Sequence context modulates the stability of a GxxxG-mediated transmembrane helix-helix dimer

To quantify the relationship between sequence and transmembrane dimer stability, a systematic mutagenesis and thermodynamic study of the protein-protein interaction residues in the glycophorin A transmembrane helix-helix dimer was carried out. The results demonstrate that the glycophorin A transmembrane sequence dimerizes when its GxxxG motif is abolished by mutation to large aliphatic residues, suggesting that the sequence encodes an intrinsic propensity to self-associate independent of a GxxxG motif. In the presence of an intact GxxxG motif, the glycophorin A dimer stability can be modulated over a span of -0.5 kcal mol(-1) to +3.2 kcal mol(-1) by mutating the surrounding sequence context. Thus, these flanking residues play an active role in determining the transmembrane dimer stability. To assess the structural consequences of the thermodynamic effects of mutations, molecular models of mutant transmembrane domains were constructed, and a structure-based parameterization of the free energy change due to mutation was carried out. The changes in association free energy for glycophorin A mutants can be explained primarily by changes in packing interactions at the protein-protein interface. The energy cost of removing favorable van der Waals interactions was found to be 0.039 kcal mol(-1) per A2 of favorable occluded surface area. The value corresponds well with estimates for mutations in bacteriorhodopsin as well as for those mutations in the interiors of soluble proteins that create packing defects.     

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.     

Intermonomer Hydrogen Bonds Enhance GxxxG-Driven Dimerization of the BNIP3 Transmembrane Domain: Roles for Sequence Context in Helix-Helix Association in Membranes

We determined the sequence dependence of human BNIP3 transmembrane domain dimerization using the biological assay TOXCAT. Mutants in which intermonomer hydrogen bonds between Ser172 and His173 are abolished show moderate interaction, indicating that side-chain hydrogen bonds contribute to dimer stability but are not essential to dimerization. Mutants in which a GxxxG motif composed of Gly180 and Gly184 has been abolished show little or no interaction, demonstrating the critical nature of the GxxxG motif to BNIP3 dimerization. These findings show that side-chain hydrogen bonds can enhance the intrinsic dimerization of a GxxxG motif and that sequence context can control how hydrogen bonds influence helix-helix interactions in membranes. The dimer interface mapped by TOXCAT mutagenesis agrees closely with the interfaces observed in the NMR structure and inferred from mutational analysis of dimerization on SDS-PAGE, showing that the native dimer structure is retained in detergents. We show that TOXCAT and SDS-PAGE give complementary and consistent information about BNIP3 transmembrane domain dimerization: TOXCAT is insensitive to mutations that have modest effects on self-association in detergents but readily discriminates among mutations that completely disrupt detergent-resistant dimerization. The close agreement between conclusions reached from TOXCAT and SDS-PAGE data for BNIP3 suggests that accurate estimates of the relative effects of mutations on native-state protein-protein interactions can be obtained even when the detergent environment is strongly disruptive.     

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.     

The GxxxG-containing transmembrane domain of the CCK4 oncogene does not encode preferential self-interactions

The recently cloned colon carcinoma kinase 4 (CCK4) oncogene contains an evolutionarily conserved GxxxG motif in its single transmembrane domain (TMD). It has previously been suggested that this pairwise glycine motif may provide a strong driving force for transmembrane helix-helix interactions. Since CCK4 is thought to represent a new member of the receptor tyrosine kinase family, interactions between the TMDs may be important in receptor self-association and activation of signal transduction pathways. To determine whether this conserved CCK4 TMD can drive protein-protein interactions, we have carried out a thermodynamic study using the TMD expressed as a Staphylococcal nuclease (SN) fusion protein. Similar SN-TMD fusion proteins have been used to determine the sequence specificity and thermodynamics of transmembrane helix-helix interactions in a number of membrane proteins, including glycophorin A. Using sedimentation equilibrium in C14 betaine micelles, we discovered that the CCK4 TMD is unable to drive strong protein-protein interactions. At high protein/detergent ratios, the SN-CCK4 fusion protein will dimerize, but a stochastic model for protein association in micelles can explain the observed dimer population. For low-affinity interactions such as the one studied here, an understanding of this discrete stochastic distribution of membrane proteins in micelles is important for distinguishing between preferential and random self-interactions, which can both influence the oligomeric population. The lack of a thermodynamically meaningful self-association propensity for the CCK4 TMDs demonstrates that a GxxxG motif is not sufficient to drive transmembrane helix-helix interactions.     

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.     

