Structural Mechanism of Ionic Conductivity of the TRPV1 Channel.

The so-called "hydrophobic gating" is widely discussed as a putative mechanism to control water and ion conduction via ion channels. This effect can occur in narrow areas of the channels pore lined by non-polar residues. In the closed state of the channel, such regions may spontaneously transit to a dehydrated state to block water and ions transport without full pore occlusion. In the open state, the hydrophobic gate is wide enough to provide sustainable hydration and conduction. Apparently, the transport through the open hydrophobic gate may by facilitated by some polar residues that assist polar/charged substances to overcome the energy barrier created by nonpolar environment. In this work, we investigated the behavior of Na+ ions and their hydration shells in the open pore of the rat TRPV1 ion channel by molecular dynamics simulations. We show that polar protein groups coordinate water molecules in such a way as to restore the hydration shell of ions in the hydrophobic gate that ensures ion transport through the gate in a fully hydrated state.

Dielectric-Dependent Strength of Interlipid Hydrogen Bonding in Biomembranes: Model Case Study.

Atomistic aspects of the structural organization, dynamics, and functioning of hydrated lipid bilayers-model cell membranes-are primarily governed by the fine balance of intermolecular interactions between all constituents of these systems. Besides the hydrophobic effect, which shapes the overall skeleton of lipid membranes, a very important contribution to their behavior is made by hydrogen bonds (H-bonds) between lipid head groups. The latter determine crucial phenomena in cell membranes, such as dynamic ultrananodomain organization, hydration, and fine-tuning of microscopic physicochemical properties that allow the membrane to adapt quickly when binding/insertion external agents (proteins, etc.). The characteristics of such H-bonds (strength, spatial localization, etc.) dramatically depend on the local polarity properties of the lipid-water environment. In this work, we calculated free energies of H-bonded complexes between typical donor (NH3+, NH, OH) and acceptor (C═O, OH, COO-, COOH) groups of lipids in vacuo and in a set of explicit solvents with dielectric constants (ε) from 1 to 78.3, which mimic membrane environment at different depths. This was done using Monte Carlo simulations and an assessment of the corresponding potential of mean force profiles. The strongest H-bonded complexes were observed in the nonpolar environment, and their strength increased sharply with decreasing ε below 17. When ε changed, the largest free energy gain (>10.8 kcal/mol) was observed for pairs of acceptors C═O and O(H) with donor NH3+. The complexation of the same acceptors with NH donor in this range of ε values was rather less sensitive to the environmental polarity, by ∼1.5 kcal/mol. Dielectric-dependent interactions of polar lipid groups with water were evaluated as well. The results explain the delicate balance that determines the unique pattern of H-bonds for a particular lipid bilayer. Understanding the factors that regulate the propensity for H-bonding in lipid bilayers provides a fundamental basis for the rational design of new membrane nano objects with predefined properties.

Interaction of linear cationic peptides with phospholipid membranes and polymers of sialic acid.

Polysialic acid (PSA) is a natural anionic polymer typically occurring on the outer surface of cell membranes. PSA is involved in cell signaling and intermolecular interactions with proteins and peptides. The antimicrobial potential of peptides is usually evaluated in model membranes consisting of lipid bilayers but devoid of either PSA or its analogs. The goal of this work was to investigate the possible effect of PSA on the structure of melittin (Mlt) and latarcins Ltc1K, Ltc2a, and the activity of these peptides with respect to model membranes. These peptides are linear cationic ones derived from the venom of bee (Mlt) and spider (both latarcins). The length of each of the peptides is 26 amino acid residues, and they all have antimicrobial activity. However, they differ with respect to conformational mobility, hydrophobic characteristics, and overall charge. In this work, using circular dichroism spectroscopy, we show that the peptides adopt an α-helical conformation upon interaction with either PSA or phospholipid liposomes formed of either zwitterionic or anionic phospholipids or their mixtures. The extent of helicity depends on the amino acid sequence and properties of the medium. Based on small angle X-ray scattering data and the analysis of the fluorescence spectrum of the Trp residue in Mlt, we conclude that the peptide forms an oligomeric complex consisting of α-helical Mlt and several PSA molecules. Both latarcins, unlike Mlt, the most hydrophobic of the peptides, interact weakly with zwitterionic liposomes. However, they bind anionic liposomes or those composed of anionic/zwitterionic lipid mixtures. Latarcin Ltc1K forms associates on liposomes composed of zwitterionic/anionic lipid mixture. The structure of the peptide associates is either disordered or of ß-sheet conformation. In all other cases the studied peptides adopt predominately α-helical conformation. In addition, we demonstrate that PSA inhibits membranolytic activity of Mlt and latarcin Ltc1K. These data suggest that the peptides, due to their high conformational lability, can vary structural and amphiphilic properties in the presence of PSA. As a result, various scenarios of the interaction of the peptides with membranes, whose surface is abundant with anionic polysaccharides, can take place. This can account for difficulties in understanding the structure-functional relationships in interactions of linear cationic peptides with biological membranes.

