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.     

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.     

Influence of proline residues in transmembrane helix packing

Integral membrane proteins often contain proline residues in their alpha-helical transmembrane (TM) fragments, which may strongly influence their folding and association. Pro-scanning mutagenesis of the helical domain of glycophorin A (GpA) showed that replacement of the residues located at the center abrogates helix packing while substitution of the residues forming the ending helical turns allows dimer formation. Synthetic TM peptides revealed that a point mutation of one of the residues of the dimerization motif (L75P) located at the N-terminal helical turn of the GpA TM fragment, adopts a secondary structure and oligomeric state similar to the wild-type sequence in detergents. In addition, both glycosylation mapping in biological membranes and molecular dynamics showed that the presence of a proline residue at the lipid/water interface has as an effect the extension of the helical end. Thus, helix packing can be an important factor that determines appearance of proline in TM helices. Membrane proteins might accumulate proline residues at the two ends of their TM segments in order to modulate the exposition of key amino acid residues at the interface for molecular recognition events while allowing stable association and native folding.     

Self-association of models of transmembrane domains of ErbB receptors in a lipid bilayer

Association of transmembrane (TM) helices is facilitated by the close packing of small residues present along the amino-acid sequence. Extensive studies have established the role of such small residue motifs (GxxxG) in the dimerization of Glycophorin A (GpA) and helped to elucidate the association of TM domains in the epidermal growth factor family of receptors (ErbBs). Although membrane-mediated interactions are known to contribute under certain conditions to the dimerization of proteins, their effect is often considered nonspecific, and any potential dependence on protein sequence has not been thoroughly investigated. We recently reported that the association of GpA is significantly assisted by membrane-induced contributions as quantified in different lipid bilayers. Herein we extend our studies to explore the origin of these effects and quantify their magnitude using different amino-acid sequences in the same lipid environment. Using a coarse-grained model that accounts for amino-acid specificity, we perform extensive parallel Monte Carlo simulations of ErbB homodimerization in dipalmitoyl-phosphatidylcholine lipid bilayers. A detailed characterization of dimer formation and estimates of the free energy of association reveal that the TM domains show a significant affinity to self-associate in lipid bilayers, in qualitative agreement with experimental findings. The presence of GxxxG motifs enhances favorable protein-protein interactions at short separations. However, the lipid-induced attraction presents a more complex character than anticipated. Depending on the interfacial residues, lipid-entropic contributions support a decrease of separation or a parallel orientation to the membrane normal, with important implications for protein function.     

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.     

The composition rather than position of polar residues (QxxS) drives aspartate receptor transmembrane domain dimerization in vivo

Transmembrane (TM) helix association is an important process affecting the function of many integral membrane proteins. Consequently, aberrations in this process are associated with diseases. Unfortunately, our knowledge of the factors that control this oligomerization process in the membrane milieu is limited at best. Previous studies have shown a role for polar residues in the assembly of synthetic peptides in vitro and the association of de novo-designed TM helices in vivo. Here we examined, for the first time, the involvement of polar residues in the dimerization of a biological TM domain in its natural environment. We analyzed both the involvement of polar residues in the dimerization process and whether their influence is position-dependent. For this purpose, we used the TM domain of the Escherichia coli aspartate receptor (Tar) and 10 single and double mutants. Polar to nonpolar mutations in the sequence demonstrated the role of the QxxS motif in the dimerization of the Tar TM domain. Moreover, creating a GxxxG motif, instead of the polar motif, almost completely abolished dimerization. Swapping positions between two wild-type polar residues did not affect dimerization, implying a similar contribution from both positions. Interestingly, mutants that contain two identical strong polar residues, EE and QQ, demonstrated a substantially higher level of dimerization than a QE mutant, although all three TM domains contain two strong polar residues. This result suggests that, in addition to the polarity of the residues, the formation of symmetric bonds also plays a role in dimer stability. The results of this study may facilitate a rational modulation of membrane protein function for therapeutic purposes.     

Polar residue tagging of transmembrane peptides

Studies that focus on packing interactions between transmembrane (TM) helices in membrane proteins would greatly benefit from the ability to investigate their association and packing interactions in multi-spanning TM domains. However, the production, purification, and characterization of such units have been impeded by their high intrinsic hydrophobicity. We describe the polar tagging approach to biophysical analysis of TM segment peptides, where incorporation of polar residues of suitable type and number at one or both peptide N- and C-termini can serve to counterbalance the apolar nature of a native TM segment, and render it aqueous-soluble. Using the native TM sequences of the human erythrocyte protein glycophorin A (GpA) and bacteriophage M13 major coat protein (MCP), properties of tags such as Lys, His, Asp, sarcosine, and Pro-Gly are evaluated, and general procedures for tagging a given TM segment are presented. Gel-shift assays on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) establish that various tagged GpA TM segments spontaneously insert into micellar membranes, and exhibit native TM dimeric states. Sedimentation equilibrium analytical centrifugation is used to confirm that Lys-tagged GpA peptides retain the native dimer state. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy studies on Lys-tagged TM MCP peptides selectively enriched with N-15 illustrate the usefulness of this system for evaluating monomer-dimer equilibria in micelle environments. The overall results suggest that polar-tagging of hydrophobic (TM) peptides approach constitutes a valuable tool for the study of protein-protein interactions in membranes.     