Changes in apparent free energy of helix-helix dimerization in a biological membrane due to point mutations

We present an implementation of the TOXCAT membrane protein self-association assay that measures the change in apparent free energy of transmembrane helix dimerization caused by point mutations. Quantifying the reporter gene expression from cells carrying wild-type and mutant constructs shows that single point mutations that disrupt dimerization of the transmembrane domain of glycophorin A reproducibly lower the TOXCAT signal more than 100-fold. Replicate cultures can show up to threefold changes in the level of expression of the membrane bound fusion construct, and correcting for these variations improves the precision of the calculated apparent free energy change. The remarkably good agreement between our TOXCAT apparent free energy scale and free energy differences from sedimentation equilibrium studies for point mutants of the glycophorin A transmembrane domain dimer indicate that sequence changes usually affect membrane helix-helix interactions quite similarly in these two very different environments. However, the effects of point mutations at threonine 87 suggest that intermonomer polar contacts by this side-chain contribute significantly to dimer stability in membranes but not in detergents. Our findings demonstrate that a comparison of quantitative measurements of helix-helix interactions in biological membranes and genuine thermodynamic data from biophysical measurements on purified proteins can elucidate how changes in the lipidic environment modulate membrane protein stability.     

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.     

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.     

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.     

Mapping the Homodimer Interface of an Optimized,Artificial, Transmembrane Protein Activator of the Human Erythropoietin Receptor

Transmembrane proteins constitute a large fraction of cellular proteins, and specific interactions involving membrane-spanning protein segments play an important role in protein oligomerization, folding, and function. We previously isolated an artificial, dimeric, 44-amino acid transmembrane protein that activates the human erythropoietin receptor (hEPOR) in trans. This artificial protein supports limited erythroid differentiation of primary human hematopoietic progenitor cells in vitro, even though it does not resemble erythropoietin, the natural ligand of this receptor. Here, we used a directed-evolution approach to explore the structural basis for the ability of transmembrane proteins to activate the hEPOR. A library that expresses thousands of mutants of the transmembrane activator was screened for variants that were more active than the original isolate at inducing growth factor independence in mouse cells expressing the hEPOR. The most active mutant, EBC5-16, supports erythroid differentiation in human cells with activity approaching that of EPO, as assessed by cell-surface expression of glycophorin A, a late-stage marker of erythroid differentiation. EBC5-16 contains a single isoleucine to serine substitution at position 25, which increases its ability to form dimers. Genetic studies confirmed the importance of dimerization for activity and identified the residues constituting the homodimer interface of EBC5-16. The interface requires a GxxxG dimer packing motif and a small amino acid at position 25 for maximal activity, implying that tight packing of the EBC5-16 dimer is a crucial determinant of activity. These experiments identified an artificial protein that causes robust activation of its target in a natural host cell, demonstrated the importance of dimerization of this protein for engagement of the hEPOR, and provided the framework for future structure-function studies of this novel mechanism of receptor activation.     

Structural model for an AxxxG-mediated dimer of surfactant-associated protein C.

The pulmonary surfactant prevents alveolar collapse and is required for normal pulmonary function. One of the important components of the surfactant besides phospholipids is surfactant-associated protein C (SP-C). SP-C shows complex oligomerization behavior and a transition to beta-amyloid-like fibril structures, which are not yet fully understood. Besides this nonspecific oligomerization, MS and chemical cross-linking data combined with CD spectra provide evidence of a specific, mainly alpha-helical, dimer at low to neutral pH. Furthermore, resistance to CNBr cleavage and dual NMR resonances of porcine and human recombinant SP-C with Met32 replaced by isoleucine point to a dimerization site located at the C-terminus of the hydrophobic alpha-helix of SP-C, where a strictly conserved heptapeptide sequence is found. Computational docking of two SP-C helices, described here, reveals a dimer with a helix-helix interface that strikingly resembles that of glycophorin A and is mediated by an AxxxG motif similar to the experimentally determined GxxxG pattern of glycophorin A. It is highly likely that mature SP-C adopts such a dimeric structure in the lamellar bilayer systems found in the surfactant. Dimerization has been shown in previous studies to have a role in sorting and trafficking of SP-C and may also be important to the surfactant function of this protein.     

A trimerizing GxxxG motif is uniquely inserted in the severe acute respiratory syndrome (SARS) coronavirus spike protein transmembrane domain

In an attempt to understand what distinguishes severe acute respiratory syndrome (SARS) coronavirus (SCoV) from other members of the coronaviridae, we searched for elements that are unique to its proteins and not present in any other family member. We identified an insertion of two glycine residues, forming the GxxxG motif, in the SCoV spike protein transmembrane domain (TMD), which is not found in any other coronavirus. This surprising finding raises an "oligomerization riddle": the GxxxG motif is a known dimerization signal, while the SCoV spike protein is known to be trimeric. Using an in vivo assay, we found that the SCoV spike protein TMD is oligomeric and that this oligomerization is driven by the GxxxG motif. We also found that the GxxxG motif contributes toward the trimerization of the entire spike protein; in that, mutations in the GxxxG motif decrease trimerization of the full-length protein expressed in mammalian cells. Using molecular modeling, we show that the SCoV spike protein TMD adopts a distinct and unique structure as opposed to all other coronaviruses. In this unique structure, the glycine residues of the GxxxG motif are facing each other, enhancing helix-helix interactions by allowing for the close positioning of the helices. This unique orientation of the glycine residues also stabilizes the trimeric bundle during multi-nanosecond molecular dynamics simulation in a hydrated lipid bilayer. To the best of our knowledge, this is the first demonstration that the GxxxG motif can potentiate other oligomeric forms beside a dimer. Finally, according to recent studies, the stabilization of the trimeric bundle is linked to a higher fusion activity of the spike protein, and the possible influence of the GxxxG motif on this feature is discussed.     