Computer simulations and modeling-assisted ToxR screening in deciphering 3D structures of transmembrane alpha-helical dimers: ephrin receptor A1.

Membrane-spanning segments of numerous proteins (e.g. receptor tyrosine kinases) represent a novel class of pharmacologically important targets, whose activity can be modulated by specially designed artificial peptides, the so-called interceptors. Rational construction of such peptides requires understanding of the main factors driving peptide-peptide association in lipid membranes. Here we present a new method for rapid prediction of the spatial structure of transmembrane (TM) helix-helix complexes. It is based on computer simulations in membrane-like media and subsequent refinement/validation of the results using experimental studies of TM helix dimerization in a bacterial membrane by means of the ToxR system. The approach was applied to TM fragments of the ephrin receptor A1 (EphA1). A set of spatial structures of the dimer was proposed based on Monte Carlo simulations in an implicit membrane followed by molecular dynamics relaxation in an explicit lipid bilayer. The resulting models were employed for rational design of wild-type and mutant genetic constructions for ToxR assays. The computational and the experimental data are self-consistent and provide an unambiguous spatial model of the TM dimer of EphA1. The results of this work can be further used to develop new biologically active 'peptide interceptors' specifically targeting membrane domains of proteins.

[Molecular docking: role of intermolecular contacts in formation of complexes of proteins with nucleotides and peptides].

Knowledge of 3D-structure of protein-ligand complex is a major prerequisite for understanding the functioning mechanism of cellular proteins and membrane receptors. This is also of a great help in rational drug design projects. In the present paper we briefly review the molecular docking approaches used to predict possible orientation of a ligand in the protein binding site. The recent trends to improve the accuracy and efficiency of docking algorithms are demonstrated with the results obtained in Laboratory of Biomolecular Modeling. Particular attention is paid to protein-ligand hydrophobic and stacking interactions responsible for molecular recognition of ligand fragments. Such type of interactions are not always adequately represented in scoring criteria of docking applications that leads to mismatch in 3D-structure complexes predictions. That is why further inquiry of methods to account for these interactions is now the area of active research.

[Prediction of the spatial structure of proteins: emphasis on membrane targets].

Knowledge of the spatial structure of proteins is a prerequisite for both awareness of their functional mechanisms and the framework for rational drug discovery and design. Meanwhile, direct structural determination is often hampered or impractical due to the complexity, expensiveness, and limited capabilities of experimental techniques. These issues are especially pronounced for integral membrane proteins. On numerous occasions, the theoretical prediction of protein structures may facilitate the process by exploiting physical or empirical principles. This paper surveys modern techniques for the prediction of the spatial structure of proteins using computer algorithms, and the main emphasis is placed on the most "complex" targets - membrane proteins (MPs). The first part of the review describes de novo methods based on empirical physical principles; in the second part, a comparative modeling philosophy, which accounts for the structure of related proteins, is described. Special focus is made regarding pharmacologically relevant classes of G-coupled receptors, receptor tyrosine ki-nases, and other MPs. Algorithms for the assessment of the models quality and potential fields of application of computer models are discussed.

Ligand-specific scoring functions: improved ranking of docking solutions.

Molecular docking is a powerful computational method that has been widely used in many biomolecular studies to predict geometry of a protein-ligand complex. However, while its conformational search algorithms are usually able to generate correct conformation of a ligand in the binding site, the scoring methods often fail to discriminate it among many false variants. We propose to treat this problem by applying more precise ligand-specific scoring filters to re-rank docking solutions. In this way specific features of interactions between protein and different types of compounds can be implicitly taken into account. New scoring functions were constructed including hydrogen bonds, hydrophobic and hydrophilic complementarity terms. These scoring functions also discriminate ligands by the size of the molecule, the total hydrophobicity, and the number of peptide bonds for peptide ligands. Weighting coefficients of the scoring functions were adjusted using a training set of 60 protein-ligand complexes. The proposed method was then tested on the results of docking obtained for an additional 70 complexes. In both cases the success rate was 5-8% better compared to the standard functions implemented in popular docking software.

Probing the effect of membrane contents on transmembrane protein-protein interaction using solution NMR and computer simulations.

