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.     

Interhelical hydrogen bonding drives strong interactions in membrane proteins

Polar residues in transmembrane alpha-helices may strongly influence the folding or association of integral membrane proteins. To test whether a motif that promotes helix association in a soluble protein could do the same within a membrane, we designed a model transmembrane helix based on the GCN4 leucine zipper. We found in both detergent micelles and biological membranes that helix association is driven strongly by asparagine, independent of the rest of the hydrophobic leucine and/or valine sequence. Hydrogen bonding between membrane helices gives stronger associations than the packing of surfaces in glycophorin A helices, creating an opportunity to stabilize structures, but also implying a danger that non-specific interactions might occur. Thus, membrane proteins may fold to avoid exposure of strongly hydrogen bonding groups at their lipid exposed surfaces.     

Implications of threonine hydrogen bonding in the glycophorin A transmembrane helix dimer

The transmembrane helix of glycophorin A contains a seven-residue motif, LIxxGVxxGVxxT, that mediates protein dimerization. Threonine is the only polar amino acid in this motif with the potential to stabilize the dimer through hydrogen-bonding interactions. Polarized Fourier transform infrared spectroscopy is used to establish a robust protocol for incorporating glycophorin A transmembrane peptides into membrane bilayers. Analysis of the dichroic ratio of the 1655-cm(-1) amide I vibration indicates that peptides reconstituted by detergent dialysis have a transmembrane orientation with a helix crossing angle of <35 degrees. Solid-state nuclear magnetic resonance spectroscopy is used to establish high resolution structural restraints on the conformation and packing of Thr-87 in the dimer interface. Rotational resonance measurement of a 2.9-A distance between the gamma-methyl and backbone carbonyl carbons of Thr-87 is consistent with a gauche- conformation for the chi1 torsion angle. Rotational-echo double-resonance measurements demonstrate close packing (4.0 +/- 0.2 A) of the Thr-87 gamma-methyl group with the backbone nitrogen of Ile-88 across the dimer interface. The short interhelical distance places the beta-hydroxyl of Thr-87 within hydrogen-bonding range of the backbone carbonyl of Val-84 on the opposing helix. These results refine the structure of the glycophorin A dimer in membrane bilayers and highlight the complementary role of small and polar residues in the tight association of transmembrane helices in membrane proteins.     

Energetics and stability of transmembrane helix packing: a replica-exchange simulation with a knowledge-based membrane potential

The energetics and stability of the packing of transmembrane helices were investigated by Monte Carlo simulations with the replica-exchange method. The helices were modeled with a united atom representation, and the CHARMM19 force field was employed. Based on known experimental structures of membrane proteins, an implicit knowledge-based potential was developed to describe the helix-membrane interactions at the residue level, whose validity was tested through prediction of the orientations when single helices were inserted into a membrane. Two systems were studied in this article, namely the glycophorin A dimer, and helices A and B of Bacteriorhodopsin. For the glycophorin A dimer, the most stable structure (0.5 A away from the experimental structure) is mainly stabilized by the favorable helix-helix interactions, and has the most population regardless of the helix-membrane interaction. However, for helices A and B of Bacteriorhodopsin, it was found that the packing determined by helix-helix interactions is nonspecific, and a native-like structure (0.2 A from the experimental one) can be identified from several structural analogs as the most stable one only after applying the membrane potential. Our results suggest that the contribution from the helix-membrane interaction could be critical in the correct packing of transmembrane helices in the membrane.     

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.     

The stability of transmembrane helix interactions measured in a biological membrane

Despite some promising progress in the understanding of membrane protein folding and assembly, there is little experimental information regarding the thermodynamic stability of transmembrane helix interactions and even less on the stability of transmembrane helix-helix interactions in a biological membrane. Here we describe an approach that allows quantitative measurement of transmembrane helix interactions in a biological membrane, and calculation of changes in the interaction free energy resulting from substitution of single amino acids. Dimerization of several variants of the glycophorin A transmembrane domain are characterized and compared to the wild-type (wt) glycophorin A transmembrane helix dimerization. The calculated DeltaDeltaG(app) values are further compared with values found in the literature. In addition, we compare interactions between the wt glycophorin A transmembrane domain and helices in which critical glycine residues are replaced by alanine or serine, respectively. The data demonstrate that replacement of the glycine residues by serine is less destabilizing than replacement by alanine with a DeltaDeltaG(app) value of about 0.4 kcal/mol. Our study comprises the first measurement of a transmembrane helix interaction in a biological membrane, and we are optimistic that it can be further developed and applied.     

