Transmembrane helix-helix interactions: comparative simulations of the glycophorin a dimer

The glycophorin helix dimer is a paradigm for the exploration of helix-helix interactions in integral membrane proteins. Two NMR structures of the dimer are known, one in a detergent micelle and one in a lipid bilayer. Multiple (4 x 50 ns) molecular dynamics simulations starting from each of the two NMR structures, with each structure in either a dodecyl phosphocholine (DPC) micelle or a dimyristoyl phosphatidylcholine (DMPC) bilayer, have been used to explore the conformational dynamics of the helix dimer. Analysis of the helix-helix interaction, mediated by the GxxxG sequence motif, suggests convergence of the simulations to a common model. This is closer to the NMR structure determined in a bilayer than to micelle structure. The stable dimer interface in the final simulation model is characterized by (i) Gly/Gly packing and (ii) Thr/Thr interhelix H-bonds. These results demonstrate the ability of extended molecular dynamics simulations in a lipid bilayer environment to refine membrane protein structures or models derived from experimental data obtained in protein/detergent micelles.     

The balance of weak and strong interactions in genetic networks

Genetic interactions are being quantitatively characterized in a comprehensive way in several model organisms. These data are then globally represented in terms of genetic networks. How are interaction strengths distributed in these networks? And what type of functional organization of the underlying genomic systems is revealed by such distribution patterns? Here, I found that weak interactions are important for the structure of genetic buffering between signaling pathways in Caenorhabditis elegans, and that the strength of the association between two genes correlates with the number of common interactors they exhibit. I also determined that this network includes genetic cascades balancing weak and strong links, and that its hubs act as particularly strong genetic modifiers; both patterns also identified in Saccharomyces cerevisae networks. In yeast, I further showed a relation, although weak, between interaction strengths and some phenotypic/evolutionary features of the corresponding target genes. Overall, this work demonstrates a non-random organization of interaction strengths in genetic networks, a feature common to other complex networks, and that could reflect in this context how genetic variation is eventually influencing the phenotype.     

A mutational study of transmembrane helix-helix interactions

Diverse methods have been developed and applied in the recent years to study interaction of transmembrane alpha-helices and often interaction of single transmembrane helices is followed on SDS-gels. Here we compare two measurements of the stability of a transmembrane helix-helix interaction, and the stability of the PsbF transmembrane helix dimer was determined in a biological membrane as well as in SDS. The observations described in this study demonstrate that the environment, in which a transmembrane helix interaction is studied, can be very critical and detergent properties can significantly influence transmembrane helix interactions, especially, when the transmembrane domain contains strongly polar residues.     

Mapping the energy surface of transmembrane helix-helix interactions

Transmembrane helices are no longer believed to be just hydrophobic segments that exist solely to anchor proteins to a lipid bilayer, but rather they appear to have the capacity to specify function and structure. Specific interactions take place between hydrophobic segments within the lipid bilayer whereby subtle mutations that normally would be considered innocuous can result in dramatic structural differences. That such specificity takes place within the lipid bilayer implies that it may be possible to identify the most favorable interaction surface of transmembrane alpha-helices based on computational methods alone, as shown in this study. Herein, an attempt is made to map the energy surface of several transmembrane helix-helix interactions for several homo-oligomerizing proteins, where experimental data regarding their structure exist (glycophorin A, phospholamban, Influenza virus A M2, Influenza virus C CM2, and HIV vpu). It is shown that due to symmetry constraints in homo-oligomers the computational problem can be simplified. The results obtained are mostly consistent with known structural data and may additionally provide a view of possible alternate and intermediate configurations.     