The interaction between the secondary structure elements is the key process, determining the spatial structure and activity of a membrane protein. Transmembrane (TM) helix-helix interaction is known to be especially important for the function of so-called type I or bitopic membrane proteins. In the present work, we present the approach to study the helix-helix interaction in the TM domains of membrane proteins in various lipid environment using solution NMR spectroscopy and phospholipid bicelles. The technique is based on the ability of bicelles to form particles with the size, depending on the lipid/detergent ratio. To implement the approach, we report the experimental parameters of "ideal bicelle" models for four kinds of zwitterionic phospholipids, which can be also used in other structural studies. We show that size of bicelles and type of the rim-forming detergent do not affect substantially the spatial structure and stability of the model TM dimer. On the other hand, the effect of bilayer thickness on the free energy of the dimer is dramatic, while the structure of the protein is unchanged in various lipids with fatty chains having a length from 12 to 18 carbon atoms. The obtained data is analyzed using the computer simulations to find the physical origin of the observed effects.

Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors.

Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3ß2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure and pharmacology of a recombinant analogue of SLURP-2. Nuclear magnetic resonance spectroscopy revealed a 'three-finger' fold of SLURP-2 with a conserved ß-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the α3, α4, α5, α7, ß2, and ß4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at α4ß2 and α3ß2-nAChRs (IC50 ~0.17 and >3 µM, respectively) expressed in Xenopus oocytes. In contrast, at α7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations <1 µM but induces inhibition at higher concentrations. SLURP-2 allosterically interacts with human M1 and M3 muscarinic acetylcholine receptors (mAChRs) that are overexpressed in CHO cells. SLURP-2 was found to promote the proliferation of human oral keratinocytes via interactions with α3ß2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the 'classical' orthosteric agonist/antagonist binding sites at α7 and α3ß2-nAChRs.

Functional characterization of sensory rhodopsin II from Halobacterium salinarum expressed in Escherichia coli.

Sensory rhodopsin II (SRII) from Halobacterium salinarum is heterologously expressed in Escherichia coli with a yield of 3-4 mg of purified SRII per liter cell culture. UV/Vis absorption spectroscopy display bands characteristic for native SRII. The resonance Raman spectrum provides evidence for a strongly hydrogen-bonded Schiff base like in mammalian rhodopsin but unlike to the homologous pSRII from Natronobacterium pharaonis. Laser flash spectroscopy indicates that SRII in detergent as well as after reconstitution into polar lipids shows its typical photochemical properties with prolonged photocycle kinetics. The first functional heterologous expression of SRII from H. salinarum provides the basis for studies with its cognate transducer HtrII to investigate the molecular processes involved in phototransduction as well as in chemotransduction.

Role of the Lipid Environment in the Dimerization of Transmembrane Domains of Glycophorin A.

An efficient computational approach is developed to quantify the free energy of a spontaneous association of the α-helices of proteins in the membrane environment. The approach is based on the numerical decomposition of the free energy profiles of the transmembrane (TM) helices into components corresponding to protein-protein, protein-lipid, and protein-water interactions. The method was tested for the TM segments of human glycophorin A (GpA) and two mutant forms, Gly83Ala and Thr87Val. It was shown that lipids make a significant negative contribution to the free energy of dimerization, while amino acid residues forming the interface of the helix-helix contact may be unfavorable in terms of free energy. The detailed balance between different energy contributions is highly dependent on the amino acid sequence of the TM protein segment. The results show the dominant role of the environment in the interaction of membrane proteins that is changing our notion of the driving force behind the spontaneous association of TM α-helices. Adequate estimation of the contribution of the water-lipid environment thus becomes an extremely urgent task for a rational design of new molecules targeting bitopic membrane proteins, including receptor tyrosine kinases.

Amino acid residue: is it structural or functional?

A new approach is suggested for delineating the structural and functional amino acid residues in proteins with known three-dimensional structure, basing on the involvement of residues in intramolecular hydrophobic and hydrophilic interactions and additional information about the conservativity of the residues. The approach is applied to the families of homologous neurotoxins and cardiotoxins. The results obtained concerning the role of amino acid residues in both families of toxins accord well with the similarity of their fold, but different mechanisms of action. Current approach can be used for detailed characterization of protein spatial structures, as well as for rational protein engineering.

Retinal Schiff base position relative to the surfaces of photoreceptor disk.

Surface-enhanced Raman spectra of native and photobleached bovine rod outer segment disks as well as inside-out (inverted) photoreceptor disks adsorbed on silver hydrosol have been analyzed. Surface-enhanced spectra of inverted disks and disk-monoclonal antibody complexes reveal the short-range mechanism of enhancement. The distance between retinal Schiff base and the cytoplasmic side of native disk has been shown to be 5-10 A.

Binding of monovalent cations induces large changes in the secondary structure of Na+,K+-ATPase as probed by Raman spectroscopy.

