptides: molecular modeling study of analogs of fusion peptide of influenza virus hemagglutinin

Nine analogs of fusion peptide of influenza virus hemagglutinin whose membrane perturbation activity has been thoroughly tested [Murata et al. (1992) Biochemistry 31, 1986–1992; Murata et al. (1993) Biophys. J. 64, 724–734] were characterized by molecular modeling techniques with the aim of delineating any specific structural and/or hydrophobic properties inherent in peptides with fusogenic activity. It was shown that, regardless of characteristics common to all analogs (peripheral disposition at the water‐lipid interface, amphiphilic nature, α‐helical structure, etc.), only fusion active peptides reveal a specific ‘tilted oblique‐oriented’ pattern of hydrophobicity on their surfaces and a certain depth of penetration to the non‐polar membrane core. The conclusion was reached that these factors are among the most important for the specific destabilization of a bilayer, which is followed by membrane fusion.

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.

Structure elucidation of dimeric transmembrane domains of bitopic proteins.

The interaction between transmembrane helices is of a great interest because it directly determines biological activity of a membrane protein. Either destroying or enhancing such interactions can result in many diseases related to dysfunction of different tissues in human body. One much studied form of membrane proteins known as bitopic protein is a dimer containing two membrane-spanning helices associating laterally. Establishing structure-function relationship as well as rational design of new types of drugs targeting membrane proteins requires precise structural information about this class of objects. At present time, to investigate spatial structure and internal dynamics of such transmembrane helical dimers, several strategies were developed based mainly on a combination of NMR spectroscopy, optical spectroscopy, protein engineering and molecular modeling. These approaches were successfully applied to homo- and heterodimeric transmembrane fragments of several bitopic proteins, which play important roles in normal and in pathological conditions of human organism.

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.

HER2 Transmembrane Domain Dimerization Coupled with Self-Association of Membrane-Embedded Cytoplasmic Juxtamembrane Regions.

Receptor tyrosine kinases of the human epidermal growth factor receptor (HER or ErbB) family transduce biochemical signals across plasma membrane, playing a significant role in vital cellular processes and in various cancers. Inactive HER/ErbB receptors exist in equilibrium between the monomeric and unspecified pre-dimerized states. After ligand binding, the receptors are involved in strong lateral dimerization with proper assembly of their extracellular ligand-binding, single-span transmembrane, and cytoplasmic kinase domains. The dimeric conformation of the HER2 transmembrane domain that is believed to support the cytoplasmic kinase domain configuration corresponding to the receptor active state was previously described in lipid bicelles. Here we used high-resolution NMR spectroscopy in another membrane-mimicking micellar environment and identified an alternative HER2 transmembrane domain dimerization coupled with self-association of membrane-embedded cytoplasmic juxtamembrane region. Such a dimerization mode appears to be capable of effectively inhibiting the receptor kinase activity. This finding refines the molecular mechanism regarding the signal propagation steps from the extracellular to cytoplasmic domains of HER/ErbB receptors.

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.