Helix packing moments reveal diversity and conservation in membrane protein structure

Helical membrane proteins are more tightly packed and the packing interactions are more diverse than those found in helical soluble proteins. Based on a linear correlation between amino acid packing values and interhelical propensity, we propose the concept of a helix packing moment to predict the orientation of helices in helical membrane proteins and membrane protein complexes. We show that the helix packing moment correlates with the helix interfaces of helix dimers of single pass membrane proteins of known structure. Helix packing moments are also shown to help identify the packing interfaces in membrane proteins with multiple transmembrane helices, where a single helix can have multiple contact surfaces. Analyses are described on class A G protein-coupled receptors (GPCRs) with seven transmembrane helices. We show that the helix packing moments are conserved across the class A family of GPCRs and correspond to key structural contacts in rhodopsin. These contacts are distinct from the highly conserved signature motifs of GPCRs and have not previously been recognized. The specific amino acid types involved in these contacts, however, are not necessarily conserved between subfamilies of GPCRs, indicating that the same protein architecture can be supported by a diverse set of interactions. In GPCRs, as well as membrane channels and transporters, amino acid residues with small side-chains (Gly, Ala, Ser, Cys) allow tight helix packing by mediating strong van der Waals interactions between helices. Closely packed helices, in turn, facilitate interhelical hydrogen bonding of both weakly polar (Ser, Thr, Cys) and strongly polar (Asn, Gln, Glu, Asp, His, Arg, Lys) amino acid residues. We propose the use of the helix packing moment as a complementary tool to the helical hydrophobic moment in the analysis of transmembrane sequences.     

Helix packing in polytopic membrane proteins: role of glycine in transmembrane helix association.

The nature and distribution of amino acids in the helix interfaces of four polytopic membrane proteins (cytochrome c oxidase, bacteriorhodopsin, the photosynthetic reaction center of Rhodobacter sphaeroides, and the potassium channel of Streptomyces lividans) are studied to address the role of glycine in transmembrane helix packing. In contrast to soluble proteins where glycine is a noted helix breaker, the backbone dihedral angles of glycine in transmembrane helices largely fall in the standard alpha-helical region of a Ramachandran plot. An analysis of helix packing reveals that glycine residues in the transmembrane region of these proteins are predominantly oriented toward helix-helix interfaces and have a high occurrence at helix crossing points. Moreover, packing voids are generally not formed at the position of glycine in folded protein structures. This suggests that transmembrane glycine residues mediate helix-helix interactions in polytopic membrane proteins in a fashion similar to that seen in oligomers of membrane proteins with single membrane-spanning helices. The picture that emerges is one where glycine residues serve as molecular notches for orienting multiple helices in a folded protein complex.     

Analysis and prediction of helix-helix interactions in membrane channels and transporters

Membrane proteins span a large variety of different functions such as cell-surface receptors, redox proteins, ion channels, and transporters. Proteins with functional pores show different characteristics of helix-helix packing as other helical membrane proteins. We found that the helix-helix contacts of 13 nonhomologous high-resolution structures of membrane channels and transporters are mainly accomplished by weakly polar amino acids (G > S > T > F) that preferably create contacts every fourth residue, typical for right-handed helix crossings. There is a strong correlation between the now available biological hydrophobicity scale and the propensities of the weakly polar and hydrophobic residues to be buried at helix-helix interfaces or to be exposed to the lipids in membrane channels and transporters. The polar residues, however, make no major contribution towards the packing of their transmembrane helices, and are therefore subsumed to be primarily exposed to the polar milieu during the folding process. The contact formation of membrane channels and transporters is therefore ruled by the solubility of the residues, which we suppose to be the driving force for the assembly of their transmembrane helices. By contrast, in 14 nonhomologous high-resolution structures of other membrane protein coils, also large and polar amino acids (D > S > M > Q) create characteristic contacts every 3.5th residues, which is a signature for left-handed helix crossings. Accordingly, it seems that dependent on the function, different concepts of folding and stabilization are realized for helical membrane proteins. Using a sequence-based matrix prediction method these differences are exploited to improve the prediction of buried and exposed residues of transmembrane helices significantly. When the sequence motifs typical for membrane channels and transporters were applied for the prediction of helix-helix contacts the quality of prediction rises by 16% to an average value of 76%, compared to the same approach when only single amino acid positions are taken into account.     