Structure of the transmembrane dimer interface of glycophorin A in membrane bilayers

The hydrophobic transmembrane domain of glycophorin A contains a sequence motif that mediates dimerization in membrane environments. Long-range interhelical distance measurements using magic angle spinning NMR spectroscopy provide high-resolution structural constraints on the packing of the dimer interface in membrane bilayers. We show that direct packing contacts occur between glycine residues at positions 79 and 83 in the transmembrane sequence. Additional interhelical constraints between Ile76 and Gly79 and between Val80 and Gly83 restrict the rotational orientation and crossing angle of the interacting helices. These results refine our previously proposed structure of the glycophorin A dimer [Smith, S. O., and Bormann, B. J. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 488-491] which revealed that the methyl groups of Val80 and Val84 are packed against Gly79 and Gly83, respectively.     

A simple method for modeling transmembrane helix oligomers

We describe an effective procedure for modeling the structures of simple transmembrane helix homo-oligomers. The method differs from many previous approaches in that the only structural constraint we use to help select the correct model is the oligomerization state of the protein. The method involves the following steps: (1) perform 100-250 independent Monte Carlo energy minimizations of helix pairs to produce a large collection of well-packed structures; (2) filter the minimized structures to find those that are consistent with the expected symmetry of the oligomer; (3) cluster the structures that pass the symmetry filter; and (4) select a representative of the most populous cluster as the final prediction. We applied the method to the transmembrane helices of five proteins and compare our results to the available experimental data. Our predictions of glycophorin A, neu, the M2 channel and phospholamban resulted in a single model for each protein that agreed with the experimental results. In the case of erbB-2, however, we obtained three structurally distinct clusters of approximately equal sizes, so it was not possible to identify a clearly favored structure. This may reflect a real heterogeneity of packing modes for erbB-2, which is known to interact with different receptor subunits. Our method should be useful for obtaining structural models of transmembrane domains, improving our understanding of structure/function relationships for particular membrane proteins.     

Folding elastic transmembrane helices to fit in a low-resolution image by electron microscopy

Structure prediction of membrane proteins could be constrained and thereby improved by introducing data of the observed molecular shape. We studied a coarse-grained molecular model that relied on residue-based dummy atoms to fold the transmembrane helices of a protein in the observed molecular shape. Based on the inter-residue potential, the α-helices were folded to contact each other in a simulated annealing protocol to search optimized conformation. Fitting the model into a three-dimensional volume was tested for proteins with known structures and resulted in a fairly reasonable arrangement of helices. In addition, the constraint to the packing transmembrane helix with the two-dimensional region was tested and found to work as a very similar folding guide. The obtained models nicely represented α-helices with the desired slight bend. Our structure prediction method for membrane proteins well demonstrated reasonable folding results using a low-resolution structural constraint introduced from recent cell-surface imaging techniques.     

Polar/Ionizable Residues in Transmembrane Segments: Effects on Helix-Helix Packing

The vast majority of membrane proteins are anchored to biological membranes through hydrophobic α-helices. Sequence analysis of high-resolution membrane protein structures show that ionizable amino acid residues are present in transmembrane (TM) helices, often with a functional and/or structural role. Here, using as scaffold the hydrophobic TM domain of the model membrane protein glycophorin A (GpA), we address the consequences of replacing specific residues by ionizable amino acids on TM helix insertion and packing, both in detergent micelles and in biological membranes. Our findings demonstrate that ionizable residues are stably inserted in hydrophobic environments, and tolerated in the dimerization process when oriented toward the lipid face, emphasizing the complexity of protein-lipid interactions in biological membranes.     

Robust Driving Forces for Transmembrane Helix Packing

The packing structures of transmembrane helices are traditionally attributed to patterns in residues along the contact surface. In this view, besides keeping the helices confined in the membrane, the bilayer has only a minor effect on the helices structure. Here, we use two different approaches to show that the lipid environment has a crucial effect in determining the cross-angle distribution of packed helices. We analyzed structural data of a membrane proteins database. We show that the distribution of cross angles of helix pairs in this database is statistically indistinguishable from the cross-angle distribution of two noninteracting helices imbedded in the membrane. These results suggest that the cross angle is, to a large extent, determined by the tilt angle of the individual helices. We test this hypothesis using molecular simulations of a coarse-grained model that contains no specific residue interactions. These simulations reproduce the same cross-angle distribution as found in the database. As the tilt angle of a helix is dominated by hydrophobic mismatch between the protein and surrounding lipids, our results indicate that hydrophobic mismatch is the dominant factor guiding the transmembrane helix packing. Other short-range forces might then fine-tune the structure to its final configuration.     

