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.     

Genetic selection for and molecular dynamic modeling of a protein transmembrane domain multimerization motif from a random Escherichia coli genomic library

In order to identify new transmembrane helix packing motifs in naturally occurring proteins, we have selected transmembrane domains from a library of random Escherichia coli genomic DNA fragments and screened them for homomultimerization via their abilities to dimerize the bacteriophage lambda cI repressor DNA-binding domain. Sequences were isolated using a modified lambda cI headpiece dimerization assay system, which was shown previously to measure transmembrane helix-helix association in the E. coli inner membrane. Screening resulted in the identification of several novel sequences that appear to mediate helix-helix interactions. One sequence, representing the predicted sixth transmembrane domain (TM6) of the E. coli protein YjiO, was chosen for further analysis. Using site-directed mutagenesis and molecular dynamics, a small set of models for YjiO TM6 multimerization interface interactions were generated. This work demonstrates the utility of combining in vivo genetic tools with computational systems for understanding membrane protein structure and assembly.     

Transmembrane helix-helix interactions are modulated by the sequence context and by lipid bilayer properties

Folding of polytopic transmembrane proteins involves interactions of individual transmembrane helices, and multiple TM helix-helix interactions need to be controlled and aligned to result in the final TM protein structure. While defined interaction motifs, such as the GxxxG motif, might be critically involved in transmembrane helix-helix interactions, the sequence context as well as lipid bilayer properties significantly modulate the strength of a sequence specific transmembrane helix-helix interaction. Structures of 11 transmembrane helix dimers have been described today, and the influence of the sequence context as well as of the detergent and lipid environment on a sequence specific dimerization is discussed in light of the available structural information. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

The position of the Gly-xxx-Gly motif in transmembrane segments modulates dimer affinity.

Although the intrinsic low solubility of membrane proteins presents challenges to their high-resolution structure determination, insight into the amino acid sequence features and forces that stabilize their folds has been provided through study of sequence-dependent helix-helix interactions between single transmembrane (TM) helices. While the stability of helix-helix partnerships mediated by the Gly-xxx-Gly (GG4) motif is known to be generally modulated by distal interfacial residues, it has not been established whether the position of this motif, with respect to the ends of a given TM segment, affects dimer affinity. Here we examine the relationship between motif position and affinity in the homodimers of 2 single-spanning membrane protein TM sequences: glycophorin A (GpA) and bacteriophage M13 coat protein (MCP). Using the TOXCAT assay for dimer affinity on a series of GpA and MCP TM segments that have been modified with either 4 Leu residues at each end or with 8 Leu residues at the N-terminal end, we show that in each protein, centrally located GG4 motifs are capable of stronger helix-helix interactions than those proximal to TM helix ends, even when surrounding interfacial residues are maintained. The relative importance of GG4 motifs in stabilizing helix-helix interactions therefore must be considered not only in its specific residue context but also in terms of the location of the interactive surface relative to the N and C termini of alpha-helical TM segments.     

GxxxG motifs, phenylalanine, and cholesterol guide the self-association of transmembrane domains of ErbB2 receptors

GxxxG motifs are common in transmembrane domains of membrane proteins and are often introduced to artificial peptides to inhibit or promote association to stable structures. The transmembrane domain of ErbB2 presents two separate such motifs that are proposed to be connected to stability and activity of the dimer. Using molecular simulations, we show that these sequences play a critical role during the recognition stage, forming transient complexes that lead to stable dimers. In pure phospholipid bilayers association occurs by contacts formed at the C-terminus promoted by the presence of phenylalanine residues. Helices subsequently rotate to eventually pack at short separations favored by lipid entropic contributions. In contrast, at intermediate cholesterol concentrations, a different pathway is followed that involves dimers with a weaker interface toward the N-terminus. However, at high cholesterol content, a switch toward the C-terminus is observed with an overall nonmonotonic change of the dimerization affinity. This conformational switch modulated by cholesterol has important implications on the thermodynamic, structural, and kinetic characteristics of helix-helix association in lipid membranes.     