Interaction of transmembrane helices by a knobs-into-holes packing characteristic of soluble coiled coils

Membrane-embedded protein domains frequently exist as α-helical bundles, as exemplified by photosynthetic reaction centers, bacteriorhodopsin, and cytochrome C oxidase. The sidechain packing between their transmembrane helices was investigated by a nearest-neighbor analysis which identified sets of interfacial residues for each analyzed helix–helix interface. For the left-handed helix–helix pairs, the interfacial residues almost exclusively occupy positions a, d, e, or g within a heptad motif (abcdefg) which is repeated two to three times for each interacting helical surface. The connectivity between the interfacial residues of adjacent helices conforms to the knobs-into-holes type of sidechain packing known from soluble coiled coils. These results demonstrate on a quantitative basis that the geometry of sidechain packing is similar for left-handed helix–helix pairs embedded in membranes and coiled coils of soluble proteins. The transmembrane helix–helix interfaces studied are somewhat less compact and regular as compared to soluble coiled coils and tolerate all hydrophobic amino acid types to similar degrees. The results are discussed with respect to previous experimental findings which demonstrate that specific interactions between transmembrane helices are important for membrane protein folding and/or oligomerization. Proteins 31:150–159, 1998. © 1998 Wiley-Liss, Inc.     

Molecular dynamics simulations of the E1/E2 transmembrane domain of the Semliki Forest virus

Transmembrane (TM) helix-helix interactions are important for virus budding and fusion. We have developed a simulation strategy that reveals the main features of the helical packing between the TM domains of the two glycoproteins E1 and E2 of the alpha-virus Semliki Forest virus and that can be extrapolated to sketch TM helical packing in other alpha-viruses. Molecular dynamics simulations were performed in wild-type and mutant peptides, both isolated and forming E1/E2 complexes. The simulations revealed that the isolated wild-type E1 peptide formed a more flexible helix than the rest of peptides and that the wild-type E1/E2 complex consists of two helices that intimately pack their N-terminals. The residues located at the interhelical interface displayed the typical motif of the left-handed coiled-coils. These were small and medium residues as Gly, Ala, Ser, and Leu, which also had the possibility to form interhelical Calpha-H...O hydrogen bonds. Results from the mutant complexes suggested that correct packing is a compromise between these residues at both E1 and E2 interhelical interfaces. This compromise allowed prediction of E1-E2 contact residues in the TM spanning domain of other alphaviruses even though the sequence identity of E2 peptides is low in this domain.     

Helical packing patterns in membrane and soluble proteins

This article presents the results of a detailed analysis of helix-helix interactions in membrane and soluble proteins. A data set of interacting pairs of helices in membrane proteins of known structure was constructed and a structure alignment algorithm was used to identify pairs of helices in soluble proteins that superimpose well with pairs of helices in the membrane-protein data set. Most helix pairs in membrane proteins are found to have a significant number of structural homologs in soluble proteins, although in some cases, primarily involving irregular helices, no close homologs exist. An analysis of geometric relationships between interacting helices in the two sets of proteins identifies some differences in the distributions of helix length, interfacial area, packing angle, and distance between the polypeptide backbones. However, a subset of soluble-protein helix pairs that are close structural homologs to membrane-protein helix pairs exhibits distributions that mirror those observed in membrane proteins. The larger average interface size and smaller distance of closest approach seen for helices in membrane proteins appears due in part to a relative enrichment of alanines and glycines, particularly as components of the AxxxA and GxxxG motifs. It is argued that membrane helices are not on average more tightly packed than helices in soluble proteins; they are simply able to approach each other more closely. This enables them to interact over longer distances, which may in turn facilitate their remaining in contact over much of the width of the lipid bilayer. The close structural similarity seen between some pairs of helices in membrane and soluble proteins suggests that packing patterns observed in soluble proteins may be useful in the modeling of membrane proteins. Moreover, there do not appear to be fundamental differences between the magnitude of the forces that drive helix packing in membrane and soluble proteins, suggesting that strategies to make membrane proteins more soluble by mutating surface residues are likely to encounter success, at least in some cases.     

