Sequence and conformational preferences at termini of α‐helices in membrane proteins: Role of the helix environment

α‐helices are amongst the most common secondary structural elements seen in membrane proteins and are packed in the form of helix bundles. These α‐helices encounter varying external environments (hydrophobic, hydrophilic) that may influence the sequence preferences at their N and C‐termini. The role of the external environment in stabilization of the helix termini in membrane proteins is still unknown. Here we analyze α‐helices in a high‐resolution dataset of integral α‐helical membrane proteins and establish that their sequence and conformational preferences differ from those in globular proteins. We specifically examine these preferences at the N and C‐termini in helices initiating/terminating inside the membrane core as well as in linkers connecting these transmembrane helices. We find that the sequence preferences and structural motifs at capping (Ncap and Ccap) and near‐helical (N' and C') positions are influenced by a combination of features including the membrane environment and the innate helix initiation and termination property of residues forming structural motifs. We also find that a large number of helix termini which do not form any particular capping motif are stabilized by formation of hydrogen bonds and hydrophobic interactions contributed from the neighboring helices in the membrane protein. We further validate the sequence preferences obtained from our analysis with data from an ultradeep sequencing study that identifies evolutionarily conserved amino acids in the rat neurotensin receptor. The results from our analysis provide insights for the secondary structure prediction, modeling and design of membrane proteins. Proteins 2014; 82:3420–3436. © 2014 Wiley Periodicals, Inc.     

Current status of membrane protein structure classification

For over 2 decades, continuous efforts to organize the jungle of available protein structures have been underway. Although a number of discrepancies between different classification approaches for soluble proteins have been reported, the classification of membrane proteins has so far not been comparatively studied because of the limited amount of available structural data. Here, we present an analysis of α‐helical membrane protein classification in the SCOP and CATH databases. In the current set of 63 α‐helical membrane protein chains having between 1 and 13 transmembrane helices, we observed a number of differently classified proteins both regarding their domain and fold assignment. The majority of all discrepancies affect single transmembrane helix, two helix hairpin, and four helix bundle domains, while domains with more than five helices are mostly classified consistently between SCOP and CATH. It thus appears that the structural constraints imposed by the lipid bilayer complicate the classification of membrane proteins with only few membrane‐spanning regions. This problem seems to be specific for membrane proteins as soluble four helix bundles, not restrained by the membrane, are more consistently classified by SCOP and CATH. Our findings indicate that the structural space of small membrane helix bundles is highly continuous such that even minor differences in individual classification procedures may lead to a significantly different classification. Membrane proteins with few helices and limited structural diversity only seem to be reasonably classifiable if the definition of a fold is adapted to include more fine‐grained structural features such as helix–helix interactions and reentrant regions. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Structural differences between thermophilic and mesophilic membrane proteins

The evolutionary adaptations of thermophilic water‐soluble proteins required for maintaining stability at high temperature have been extensively investigated. Little is known about the adaptations in membrane proteins, however. Here, we compare many properties of mesophilic and thermophilic membrane protein structures, including side‐chain burial, packing, hydrogen bonding, transmembrane kinks, loop lengths, hydrophobicity, and other sequence features. Most of these properties are quite similar between mesophiles and thermophiles although we observe a slight increase in side‐chain burial and possibly a slight decrease in the frequency of transmembrane kinks in thermophilic membrane protein structures. The most striking difference is the increased hydrophobicity of thermophilic transmembrane helices, possibly reflecting more stringent hydrophobicity requirements for membrane partitioning at high temperature. In agreement with prior work examining transmembrane sequences, we find that thermophiles have an increase in small residues (Gly, Ala, Ser, and Val) and a strong suppression of Cys. We also find a relative dearth of most strongly polar residues (Asp, Asn, Glu, Gln, and Arg). These results suggest that in thermophiles, there is significant evolutionary pressure to offload destabilizing polar amino acids, to decrease the entropy cost of side chain burial, and to eliminate thermally sensitive amino acids.     