GXXXG and AXXXA: common alpha-helical interaction motifs in proteins, particularly in extremophiles

The GXXXG motif is a frequently occurring sequence of residues that is known to favor helix-helix interactions in membrane proteins. Here we show that the GXXXG motif is also prevalent in soluble proteins whose structures have been determined. Some 152 proteins from a non-redundant PDB set contain at least one alpha-helix with the GXXXG motif, 41 +/- 9% more than expected if glycine residues were uniformly distributed in those alpha-helices. More than 50% of the GXXXG-containing alpha-helices participate in helix-helix interactions. In fact, 26 of those helix-helix interactions are structurally similar to the helix-helix interaction of the glycophorin A dimer, where two transmembrane helices associate to form a dimer stabilized by the GXXXG motif. As for the glycophorin A structure, we find backbone-to-backbone atomic contacts of the C alpha-H...O type in each of these 26 helix-helix interactions that display the stereochemical hallmarks of hydrogen bond formation. These glycophorin A-like helix-helix interactions are enriched in the general set of helix-helix interactions containing the GXXXG motif, suggesting that the inferred C alpha-H...O hydrogen bonds stabilize the helix-helix interactions. In addition to the GXXXG motif, some 808 proteins from the non-redundant PDB set contain at least one alpha-helix with the AXXXA motif (30 +/- 3% greater than expected). Both the GXXXG and AXXXA motifs occur frequently in predicted alpha-helices from 24 fully sequenced genomes. Occurrence of the AXXXA motif is enhanced to a greater extent in thermophiles than in mesophiles, suggesting that helical interaction based on the AXXXA motif may be a common mechanism of thermostability in protein structures. We conclude that the GXXXG sequence motif stabilizes helix-helix interactions in proteins, and that the AXXXA sequence motif also stabilizes the folded state of proteins.     

Self-association of transmembrane alpha-helices in model membranes: importance of helix orientation and role of hydrophobic mismatch

Interactions between transmembrane helices play a key role in almost all cellular processes involving membrane proteins. We have investigated helix-helix interactions in lipid bilayers with synthetic tryptophan-flanked peptides that mimic the membrane spanning parts of membrane proteins. The peptides were functionalized with pyrene to allow the self-association of the helices to be monitored by pyrene fluorescence and Trp-pyrene fluorescence resonance energy transfer (FRET). Specific labeling of peptides at either their N or C terminus has shown that helix-helix association occurs almost exclusively between antiparallel helices. Furthermore, computer modeling suggested that antiparallel association arises primarily from the electrostatic interactions between alpha-helix backbone atoms. We propose that such interactions may provide a force for the preferentially antiparallel association of helices in polytopic membrane proteins. Helix-helix association was also found to depend on the lipid environment. In bilayers of dioleoylphosphatidylcholine, in which the hydrophobic length of the peptides approximately matched the bilayer thickness, association between the helices was found to require peptide/lipid ratios exceeding 1/25. Self-association of the helices was promoted by either increasing or decreasing the bilayer thickness, and by adding cholesterol. These results indicate that helix-helix association in membrane proteins can be promoted by unfavorable protein-lipid interactions.     

The structure of interfaces between subunits of dimeric and tetrameric proteins

The structures of the interfaces of nine dimeric and nine tetrameric proteins have been analyzed and have been seen to follow general principles. These interfaces are combinations of four structural motifs, which resemble features of monomeric proteins. These are: (i) extended beta sheet; (ii) helix-helix packing; (iii) sheet-sheet packing; and (iv) loop interactions. Other common structural features in the interfaces studied are two-fold symmetry, charged hydrogen bonds and channel formation (found only in tetramers). Monomer-monomer interfaces are intermediate in hydrophobicity and charge between the interfaces between secondary structures of monomeric proteins and the exteriors of monomeric proteins. A typical interface has one of the first three of the structural motifs at its centre and loop interactions around the outside, where most of the charge resides.     

The membrane environment modulates self-association of the human GpA TM domain—Implications for membrane protein folding and transmembrane signaling

The influence of lipid bilayer properties on a defined and sequence-specific transmembrane helix-helix interaction is not well characterized yet. To study the potential impact of changing bilayer properties on a sequence-specific transmembrane helix-helix interaction, we have traced the association of fluorescent-labeled glycophorin A transmembrane peptides by fluorescence spectroscopy in model membranes with varying lipid compositions. The observed changes of the glycophorin A dimerization propensities in different lipid bilayers suggest that the lipid bilayer thickness severely influences the monomer-dimer equilibrium of this transmembrane domain, and dimerization was most efficient under hydrophobic matching conditions. Moreover, cholesterol considerably promotes self-association of transmembrane helices in model membranes by affecting the lipid acyl chain ordering. In general, the order of the lipid acyl chains appears to be an important factor involved in determining the strength and stability of transmembrane helix-helix interactions. As discussed, the described influences of membrane properties on transmembrane helix-helix interactions are highly important for understanding the mechanism of transmembrane protein folding and functioning as well as for gaining a deeper insight into the regulation of signal transduction via membrane integral proteins by bilayer properties.     