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.     

Complex Patterns of Histidine, Hydroxylated Amino Acids and the GxxxG Motif Mediate High-affinity Transmembrane Domain Interactions

Specific interactions of transmembrane helices play a pivotal role in the folding and oligomerization of integral membrane proteins. The helix-helix interfaces frequently depend on specific amino acid patterns. In this study, a heptad repeat pattern was randomized with all naturally occurring amino acids to uncover novel sequence motifs promoting transmembrane domain interactions. Self-interacting transmembrane domains were selected from the resulting combinatorial library by means of the ToxR/POSSYCCAT system. A comparison of the amino acid composition of high-and low-affinity sequences revealed that high-affinity transmembrane domains exhibit position-specific enrichment of histidine. Further, sequences containing His preferentially display Gly, Ser, and/or Thr residues at flanking positions and frequently contain a C-terminal GxxxG motif. Mutational analysis of selected sequences confirmed the importance of these residues in homotypic interaction. Probing heterotypic interaction indicated that His interacts in trans with hydroxylated residues. Reconstruction of minimal interaction motifs within the context of an oligo-Leu sequence confirmed that His is part of a hydrogen bonded cluster that is brought into register by the GxxxG motif. Notably, a similar motif contributes to self-interaction of the BNIP3 transmembrane domain.     

The role of individual amino acids in the dimerization of CR4 and ACR4 transmembrane domains

CRINKLY4 is a growth factor-like plant receptor kinase designated as CR4 in Zea mays and ACR4 in Arabidopsis. Using the TOXCAT system, a genetic assay that measures helix interactions in a natural membrane environment, we have previously demonstrated that the dimerization potential of the ACR4 transmembrane (TM) domain is significantly weaker than that of CR4 TM domain, even though 13 of the 24 residues are identical. Neither of the TM domains contain the GxxxG motif that has been shown to be important for the dimerization of the TM segments of several receptors. To further investigate the relationship between protein sequence and dimerization potential, we (a) mutated each of the 11 differing residues in the CR4 TM domain to the corresponding residue of ACR4 (b) made reciprocal mutations in ACR4 and (c) made hybrids consisting of half CR4 and half ACR4 TM domains. Our results suggest that most mutations in ACR4 or CR4 TM domains have low to moderate effects on the dimerization potential and that residues in the N-terminal half of the CR4 TM domain are important for dimerization.     

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.     

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.     

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.     

Molecular Dynamics Simulations of Micelle Formation around Dimeric Glycophorin A Transmembrane Helices

Insertion and formation of membrane proteins involves the interaction of protein helices with one another in lipid environments. Researchers have studied glycophorin A (GpA) transmembrane helices embedded in sodium dodecyl sulfate (SDS) micelles to identify contacts significant for helix dimerization. However, a detailed picture of the conformation and dynamics of the GpA-SDS system cannot be obtained solely through experiment. Molecular dynamics simulations of SDS and a GpA dimer can provide an atomic-level picture of SDS aggregation and helix association. We report 2.5-ns simulations of GpA wild-type and mutants in a preformed micelle as well as a 32-ns simulation showing the formation of a complete micelle around wild-type GpA from an initially random placement of SDS molecules in an aqueous environment. In the latter case, an initial instability of GpA helices in water is reversed after the helices become surrounded by SDS. The properties of the spontaneously formed micelle surrounding the GpA are indistinguishable from those of the preformed micelle surrounding the GpA dimer.     

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.     

Complex interactions at the helix-helix interface stabilize the glycophorin A transmembrane dimer

To explore the residue interactions in the glycophorin A dimerization motif, an alanine scan double mutant analysis at the helix-helix interface was carried out. These data reveal a combination of additive and coupled effects. The majority of the double mutants are found to be equally or slightly more stable than would be predicted by the sum of the energetic cost of the single-point mutants. The proximity of the mutated sites is not related to the presence of coupling between those sites. Previous studies reveal that a single face of the glycophorin A monomer contains a specific glycine-containing motif (GxxxG) that is thought to be a driving force for the association of transmembrane helices. Double mutant cycles suggest that the relationship of the GxxxG motif to the remainder of the helix-helix interface is complex. Sequences containing mutations that abolish the GxxxG motif retain an ability to dimerize, while a sequence containing a GxxxG motif appears unable to form dimers. The energetic effects of weakly coupled and additive double mutants can be explained by changes in van der Waals interactions at the dimer interface. These results emphasize the fact that the sequence context of the dimer interface modulates the strength of the glycophorin A GxxxG-mediated transmembrane dimerization reaction.     