Raman spectra of active Na+,K+-ATPase from pig kidney in media containing Na+ (E1), K+ (E2) or without exogenous ions (E1 conformation) were recorded in order to calculate the changes in the enzyme's secondary structure induced by binding of monovalent cations. It is demonstrated that: (i) K+ binding to the E1 form of the enzyme leads to conversion of approximately 100 peptide groups from the beta-structure to alpha-helical conformation; (ii) the transition is reversible and fully reproducible in the E1----E2----E1 and E2----E1----E2 experimental schemes. Predictional calculations revealed polypeptide chain segments involved in the alpha----beta transformations. These segments reside mainly in the two highly conserved regions of the alpha-subunit in the cytoplasmic domain of Na+,K+-ATPase. A possible role for the beta-subunit is discussed.

Study of ATP binding in the active site of Na+,K(+)-ATPase as probed by ultraviolet resonance Raman spectroscopy.

The ultraviolet resonance Raman (UV RR) spectra of functional ATP/membrane-bound Na+,K+-ATPase complexes have been obtained. The substrate binding in the enzyme active site has been shown to be accompanied with significant changes in the electronic vibrational structure of the adenine ring. From the spectral analysis of ATP, 8-Br-ATP and 6-NHMe-adenine at various pH values the conclusion was made that N1 and the NH2 group and, probably, N7 of the substrate adenine part, interact with the protein surroundings via hydrogen bonds.

Spatial structure of the M2 transmembrane segment of the nicotinic acetylcholine receptor alpha-subunit.

A synthetic peptide corresponding to the transmembrane segment M2 (residues 236-267) of the alpha-subunit of the nicotinic acetylcholine receptor from Torpedo californica has been studied by two dimensional 1H-NMR spectroscopy in a chloroform-methanol (1:1) mixture containing 0.1 M LiClO4. Reconstruction of the spatial structure of M2 from the NMR data resulted in an alpha-helix formed by residues 241-263. Distribution of the molecular hydrophobicity potential on the helix surface is very similar to that in five-helix bundles of proteins with a known three dimensional structure: two hydrophilic bands located on the opposite helix sides separated by strong hydrophobic zones.

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, alpha-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.

Differentiated analysis of the secondary structure of hydrophilic and hydrophobic regions in alpha- and beta-subunits of Na+,K+-ATPase by Raman spectroscopy.

Raman spectra of active Na+,K+-ATPase from pig kidney and membrane-bound products of its two-stage trypsinolysis, including alpha-subunit hydrophobic regions as well as the intact beta-subunit and hydrophobic regions of alpha- and beta-subunits, were measured to calculate the secondary structure of hydrophilic and hydrophobic regions of the enzyme. Consequent comparison demonstrated unambiguously that (i) membrane-bound hydrophobic parts of polypeptide chains of Na+,K+-ATPase subunits are in the alpha-helical conformation; (ii) essential contents of the alpha-helix as well as beta-sheet are estimated to form the hydrophilic (mainly cytoplasmic) domain of the Na+,K+-ATPase alpha-subunit; (iii) the exoplasmic hydrophilic domain of the beta-subunit is shown to include several antiparallel beta-pleated sheets and a small amount of the alpha-helix and unordered conformations. The model of the secondary structure organization of hydrophilic domains as well as 8 hydrophobic transmembrane segments of the enzyme molecule was proposed on the basis of experimental results and predictional calculations.

Peptides and proteins in membranes: what can we learn via computer simulations?

Membrane and membrane-active peptides and proteins play a crucial role in numerous cell processes, such as signaling, ion conductance, fusion, and others. Many of them act as highly specific and efficient drugs or drug targets, and, therefore, attract growing interest of medicinal chemists. Because of experimental difficulties with characterization of their spatial structure and mode of membrane binding, essential attention is given now to molecular modeling techniques. During the last years an important progress has been achieved in molecular dynamics (MD) and Monte Carlo (MC) simulations of peptides and proteins with explicit and/or implicit theoretical models of membranes. The first ones allow atomic-resolution studies of peptides behavior on the membrane-water interfaces. Models with implicit consideration of membrane are of a special interest because of their computational efficiency and ability to account for principal trends in protein-lipid interactions. In this approximation, the bilayer is usually treated as continuum whose properties vary along the membrane thickness, and membrane insertion is simulated using either MC or MD methods. This review surveys recent applications of both types of lipid bilayer models in computer simulations of a wide variety of peptides and proteins with different biological activities. Theoretical background of the membrane models is considered with examples of their applications to biologically relevant problems. The emphasis of the review is made on recent MC and MD computations, on structural and/or functional information, which may be obtained via molecular modeling. The approximations and shortcomings of the models, along with their perspectives in design of new membrane active drugs, are discussed.