De novo design of transmembrane helix–helix interactions and measurement of stability in a biological membrane

Membrane proteins regulate a large number of cellular functions, and have great potential as tools for manipulation of biological systems. Developing these tools requires a robust and quantitative understanding of membrane protein folding and interactions within the bilayer. With this in mind, we have designed a series of proteins to probe the net thermodynamic contribution of well-known sequence motifs to transmembrane helix-helix association in a biological membrane. The proteins were designed from first principles (de novo) using current knowledge about membrane insertion and stabilizing interaction motifs. A simple poly-Leu “scaffold” was decorated with individual helix interaction motifs (G-XXX-G, polar residues, heptad repeat) to create transmembrane helix–helix interactions of increasing strength. The GALLEX assay, an in vivo assay for measurement of transmembrane helix self-association, was combined with computational methods to characterize the relative strength and mode of interaction for each sequence. In addition, the apparent free energy contribution (ΔΔG^app) of each motif to transmembrane helix self-association was measured in a biological membrane, results that are the first of their kind for these de novo designed sequences, and suggest that the free energy barrier to overcoming weak association is quite small (< 1.4 kcal mol^− 1) in a natural membrane. By quantifying and rationalizing the contribution of key motifs to transmembrane helix association, our work offers a route to direct the design of novel sequences for use in biotechnology or synthetic biology (e.g. molecular switches) and to predict the effects of sequence modification in known transmembrane domains (for control of cellular processes).     

Statistical analysis of amino acid patterns in transmembrane helices: the GxxxG motif occurs frequently and in association with beta-branched residues at neighboring positions

To find motifs that mediate helix-helix interactions in membrane proteins, we have analyzed frequently occurring combinations of residues in a database of transmembrane domains. Our analysis was performed with a novel formalism, which we call TMSTAT, for exactly calculating the expectancies of all pairs and triplets of residues in individual sequences, taking into account differential sequence composition and the substantial effect of finite length in short segments. We found that the number of significantly over and under-represented pairs and triplets was much greater than the random expectation. Isoleucine, glycine and valine were the most common residues in these extreme cases. The main theme observed is patterns of small residues (Gly, Ala and Ser) at i and i+4 found in association with large aliphatic residues (Ile, Val and Leu) at neighboring positions (i.e. i+/-1 and i+/-2). The most over-represented pair is formed by two glycine residues at i and i+4 (GxxxG, 31.6 % above expectation, p<1x10(-33)) and it is strongly associated with the neighboring beta-branched residues Ile and Val. In fact, the GxxxG pair has been described as part of the strong interaction motif in the glycophorin A transmembrane dimer, in which the pair is associated with two Val residues (GVxxGV). GxxxG is also the major motif identified using TOXCAT, an in vivo selection system for transmembrane oligomerization motifs. In conjunction with these experimental observations, our results highlight the importance of the GxxxG+beta-branched motif in transmembrane helix-helix interactions. In addition, the special role for the beta-branched residues Ile and Val suggested here is consistent with the hypothesis that residues with constrained rotameric freedom in helical conformation might reduce the entropic cost of folding in transmembrane proteins. Additional material is available at http://engelman.csb.yale. edu/tmstat and http://bioinfo.mbb.yale. edu/tmstat.     

Discovery of a significant,nontopological preference for antiparallel alignment of helices with parallel regions in sheets

To help elucidate the role of secondary structure packing preferences in protein folding, here we present an analysis of the packing geometry observed between alpha-helices and between alpha-helices and beta-sheets in 1316 diverse, nonredundant protein structures. Finite-length vectors were fit to the alpha-carbon atoms in each of the helices and strands, and the packing angle between the vectors, Omega, was determined at the closest point of approach within each helix-helix or helix-sheet pair. Helix-sheet interactions were found in 391 of the proteins, and the distributions of Omega values were calculated for all the helix-sheet and helix-helix interactions. The packing angle preferences for helix-helix interactions are similar to those previously observed. However, analysis of helix-strand packing preferences uncovered a remarkable tendency for helices to align antiparallel to parallel regions of beta-sheets, independent of the topological constraints or prevalence of beta-alpha-beta motifs in the proteins. This packing angle preference is significantly diminished in helix interactions involving mixed and antiparallel beta-sheets, suggesting a role for helix-sheet dipole alignment in guiding supersecondary structure formation in protein folding. This knowledge of preferred packing angles can be used to guide the engineering of stable protein modules.     