Amino acid composition of parallel helix-helix interfaces

Amino acids at helix-helix parallel interfaces influence arrangement of helices and interhelical angles. Parallel interfaces in 79 proteins were considered. Location of amino acids at the positions analogous to a and d in GCN4 leucine zipper nomenclature shows that certain combinations of amino acids characteristic for parallel packing occur more often than could be expected by chance. Repeating sequence combinations occur at a and d positions of parallel helix-helix interfaces with similar values of interhelical angles not only in homologous proteins but also within the same protein and in nonhomologous proteins. Within each group of observed combinations correlation exists between the size of amino acid and magnitude of the interhelical angle.     

Molecular dynamics (MD) investigations of preformed structures of the transmembrane domain of the oncogenic Neu receptor dimer in a DMPC bilayer

The critical Val/Glu mutation in the membrane spanning domain of the rat Neu receptor confers the ability for ligand-independent signaling and leads to increased dimerization and transforming ability. There is evidence that the two transmembrane interacting helices play a role in receptor activation by imposing orientation constraints to the intracellular tyrosine kinase domains. By using MD simulations we have attempted to discriminate between correct and improper helix-helix packing by examining the structural and energetic properties of preformed left-handed and right-handed structures in a fully hydrated DMPC bilayer. The best energetic balance between the residues at the helix-helix interface and the residues exposed to the lipids is obtained for helices in symmetrical left-handed interactions packed together via Glu side chain/Ala backbone interhelical hydrogen bonds. Analyses demonstrate the importance of the ATVEG motif in helix-helix packing and point to additional contacting residues necessary for association. Our findings, all consistent with experimental data, suggest that a symmetrical left-handed structure of the helices could be the transmembrane domain configuration that promotes receptor activation and transformation. The present study may provide further insight into signal transduction mechanisms of the ErbB/Neu receptors.     

Molecular packing and packing defects in helical membrane proteins

The packing of helices spanning lipid bilayers is crucial for the stability and function of alpha-helical membrane proteins. Using a modified Voronoi procedure, we calculated packing densities for helix-helix contacts in membrane spanning domains. Our results show that the transmembrane helices of protein channels and transporters are significantly more loosely packed compared with helices in globular proteins. The observed packing deficiencies of these membrane proteins are also reflected by a higher amount of cavities at functionally important sites. The cavities positioned along the gated pores of membrane channels and transporters are noticeably lined by polar amino acids that should be exposed to the aqueous medium when the protein is in the open state. In contrast, nonpolar amino acids surround the cavities in those protein regions where large rearrangements are supposed to take place, as near the hinge regions of transporters or at restriction sites of protein channels. We presume that the observed deficiencies of helix-helix packing are essential for the helical mobility that sustains the function of many membrane protein channels and transporters.     

Thermostability of membrane protein helix-helix interaction elucidated by statistical analysis

A prerequisite for the survival of (micro)organisms at high temperatures is an adaptation of protein stability to extreme environmental conditions. In contrast to soluble proteins, where many factors have already been identified, the mechanisms by which the thermostability of membrane proteins is enhanced are almost unknown. The hydrophobic membrane environment constrains possible stabilizing factors for transmembrane domains, so that a difference might be expected between soluble and membrane proteins. Here we present sequence analysis of predicted transmembrane helices of the genomes from eight thermophilic and 12 mesophilic organisms. A comparison of the amino acid compositions indicates that more polar residues can be found in the transmembrane helices of thermophilic organisms. Particularly, the amino acids aspartic acid and glutamic acid replace the corresponding amides. Cysteine residues are found to be significantly decreased by about 70% in thermophilic membrane domains suggesting a non-specific function of most cysteine residues in transmembrane domains of mesophilic organisms. By a pair-motif analysis of the two sets of transmembrane helices, we found that the small residues glycine and serine contribute more to transmembrane helix-helix interactions in thermophilic organisms. This may result in a tighter packing of the helices allowing more hydrogen bond formation.     

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.     