Multivalent ion-mediated nucleic acid helix-helix interactions: RNA versus DNA

Ion-mediated interaction is critical to the structure and stability of nucleic acids. Recent experiments suggest that the multivalent ion-induced aggregation of double-stranded (ds) RNAs and DNAs may strongly depend on the topological nature of helices, while there is still lack of an understanding on the relevant ion-mediated interactions at atomistic level. In this work, we have directly calculated the potentials of mean force (PMF) between two dsRNAs and between two dsDNAs in Co(NH₃)₆³⁺ (Co-Hex) solutions by the atomistic molecular dynamics simulations. Our calculations show that at low [Co-Hex], the PMFs between B-DNAs and between A-RNAs are both (strongly) repulsive. However, at high [Co-Hex], the PMF between B-DNAs is strongly attractive, while those between A-RNAs and between A-DNAs are still (weakly) repulsive. The microscopic analyses show that for A-form helices, Co-Hex would become ‘internal binding’ into the deep major groove and consequently cannot form the evident ion-bridge between adjacent helices, while for B-form helices without deep grooves, Co-Hex would exhibit ‘external binding’ to strongly bridge adjacent helices. In addition, our further calculations show that, the PMF between A-RNAs could become strongly attractive either at very high [Co-Hex] or when the bottom of deep major groove is fixed with a layer of water.     

Aromatic and cation-pi interactions enhance helix-helix association in a membrane environment

The cation-pi interaction is an electrostatic attraction between a positive charge and the conjugated pi electrons of an aromatic ring. These interactions are well documented in soluble proteins and can be both structurally and functionally important. Catalyzed by observations in our laboratory that an Ala- and Ile-rich two-helix transmembrane segment tended to form SDS-resistant dimers upon the incorporation of suitably located Trp residues, here we have constructed a library of related constructs to study systematically the impact of aromatic-aromatic and cation-pi interactions on tertiary structure formation within an Escherichia coli membrane. Using the TOXCAT oligomerization assay with the hydrophobic segment AIAIAIIAZAXAIIAIAIAI, where Z = A, W, Y, or F and X = A, H, R, or K in all possible combinations of cation and/or aromatic pairings, to assess the TM-TM dependent expression of the chloramphenicol acetyltransferase reporter gene, we find that cation-pi interactions, particularly between Lys and Trp, Tyr, or Phe, as well as weakly polar interactions between pairs of aromatic residues, significantly enhance the strength of oligomerization of these hydrophobic helices, in some instances forming oligomers four times stronger than the high-affinity glycophorin A dimer. The contribution of these forces to the tertiary structure formation in designed transmembrane segments suggests that similar forces may also be a significant factor in the folding and stability of native membrane proteins.     

Standardizing the free energy change of transmembrane helix-helix interactions

Side-to-side associations of transmembrane alpha-helices are integral components of the structure and function of helical membrane proteins. A fundamental unknown in the understanding of the chemical principles driving the lateral interactions between transmembrane alpha-helices is the balance of forces arising from the polypeptide sequence versus the hydrophobic solvent. To begin to address this question, a consideration of basic thermodynamic principles has been applied to assess the experimental free energy change associated with transmembrane helix dimerization in micelles. This analysis demonstrates the ability to partition the apparent free energy of transmembrane helix-helix association into two components. The first component is a statistical energy term, which arises from the fact that there are an unequal number of reactants and products. The second component is a standard state free energy change, which informs on the molecular details of the transmembrane helix self-association reaction. The advantage of separating these two energy terms arises from the fact that extrapolation to the standard state free energy change normalizes the statistical energy term so that it applies equivalently in all experimental systems. Accompanying experimental results for the glycophorin A transmembrane alpha-helix dimer measured in micelles are well described by these theoretical components assuming an ideal-dilute solution.     

Transthyretin amyloidosis: a tale of weak interactions.