Sequence specificity in the dimerization of transmembrane alpha-helices.

While several reports have suggested a role for helix-helix interactions in membrane protein oligomerization, there are few direct biochemical data bearing on this subject. Here, using mutational analysis, we show that dimerization of the transmembrane alpha-helix of glycophorin A in a detergent environment is spontaneous and highly specific. Very subtle changes in the side-chain structure at certain sensitive positions disrupt the helix-helix association. These sensitive positions occur at approximately every 3.9 residues along the helix, consistent with their comprising the interface of a closely fit transmembranous supercoil of alpha-helices. By contrast with other reported cases of interactions between transmembrane helices, the set of interfacial residues in this case contains no highly polar groups. Amino acids with aliphatic side chains define much of the interface, indicating that precise packing interactions between the helices may provide much of the energy for association. These data highlight the potential general importance of specific interactions between the hydrophobic anchors of integral membrane proteins.     

Ionic Interactions Promote Transmembrane Helix-Helix Association Depending on Sequence Context

Folding and oligomerization of integral membrane proteins frequently depend on specific interactions of transmembrane helices. Interacting amino acids of helix-helix interfaces may form complex motifs and exert different types of molecular forces. Here, a set of strongly self-interacting transmembrane domains (TMDs), as isolated from a combinatorial library, was found to contain basic and acidic residues, in combination with polar nonionizable amino acids and C-terminal GxxxG motifs. Mutational analyses of selected sequences and reconstruction of high-affinity interfaces confirmed the cooperation of these residues in homotypic interactions. Probing heterotypic interaction indicated the presence of interhelical charge-charge interactions. Furthermore, simple motifs of an ionizable residue and GxxxG are significantly overrepresented in natural TMDs, and a specific combination of these motifs exhibits high-affinity heterotypic interaction. We conclude that intramembrane charge-charge interactions depend on sequence context. Moreover, they appear important for homotypic and heterotypic interactions of numerous natural TMDs.     

De novo design: backbone conformational constraints in nucleating helices and beta-hairpins.

A modular approach to synthetic protein design is being developed using conformationally constrained amino acid as stereochemical directors of polypeptide chain folding. An overview of studies aimed at constructing peptide helices using alpha,alpha-dialkyated residues and beta-hairpins using D-Pro as a turn nucleator is presented. The construction of helix-helix motifs and three- and four-stranded structures has been achieved using non-protein amino acids to stabilize specific elements of secondary structures.     

Analysis of the loop-helix interaction in bundle motif protein structures

Molecular dynamics simulations and energy analysis have been carried out to study the structural mobility and stability of the four -helix bundle motifs. The simulation results as well as the X-ray data show that the atomic RMS fluctuation is larger at the loop region for four representative proteins investigated: methemerythrin, cytochrome b-562, cytochrome c, and bovine somatotropin. The loop-loop, helix-helix, and loop-helix interactions are computed for the unfolded and folded proteins. In the folded and solvated protein structures the loop-helix interaction is stronger than the helix-helix interaction, especially in the electrostatic component. But the stabilization energies of both the loop-helix and the helix-helix interactions relative to the those of an unfolded structure are of the same order of magnitude. The stabilization due to protein-solvent interaction is greater in the helix region than in the loop region. The percentage of hydrophilic solvent accessible area for the four proteins studied was calculated with the method of Eisenberg and McLachlan. The percentage of the hydrophilic area is greater in the loops than in the helices. A Poisson-Boltzmann calculation shows that the potential from the loops acting on a helix is generally more negative than that from other helices.     