Higher-order interhelical spatial interactions in membrane proteins

Higher-order interactions are important for protein folding and assembly. We introduce the concept of interhelical three-body interactions as derived from Delaunay triangulation and alpha shapes of protein structures. In addition to glycophorin A, where triplets are strongly correlated with protein stability, we found that tight interhelical triplet interactions exist extensively in other membrane proteins, where many types of triplets occur far more frequently than in soluble proteins. We developed a probabilistic model for estimating the value of membrane helical interaction triplet (MHIT) propensity. Because the number of known structures of membrane proteins is limited, we developed a bootstrap method for determining the 95% confidence intervals of estimated MHIT values. We identified triplets that have high propensity for interhelical interactions and are unique to membrane proteins, e.g. AGF, AGG, GLL, GFF and others. A significant fraction (32%) of triplet types contains triplets that may be involved in interhelical hydrogen bond interactions, suggesting the prevalent and important roles of H-bond in the assembly of TM helices. There are several well-defined spatial conformations for triplet interactions on helices with similar parallel or antiparallel orientations and with similar right-handed or left-handed crossing angles. Often, they contain small residues and correspond to the regions of the closest contact between helices. Sequence motifs such as GG4 and AG4 can be part of the three-body interactions that have similar conformations, which in turn can be part of a higher-order cooperative four residue spatial motif observed in helical pairs from different proteins. In many cases, spatial motifs such as serine zipper and polar clamp are part of triplet interactions. On the basis of the analysis of the archaeal rhodopsin family of proteins, tightly packed triplet interactions can be achieved with several different choices of amino acid residues.     

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.     

Structural and functional roles of small group-conserved amino acids present on helix-H7 in the β(2)-adrenergic receptor

Sequence analysis of the class A G protein-coupled receptors (GPCRs) reveals that most of the highly conserved sites are located in the transmembrane helices. A second level of conservation exists involving those residues that are conserved as a group characterized by small and/or weakly polar side chains (Ala, Gly, Ser, Cys, Thr). These positions can have group conservation levels of up to 99% across the class A GPCRs and have been implicated in mediating helix-helix interactions in membrane proteins. We have previously shown that mutation of group-conserved residues present on transmembrane helices H2-H4 in the β(2)-adrenergic receptor (β(2)-AR) can influence both receptor expression and function. We now target the group-conserved sites, Gly315(7.42) and Ser319(7.46), on H7 for structure-function analysis. Replacing Ser319(7.46) with smaller amino acids (Ala or Gly) did not influence the ability of the mutant receptors to bind to the antagonist dihydroalprenolol (DHA) but resulted in ~15-20% agonist-independent activity. Replacement of Ser319(7.46) with the larger amino acid leucine lowered the expression of the S319L mutant and its ability to bind DHA. Both the G315A and G315S mutants also exhibited agonist-independent signaling, while the G315L mutant did not show specific binding to DHA. These data indicate that Gly315(7.42) and Ser319(7.46) are stabilizing β(2)-AR in an inactive conformation. We discuss our results in the context of van der Waals interactions of Gly315(7.42) with Trp286(6.48) and hydrogen bonding interactions of Ser319(7.46) with amino acids on H1-H2-H7 and with structural water.     

Sequence dependence of BNIP3 transmembrane domain dimerization implicates side-chain hydrogen bonding and a tandem GxxxG motif in specific helix-helix interactions

The transmembrane domain of the pro-apoptotic protein BNIP3 self-associates strongly in membranes and in detergents. We have used site-directed mutagenesis to analyze the sequence dependence of BNIP3 transmembrane domain dimerization, from which we infer the physical basis for strong and specific helix-helix interactions in this system. Hydrophobic substitutions identify six residues as critical to dimerization, and the pattern of sensitive residues suggests that the BNIP3 helices interact at a right-handed crossing angle. Based on the dimerization propensities of single point mutants, we propose that: polar residues His173 and Ser172 make inter-monomer hydrogen bonds to one another through their side-chains; Ala176, Gly180, and Gly184 form a tandem GxxxG motif that allows close approach of the helices; and Ile183 makes inter-monomer van der Waals contacts. Since neither the tandem GxxxG motif nor the hydrogen bonding pair is sufficient to drive dimerization, our results demonstrate the importance of sequence context for either hydrogen bonding or GxxxG motif involvement in BNIP3 transmembrane helix-helix interactions. In this study, hydrophobic substitutions away from the six interfacial positions have almost no effect on dimerization, confirming the expectation that hydrophobic replacements affect helix-helix interactions only if they interfere with packing or hydrogen bonding by interfacial residues. However, changes to slightly polar residues are somewhat disruptive even when located away from the interface, and the degree of disruption correlates with the decrease in hydrophobicity. Changing the hydrophobicity of the BNIP3 transmembrane domain alters its helicity and protection of its backbone amides. We suggest that polar substitutions decrease the fraction of dimer by stabilizing an unfolded monomeric state of the transmembrane span, rather than by affecting helix-helix interactions. This result has broad implications for interpreting the sequence dependence of membrane protein stability in detergents.     