Over 70 transthyretin (TTR) mutations have been associated with hereditary amyloidoses, which are all autosomal dominant disorders with adult age of onset. TTR is the main constituent of amyloid that deposits preferentially in peripheral nerve giving rise to familial amyloid polyneuropathy (FAP), or in the heart leading to familial amyloid cardiomyopathy. Since the beginning of this decade the central question of these types of amyloidoses has been why TTR is an amyloidogenic protein with clinically heterogeneous pathogenic consequences. As a result of amino acid substitutions, conformational changes occur in the molecule, leading to weaker subunit interactions of the tetrameric structure as revealed by X-ray studies of some amyloidogenic mutants. Modified soluble tetramers exposing cryptic epitopes seem to circulate in FAP patients as evidenced by antibody probes recognizing specifically TTR amyloid fibrils, but what triggers dissociation into monomeric and oligomeric intermediates of amyloid fibrils is largely unknown. Avoiding tetramer dissociation and disrupting amyloid fibrils are possible avenues of therapeutic intervention based on current molecular knowledge of TTR amyloidogenesis and fibril structure.     

Enhancing evolvability with mutation buffering mediated through multiple weak interactions

The evolutionary adaptability of a system is dependent on three organizational properties, self-organizing dynamics that are hierarchically organized, component redundancy, and multiple weak interactions [Towards high evolvability dynamics, in: G. van de Vijver, S. Salthe, M. Delpos (Eds.), Evolutionary Systems, Kluwer Academic Publishers, Dordrecht, 1998, pp. 147-169]. This study reports on the use of the dual dynamics network model as an aid in understanding the role multiple weak interactions play in enhancing evolutionary adaptability. Dual dynamics networks are self-organizing systems that consist of simple components that change local state due to the coupled influences from connected components exerting strong discrete decision-making influences and from groups of components exerting multiple weak influences [J. Theor. Biol. 193 (1998) 287]. The dual dynamics model has been enhanced to support investigations of properties relevant to a system's capacity for evolvability, such as structure-function relationships, neutrality, adaptive tolerance, and evolutionary search performance.Three network types are investigated, each utilizing a different method of coupling strong and weak influences. The results demonstrate that the manner of coupling multiple weak interactions into the systems dynamics significantly affects the structure-function maps and the consequent evolvability characteristics. Specifically it is found that a form of coupling, denoted as linear modulation, enhances evolutionary adaptability. Linear modulation coupling requires that the weak interactions be integrated with strong interactions in a manner that implies a linear ordered relation between the possible state values of the components of the systems. When coupling functions that do not imply such an ordering of local state values are used, evolutionary adaptability is decreased.     

Role of helix-helix interactions in assembly of the bacteriorhodopsin lattice.

The purple membrane of Halobacterium salinarium is a two-dimensional lattice of lipids and the integral membrane protein bacteriorhodopsin (BR). To determine whether helix-helix interactions within the membrane core stabilize this complex, we substituted amino acid residues at the helix-helix interface between BR monomers and examined the assembly of the protein into the lattice. Lattice assembly was demonstrated to fit a cooperative self-assembly model that exhibits a critical concentration in vivo. Using this model as the basis for a quantitative assay of lattice stability, bulky substitutions at the helix-helix interface between BR monomers within the membrane core were shown to be destabilizing, probably due to steric clash. Ala substitutions of two residues at the helix-helix interface also reduced stability, suggesting that the side chains of these residues participate in favorable van der Waals packing interactions. However, the stabilizing interactions were restricted to a small region of the interface, and most of the substitutions had little effect. Thus, the contribution of helix-helix interactions to lattice stability appears limited, and favorable interactions between other regions of neighboring BR monomers or between BR and lipid molecules must also contribute.     