Proline-induced distortions of transmembrane helices

Proline residues in the transmembrane (TM) alpha-helices of integral membrane proteins have long been suspected to play a key role for helix packing and signal transduction by inducing regions of helix distortion and/or dynamic flexibility (hinges). In this study we try to characterise the effect of proline on the geometric properties of TM alpha-helices. We have examined 199 transmembrane alpha-helices from polytopic membrane proteins of known structure. After examining the location of proline residues within the amino acid sequences of TM helices, we estimated the helix axes either side of a hinge and hence identified a hinge residue. This enabled us to calculate helix kink and swivel angles. The results of this analysis show that proline residues occur with a significant concentration in the centre of sequences of TM alpha-helices. In this location, they may induce formation of molecular hinges, located on average about four residues N-terminal to the proline residue. A superposition of proline-containing TM helices structures shows that the distortion induced is anisotropic and favours certain relative orientations (defined by helix kink and swivel angles) of the two helix segments.     

Structures of membrane proteins

Summary The possible conformations of integral membrane proteins are restricted by the nature of their environment. In order to satisfy the requirement of maximum hydrogen bonding, those protions of the polypeptide chain which are in contact with lipid hydrocarbon must be organized into regions of regular secondary structure. As possible models of the intramembranous regions of integral membrane proteins, three types of regular structues are discussed. Two, the alpha helix and the beta-pleated sheet, are regularly occurring structural features of soluble proteins. The third is a newly proposed class of conformations called beta helices. These helices have unique features which make them particularly well-suited to the lipid bilayer environment. The central segment of the membrane-spanning protein glycophorin can be arranged into a beta helix with a hydrophobic exterior and a polar interior containing charged amino-acid side chains. Such structures could function as transmembrane ion channels. A model of the activation process based on a hypothetical equilibrium between alpha and beta helical forms of a transmembrane protein is presented. The model can accurately reproduce the kinetics and voltage dependence of the channels in nerve.     

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

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

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.     

Comparison of helix interactions in membrane and soluble alpha-bundle proteins

Helix-helix interactions are important for the folding, stability, and function of membrane proteins. Here, two independent and complementary methods are used to investigate the nature and distribution of amino acids that mediate helix-helix interactions in membrane and soluble alpha-bundle proteins. The first method characterizes the packing density of individual amino acids in helical proteins based on the van der Waals surface area occluded by surrounding atoms. We have recently used this method to show that transmembrane helices pack more tightly, on average, than helices in soluble proteins. These studies are extended here to characterize the packing of interfacial and noninterfacial amino acids and the packing of amino acids in the interfaces of helices that have either right- or left-handed crossing angles, and either parallel or antiparallel orientations. We show that the most abundant tightly packed interfacial residues in membrane proteins are Gly, Ala, and Ser, and that helices with left-handed crossing angles are more tightly packed on average than helices with right-handed crossing angles. The second method used to characterize helix-helix interactions involves the use of helix contact plots. We find that helices in membrane proteins exhibit a broader distribution of interhelical contacts than helices in soluble proteins. Both helical membrane and soluble proteins make use of a general motif for helix interactions that relies mainly on four residues (Leu, Ala, Ile, Val) to mediate helix interactions in a fashion characteristic of left-handed helical coiled coils. However, a second motif for mediating helix interactions is revealed by the high occurrence and high average packing values of small and polar residues (Ala, Gly, Ser, Thr) in the helix interfaces of membrane proteins. Finally, we show that there is a strong linear correlation between the occurrence of residues in helix-helix interfaces and their packing values, and discuss these results with respect to membrane protein structure prediction and membrane protein stability.     

Unfolding a transmembrane helix dimer: A FRET study in mixed micelles

The exact nature of membrane protein folding and assembly is not understood in detail yet. Addition of SDS to a membrane protein dissolved in mild, non-polar detergent results in formation of mixed micelles and in subsequent denaturation of higher ordered membrane protein structures. The exact nature of this denaturation event is, however, enigmatic, and separation of an individual helix pair in mixed micelles has also not been reported yet. Here we followed unfolding of the human glycophorin A transmembrane helix dimer in mixed micelles by fluorescence spectroscopy. Energy transfer between differently labelled glycophorin A transmembrane helices decreased with increasing SDS mole fractions albeit without modifying the helicity of the peptides. The energetics and kinetics of the dimer dissociation in mixed micelles is analyzed and discussed, and the experimental data demonstrate that mixed micelles can be used as a general method to investigate unfolding of α-helical membrane proteins.     

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 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.     

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.