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.     

Transmembrane helix packing of ErbB/Neu receptor in membrane environment: a molecular dynamics study

Dimerization or oligomerization of the ErbB/Neu receptors are necessary but not sufficient for initiation of receptor signaling. The two intracellular domains must be properly oriented for the juxtaposition of the kinase domains allowing trans-phosphorylation. This suggests that the transmembrane (TM) domain acts as a guide for defining the proper orientation of the intracellular domains. Two structural models, with the two helices either in left-handed or in right-handed coiling have been proposed as the TM domain structure of the active receptor. Because experimental data do not distinguish clearly helix-helix packing, molecular dynamics (MD) simulations are used to investigate the energetic factors that drive Neu TM-TM interactions of the wild and the oncogenic receptor (Val664/Glu mutation) in DMPC or in POPC environments. MD results indicate that helix-lipid interactions in the bilayer core are extremely similar in the two environments and raise the role of the juxtamembrane residues in helix insertion and helix-helix packing. The TM domain shows a greater propensity to adopt a left-handed structure in DMPC, with helices in optimal position for strong inter-helical Hbonds induced by the Glu mutation. In POPC, the right-handed structure is preferentially formed with the participation of water in inter-helical Hbonds. The two structural arrangements of the Neu(TM) helices both with GG4 residue motif in close contact at the interface are permissible in the membrane environment. According to the hypothesis of a monomer-dimer equilibrium of the proteins it is likely that the bilayer imposes structural constraints that favor dimerization-competent structure responsible of the proper topology necessary for receptor activation.     

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.     

Role of amino acid residues in left-handed helical conformation for the conformational stability of a protein

Our previous study of six non-Gly to Gly/Ala mutant human lysozymes in a left-handed helical region showed that only one non-Gly residue at a rigid site had unfavorable strain energy as compared with Gly at the same position (Takano et al., Proteins 2001; 44:233-243). To further examine the role of left-handed residues in the conformational stability of a protein, we constructed ten Gly to Ala mutant human lysozymes. Most Gly residues in human lysozyme are located in the left-handed helix region. The thermodynamic parameters for denaturation and crystal structures were determined by differential scanning calorimetry and X-ray analysis, respectively. The difference in denaturation Gibbs energy (DeltaDeltaG) for the ten Gly to Ala mutants ranged from + 1.9 to -7.5 kJ/mol, indicating that the effect of the mutation depends on the environment of the residue. We confirm that Gly in a left-handed region is more favorable at rigid sites than non-Gly, but there is little difference in energetic cost between Gly and non-Gly at flexible sites. The present results indicate that dihedral angles in the backbone conformation and also the flexibility at the position should be considered for analyses of protein stability, and protein structural determination, prediction, and design.     

Proline residues in transmembrane alpha helices affect the folding of bacteriorhodopsin

Proline residues occur frequently in transmembrane alpha helices, which contrasts with their behaviour as helix-breakers in water-soluble proteins. The three membrane-embedded proline residues of bacteriorhodopsin have been replaced individually by alanine and glycine to give P50A, or P50G on helix B, P91A, or P91G on helix C, and P186A or P186G on helix F, and the effect on the protein folding kinetics has been investigated. The rate-limiting apoprotein folding step, which results in formation of a seven transmembrane, alpha helical state, was slower than wild-type protein for the Pro50 and Pro91 mutants, regardless of whether they were mutated to Ala or Gly. These proline residues give rise to several inter-helix contacts, which are therefore important in folding to the seven transmembrane helix state. No evidence for cis-trans isomerisations of the peptidyl prolyl bonds was found during this rate-limiting apoprotein folding step. Mutations of all three membrane-embedded proline residues affected the subsequent retinal binding and final folding to bacteriorhodopsin, suggesting that these proline residues contribute to formation of the retinal binding pocket within the helix bundle, again via helix/helix interactions. These results point to proline residues in transmembrane alpha helices being important in the folding of integral membrane proteins. The helix/helix interactions and hydrogen bonds that arise from the presence of proline residues in transmembrane alpha helices can affect the formation of transmembrane alpha helix bundles as well as cofactor binding pockets.