Insights into protein-DNA interactions through structure network analysis

Protein-DNA interactions are crucial for many cellular processes. Now with the increased availability of structures of protein-DNA complexes, gaining deeper insights into the nature of protein-DNA interactions has become possible. Earlier, investigations have characterized the interface properties by considering pairwise interactions. However, the information communicated along the interfaces is rarely a pairwise phenomenon, and we feel that a global picture can be obtained by considering a protein-DNA complex as a network of noncovalently interacting systems. Furthermore, most of the earlier investigations have been carried out from the protein point of view (protein-centric), and the present network approach aims to combine both the protein-centric and the DNA-centric points of view. Part of the study involves the development of methodology to investigate protein-DNA graphs/networks with the development of key parameters. A network representation provides a holistic view of the interacting surface and has been reported here for the first time. The second part of the study involves the analyses of these graphs in terms of clusters of interacting residues and the identification of highly connected residues (hubs) along the protein-DNA interface. A predominance of deoxyribose-amino acid clusters in beta-sheet proteins, distinction of the interface clusters in helix-turn-helix, and the zipper-type proteins would not have been possible by conventional pairwise interaction analysis. Additionally, we propose a potential classification scheme for a set of protein-DNA complexes on the basis of the protein-DNA interface clusters. This provides a general idea of how the proteins interact with the different components of DNA in different complexes. Thus, we believe that the present graph-based method provides a deeper insight into the analysis of the protein-DNA recognition mechanisms by throwing more light on the nature and the specificity of these interactions.     

Helix Interaction Tool (HIT): a web-based tool for analysis of helix-helix interactions in proteins

MOTIVATION: In many proteins, helix-helix interactions can be critical to establishing protein conformation (folding) and dynamics, as well as determining associations between protein units. However, the determination of a set of rules that guide helix-helix interaction has been elusive. In order to gain further insight into the helix-helix interface, we have developed a comprehensive package of tools for analyzing helix-helix packing in proteins. These tools are available at http://helix.gersteinlab.org. They include quantitative measures of the helix interaction surface area and helix crossing angle, as well as several methods for visualizing the helical interaction. These methods can be used for analysis of individual protein conformations or to gain insight into dynamic changes in helix interactions. For the latter purpose, a direct interface from entries in the Molecular Motions Database to the HIT site has been provided.     

Peptides as transmembrane segments: decrypting the determinants for helix-helix interactions in membrane proteins

Although the structural analysis of membrane proteins is advancing, an understanding of the basic principles that underlie their folding and assembly remains limited because of the high insolubility intrinsic to these molecules and concomitant challenges in obtaining crystals. Fortunately, from an experimental standpoint, membrane protein folding can be approximated as the rigid-body docking of pre-formed alpha-helical transmembrane segments one with another to form the final functional protein structure. Peptides derived from the sequences of native alpha-helical transmembrane segments and those that mimic their properties are therefore valuable in the experimental evaluation of protein folding within the membrane. Here we present an overview of the progress made in our laboratory and elsewhere in using peptide models toward defining the sequence requirements and forces stabilizing membrane protein folds.     

Lipid solvation effects contribute to the affinity of Gly-xxx-Gly motif-mediated helix-helix interactions

Interactions between transmembrane helices are mediated by the concave Gly-xxx-Gly motif surface. Whether Gly residues per se are sufficient for selection of this motif has not been established. Here, we used the in vivo TOXCAT assay to measure the relative affinities of all 18 combinations of Gly, Ala, and Ser "small-xxx-small" mutations in glycophorin A (GpA) and bacteriophage M13 major coat protein (MCP) homodimers. Affinity values were compared with the accessibility to a methylene-sized probe of the total surface area of each helix monomer as a measure of solvation by membrane components. A strong inverse correlation was found between nonpolar-group lipid accessibility and dimer affinity (R = 0.75 for GpA, p = 0.013, and R = 0.81 for MCP, p = 0.004), suggesting that lipid as a poor membrane protein solvent, conceptually analogous to water in soluble protein folding, can contribute to dimer stability and help to define helix-helix interfaces.     