Computation and mutagenesis suggest a right-handed structure for the synaptobrevin transmembrane dimer.

Biological membrane fusion involves a highly precise and ordered set of protein-protein interactions. Synaptobrevin is a key player in this process. Mutagenesis studies of its single transmembrane segment suggest that it dimerizes in a sequence specific manner. Using the computational methods developed for the successful structure prediction of the glycophorin A transmembrane dimer, we have calculated a structural model for the synaptobrevin dimer. Our computational search yields a well-populated cluster of right-handed structures consistent with the experimentally determined dimerization motif. The three-dimensional structure contains an interface formed primarily by leucine and isoleucine side-chain atoms and has no interhelical hydrogen bonds. The model is the first three-dimensional picture of the synaptobrevin transmembrane dimer and provides a basis for further focused experimentation on its structure and association thermodynamics.     

Modulation of glycophorin A transmembrane helix interactions by lipid bilayers: molecular dynamics calculations

Starting from the glycophorin A dimer structure determined by NMR, we performed simulations of both dimer and monomer forms in explicit lipid bilayers with constant normal pressure, lateral area, and temperature using the CHARMM potential. Analysis of the trajectories in four different lipids reveals how lipid chain length and saturation modulate the structural and energetic properties of transmembrane helices. Helix tilt, helix-helix crossing angle, and helix accessible volume depend on lipid type in a manner consistent with hydrophobic matching concepts: the most relevant lipid property appears to be the bilayer thickness. Although the net helix-helix interaction enthalpy is strongly attractive, analysis of residue-residue interactions reveals significant unfavorable electrostatic repulsion between interfacial glycine residues previously shown to be critical for dimerization. Peptide volume is nearly conserved upon dimerization in all lipid types, indicating that the monomeric helices pack equally well with lipid as dimer helices do with one another. Enthalpy calculations indicate that the helix-environment interaction energy is lower in the dimer than in the monomer form, when solvated by unsaturated lipids. In all lipid environments there is a marked preference for lipids to interact with peptide predominantly through one rather than both acyl chains. Although our trajectories are not long enough to allow a full thermodynamic treatment, these results demonstrate that molecular dynamics simulations are a powerful method for investigating the protein-protein, protein-lipid, and lipid-lipid interactions that determine the structure, stability and dynamics of transmembrane alpha-helices in membranes.     

Role of Side-Chain Conformational Entropy in Transmembrane Helix Dimerization of Glycophorin A

Dimerization of the transmembrane domain of glycophorin A is mediated by a seven residue motif LIxxGVxxGVxxT through a combination of van der Waals and hydrogen bonding interactions. One of the unusual features of the motif is the large number of β-branched amino acids that may limit the entropic cost of dimerization by restricting side-chain motion in the monomeric transmembrane helix. Deuterium NMR spectroscopy is used to characterize the dynamics of fully deuterated Val80 and Val84, two essential amino acids of the dimerization motif. Deuterium spectra of the glycophorin A transmembrane dimer were obtained using synthetic peptides corresponding to the transmembrane sequence containing either perdeuterated Val80 or Val84. These data were compared with spectra of monomeric glycophorin A peptides deuterated at Val84. In all cases, the deuterium line shapes are characterized by fast methyl group rotation with virtually no motion about the C_α-C_β bond. This is consistent with restriction of the side chain in both the monomer and dimer due to intrahelical packing interactions involving the β-methyl groups, and indicates that there is no energy cost associated with dimerization due to loss of conformational entropy. In contrast, deuterium NMR spectra of Met81 and Val82, in the lipid interface, reflected greater motional averaging and fast exchange between different side-chain conformers.     

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.     

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.     

Influence of the C-terminus of the glycophorin A transmembrane fragment on the dimerization process

The monomer-dimer equilibrium of the glycophorin A (GpA) transmembrane (TM) fragment has been used as a model system to investigate the amino acid sequence requirements that permit an appropriate helix-helix packing in a membrane-mimetic environment. In particular, we have focused on a region of the helix where no crucial residues for packing have been yet reported. Various deletion and replacement mutants in the C-terminal region of the TM fragment showed that the distance between the dimerization motif and the flanking charged residues from the cytoplasmic side of the protein is important for helix packing. Furthermore, selected GpA mutants have been used to illustrate the rearrangement of TM fragments that takes place when leucine repeats are introduced in such protein segments. We also show that secondary structure of GpA derivatives was independent from dimerization, in agreement with the two-stage model for membrane protein folding and oligomerization.     

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.