How do helix–helix interactions help determine the folds of membrane proteins? Perspectives from the study of homo‐oligomeric helical bundles

The final, structure-determining step in the folding of membrane proteins involves the coalescence of preformed transmembrane helices to form the native tertiary structure. Here, we review recent studies on small peptide and protein systems that are providing quantitative data on the interactions that drive this process. Gel electrophoresis, analytical ultracentrifugation, and fluorescence resonance energy transfer (FRET) are useful methods for examining the assembly of homo-oligomeric transmembrane helical proteins. These methods have been used to study the assembly of the M2 proton channel from influenza A virus, glycophorin, phospholamban, and several designed membrane proteins-all of which have a single transmembrane helix that is sufficient for association into a transmembrane helical bundle. These systems are being studied to determine the relative thermodynamic contributions of van der Waals interactions, conformational entropy, and polar interactions in the stabilization of membrane proteins. Although the database of thermodynamic information is not yet large, a few generalities are beginning to emerge concerning the energetic differences between membrane and water-soluble proteins: the packing of apolar side chains in the interior of helical membrane proteins plays a smaller, but nevertheless significant, role in stabilizing their structure. Polar, hydrogen-bonded interactions occur less frequently, but, nevertheless, they often provide a strong driving force for folding helix-helix pairs in membrane proteins. These studies are laying the groundwork for the design of sequence motifs that dictate the association of membrane helices.     

Motifs of two small residues can assist but are not sufficient to mediate transmembrane helix interactions

Both experimental and statistical searches for specific motifs that mediate transmembrane helix-helix interactions showed that two glycine residues separated by three intervening residues (GxxxG) provide a framework for specific interactions. Further work suggested that other motifs of small residues can mediate the interaction of transmembrane domains, so that the AxxxA-motif could also drive strong interactions of alpha-helices in soluble proteins. Thus, all these data indicate that a motif of two small residues in a distance of four might be enough to provide a framework for transmembrane helix-helix interaction. To test whether GxxxG is equivalent to (small)xxx(small), we investigated the effect of a substitution of either of the two Gly residues in the glycophorin A GxxxG-motif by Ala or Ser using the recently developed GALLEX system. The results of this mutational study demonstrate that, while a replacement of either of the two Gly by Ala strongly disrupts GpA homo-dimerization, the mutation to Ser partly stabilizes a dimeric structure. We suggest that the Ser residue can form a hydrogen bond with a backbone carbonyl group of the adjacent helix stabilizing a preformed homo-dimer. While (small)xxx(small) serves as a useful clue, the context of adjacent side-chains is essential for stable helix interaction, so each case must be tested.     

Three-dimensional structure of the two peptides that constitute the two-peptide bacteriocin plantaricin EF

The three-dimensional structures of the two peptides plantaricin E (plnE; 33 residues) and plantaricin F (plnF; 34 residues) constituting the two-peptide bacteriocin plantaricin EF (plnEF) have been determined by nuclear magnetic resonance (NMR) spectroscopy in the presence of DPC micelles. PlnE has an N-terminal alpha-helix (residues 10-21), and a C-terminal alpha-helix-like structure (residues 25-31). PlnF has a long central alpha-helix (residues 7-32) with a kink of 38+/-7 degrees at Pro20. There is some flexibility in the helix in the kink region. Both helices in plnE are amphiphilic, while the helix in plnF is polar in its N-terminal half and amphiphilic in its C-terminal half. The alpha-helical content obtained by NMR spectroscopy is in agreement with CD studies. PlnE has two GxxxG motifs which are putative helix-helix interaction motifs, one at residues 5 to 9 and one at residues 20 to 24, while plnF has one such motif at residues 30 to 34. The peptides are flexible in these GxxxG regions. It is suggested that the two peptides lie parallel in a staggered fashion relative to each other and interact through helix-helix interactions involving the GxxxG motifs.     

Influence of hydrophobic matching on association of model transmembrane fragments containing a minimised glycophorin A dimerisation motif

The principles that govern the folding and packing of membrane proteins are still not completely understood. In the present work, we have revisited the glycophorin A (GpA) dimerisation motif that mediates transmembrane (TM) helix association, one of the best-suited models of membrane protein oligomerisation. By using artificial polyleucine TM segments we have demonstrated in this study that a pattern of only five amino acids (GVxxGVxxT) promotes specific dimerisation. Further, we have used this minimised GpA motif to assess the influence of hydrophobic matching on the TM helix packing process in detergent micelles and found that this factor modulates helix-helix association and/or dissociation between TM fragments.