Computer modeling of polyleucine-based coiled coil dimers in a realistic membrane environment: insight into helix-helix interactions in membrane proteins

Simulated annealing was performed to model parallel dimers of alpha-helical transmembrane peptides with the sequence L(11)XL(12), predicting left-handed coiled coil geometry in all cases. Insertion of peptides containing threonine, asparagine, alanine, phenylalanine, and leucine in position 12 into realistic model membranes showed these structures were stable for 20 ns of molecular dynamics simulation time. Threonine could participate in intermolecular hydrogen bonds, but predominantly formed hydrogen bonds to the backbone of the helix it resided on. These hydrogen bonds, although infrequent, appeared to promote closer association of polyleucine helices. Asparagine participated in multiple, rapidly fluctuating intermolecular and intramolecular hydrogen bonds, and may have slightly destabilized optimum van der Waals packing in favor of optimum hydrogen bonding. Coordinated rotations of transmembrane helices about their axes were observed, indicating helices may rotate around one another during the folding of membrane proteins or other processes. These rotations were inhibited by phenylalanine, suggesting a role for bulky residues in modulating membrane protein dynamics.     

The GxxxG motif: a framework for transmembrane helix-helix association

In order to identify strong transmembrane helix packing motifs, we have selected transmembrane domains exhibiting high-affinity homo-oligomerization from a randomized sequence library based on the right-handed dimerization motif of glycophorin A. Sequences were isolated using the TOXCAT system, which measures transmembrane helix-helix association in the Escherichia coli inner membrane. Strong selection was applied to a large range of sequences ( approximately 10(7) possibilities) and resulted in the identification of sequence patterns that mediate high-affinity helix-helix association. The most frequent motif isolated, GxxxG, occurs in over 80% of the isolates. Additional correlations suggest that flanking residues act in concert with the GxxxG motif, and that size complementarity is maintained at the interface, consistent with the idea that the identified sequence patterns represent packing motifs. The convergent identification of similar sequence patterns from an analysis of the transmembrane domains in the SwissProt sequence database suggests that these packing motifs are frequently utilized in naturally occurring helical membrane proteins.     

Hydrophobic matching of HIV-1 Vpu transmembrane helix-helix interactions is optimized for subcellular location

The HIV-1 accessory protein Vpu mediates the downregulation of several host cell proteins, an activity that is critical for viral replication in vivo. As the first step in directing cell-surface proteins to internal cellular compartments, and in many cases degradation, Vpu binds a subset of its target proteins through their transmembrane domains. Each of the known targets of Vpu are synthesized in the ER, and must traverse the different membrane environments found along the secretory pathway, thus it is important to consider how membrane composition might influence the interactions between Vpu and its targets. We have used Förster resonance energy transfer (FRET) to measure the oligomerization of Vpu with the transmembrane domains of target proteins in model membranes of varying lipid composition. Our data show that both lipid bilayer thickness and acyl chain order can significantly influence monomer-oligomer equilibria within the Vpu-target system. Changes in oligomerization levels were found to be non-specific with no single Vpu-target interaction being favored under any condition. Our analysis of the influence of the membrane environment on the strength of helix-helix interactions between Vpu and its targets in vitro suggests that the strength of Vpu-target interactions in vivo will be partially dependent on the membrane environment found in specific membrane compartments.     

The affinity of GXXXG motifs in transmembrane helix-helix interactions is modulated by long-range communication

Sequence motifs are responsible for ensuring the proper assembly of transmembrane (TM) helices in the lipid bilayer. To understand the mechanism by which the affinity of a common TM-TM interactive motif is controlled at the sequence level, we compared two well characterized GXXXG motif-containing homodimers, those formed by human erythrocyte protein glycophorin A (GpA, high-affinity dimer) and those formed by bacteriophage M13 major coat protein (MCP, low affinity dimer). In both constructs, the GXXXG motif is necessary for TM-TM association. Although the remaining interfacial residues (underlined) in GpA (LIXXGVXXGVXXT) differ from those in MCP (VVXXGAXXGIXXF), molecular modeling performed here indicated that GpA and MCP dimers possess the same overall fold. Thus, we could introduce GpA interfacial residues, alone and in combination, into the MCP sequence to help decrypt the determinants of dimer affinity. Using both in vivo TOXCAT assays and SDS-PAGE gel migration rates of synthetic peptides derived from TM regions of the proteins, we found that the most distal interfacial sites, 12 residues apart (and approximately 18 A in structural space), work in concert to control TM-TM affinity synergistically.