Energetics of the native and non-native states of the glycophorin transmembrane helix dimer

Using an implicit membrane model (IMM1), we examine whether the structure of the transmembrane domain of Glycophorin A (GpA) could be predicted based on energetic considerations alone. The energetics of native GpA shows that van der Waals interactions make the largest contribution to stability. Although specific electrostatic interactions are stabilizing, the overall electrostatic contribution is close to zero. The GXXXG motif contributes significantly to stability, but residues outside this motif contribute almost twice as much. To generate non-native states a global conformational search was done on two segments of GpA: an 18-residue peptide (GpA74-91) that is embedded in the membrane and a 29-residue peptide (GpA70-98) that has additional polar residues flanking the transmembrane region. Simulated annealing was done on a large number of conformations generated from parallel, antiparallel, left- and right-handed starting structures by rotating each helix at 20 degrees intervals around its helical axis. Several crossing points along the helix dimer were considered. For 18-residue parallel topology, an ensemble of native-like structures was found at the lowest effective energy region; the effective energy is lowest for a right-handed structure with an RMSD of 1.0 A from the solid-state NMR structure with correct orientation of the helices. For the 29-residue peptide, the effective energies of several left-handed structures were lower than that of the native, right-handed structure. This could be due to deficiencies in modeling the interactions between charged sidechains and/or omission of the sidechain entropy contribution to the free energy. For 18-residue antiparallel topology, both IMM1 and a Generalized Born model give effective energies that are lower than that of the native structure. In contrast, the Poisson-Boltzmann solvation model gives lower effective energy for the parallel topology, largely because the electrostatic solvation energy is more favorable for the parallel structure. IMM1 seems to underestimate the solvation free energy advantage when the CO and NH dipoles just outside the membrane are parallel. This highlights the importance of electrostatic interactions even when these are not obvious by looking at the structures.     

Helix-helix interactions in membrane proteins: coarse-grained simulations of glycophorin a helix dimerization

Oligomerization of transmembrane (TM) helices is a key stage in the folding of membrane proteins. Glycophorin A (GpA) is a well-documented test system for this process. Coarse-grained molecular dynamics (CG-MD) allows us to simulate the self-assembly of TM helices into dimers, for both wild-type (WT) and mutant GpA sequences. For the WT sequences, dimers formed rapidly and remained stable in all simulations. The resultant dimers exhibited right-handed crossing and the same interhelix contacts as in NMR structures. Simulations of disruptive mutants revealed the dimers were less stable, with values of DeltaDelta G dimerization consistent with experimental data. The dimers of disruptive mutants were distorted relative to the WT and showed left-handed crossing of their helices. CG-MD can therefore be used to explore the interactions of TM helices, an important stage in the folding of membrane proteins. In particular, CG-MD has been shown to be sensitive enough to detect disruptions introduced by mutation. Future refinement of such models via atomistic simulations will enable a multiscale approach to predict the folding of membrane proteins.     

Beta-branched residues adjacent to GG4 motifs promote the efficient association of glycophorin A transmembrane helices

Protein transmenembrane (TM) segments participating in helix-helix packing commonly contain small residue patterns (termed GG4 or "small-xxx-small" motifs) at i and i + 4 positions. Within many TM segments - such as the glycophorin A (GpA) sequence L75IxxGVxxGVxxT87- the G17y-xxx-Gly83 motif often occurs in combination with large, usually beta3-branched aliphatic residues at adjacent positions, typified here by Val30 and Val84 residues. To explore the importance of local P-branched character on GpA dimerization, we made systematic replacements to all 16 combinations of single or double Ile, Leu, and AIa residues at GpA TM Val/Val positions 80 and 84. Using the TOXCAT system to assay self-oligomerization in the Escherichia coli inner membrane--we observed that (i) combinations of Val and lie residues maintained, or improved dimerization levels; (ii) single Ala or Leu mutant combinations with Val or Ile maintained near-wild type dimerization affinities; and (iii) in the absence of beta-branching, i.e., Leu/Leu, Ala/Ala and Ala/Leu combinations, GpA dimerization was significantly diminished. An apparent capacity of lle-containing mutants to increase GpA dimerization versus WT likely arises from improved van der Waals packing (vs. Val) within the locus of helix contact, consistent with correlations we noted in lipid accessibility measurements. Examination of several synthetic peptides with sequences corresponding to selected GpA mutants (VV VI, IV II, and LL) confirmed their dimerization on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The overall results reinforce the importance of a beta-branch-containing "ridge" residue to complement a "small-xxx-small groove" in promotion of TM-TM interactions.