Evaluation of transmembrane helix predictions in 2014

Experimental structure determination continues to be challenging for membrane proteins. Computational prediction methods are therefore needed and widely used to supplement experimental data. Here, we re‐examined the state of the art in transmembrane helix prediction based on a nonredundant dataset with 190 high‐resolution structures. Analyzing 12 widely‐used and well‐known methods using a stringent performance measure, we largely confirmed the expected high level of performance. On the other hand, all methods performed worse for proteins that could not have been used for development. A few results stood out: First, all methods predicted proteins in eukaryotes better than those in bacteria. Second, methods worked less well for proteins with many transmembrane helices. Third, most methods correctly discriminated between soluble and transmembrane proteins. However, several older methods often mistook signal peptides for transmembrane helices. Some newer methods have overcome this shortcoming. In our hands, PolyPhobius and MEMSAT‐SVM outperformed other methods. Proteins 2015; 83:473–484. © 2014 Wiley Periodicals, Inc.     

Proline kinks in transmembrane alpha-helices

Integral membrane proteins often contain proline residues in their presumably alpha-helical transmembrane segments. This is in marked contrast to globular proteins, where proline is rarely found inside alpha-helices. Proline residues cause kinks in helices, and, in addition to leaving the i-4 backbone carbonyl without its normal hydrogen bond donor, also sterically prevent the (i-3)-carbonyl-(i + l)-amide backbone hydrogen bond from forming. Here, some structural aspects of proline kinks in transmembrane helices are discussed on the basis of an analysis of Pro-kinked helices in the photosynthetic reaction center and bacteriorhodopsin, as well as results from an analysis of Pro-containing transmembrane segments identified in the NBRF Protein Sequence Databank.     

Prediction of buried helices in multispan alpha helical membrane proteins

Analysis of a database of structures of membrane proteins shows that membrane proteins composed of 10 or more transmembrane (TM) helices often contain buried helices that are inaccessible to phospholipids. We introduce a method for identifying TM helices that are least phospholipid accessible and for prediction of fully buried TM helices in membrane proteins from sequence information alone. Our method is based on the calculation of residue lipophilicity and evolutionary conservation. Given that the number of buried helices in a membrane protein is known, our method achieves an accuracy of 78% and a Matthew's correlation coefficient of 0.68. A server for this tool (RANTS) is available online at http://gila.bioengr.uic.edu/lab/.     

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.     

Structural comparison and classification of alpha‐helical transmembrane domains based on helix interaction patterns

Structural classification of membrane proteins is still in its infancy due to the relative paucity of available three‐dimensional structures compared with soluble proteins. However, recent technological advances in protein structure determination have led to a significant increase in experimentally known membrane protein folds, warranting exploration of the structural universe of membrane proteins. Here, a new and completely membrane protein specific structural classification system is introduced that classifies α‐helical membrane proteins according to common helix architectures. Each membrane protein is represented by a helix interaction graph depicting transmembrane helices with their pairwise interactions resulting from individual residue contacts. Subsequently, proteins are clustered according to similarities among these helix interaction graphs using a newly developed structural similarity score called HISS. As HISS scores explicitly disregard structural properties of loop regions, they are more suitable to capture conserved transmembrane helix bundle architectures than other structural similarity scores. Importantly, we are able to show that a classification approach based on helix interaction similarity closely resembles conventional structural classification databases such as SCOP and CATH implying that helix interactions are one of the major determinants of α‐helical membrane protein folds. Furthermore, the classification of all currently available membrane protein structures into 20 recurrent helix architectures and 15 singleton proteins demonstrates not only an impressive variability of membrane helix bundles but also the conservation of common helix interaction patterns among proteins with distinctly different sequences. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Conserved positioning of proline residues in membrane-spanning helices of ion-channel proteins.

Proline residues are a common feature of known and putative transmembrane helices of transport proteins. We find considerable consistency in the positioning of these residues within the structures. The proline residues are usually found on the hydrophilic (interior) faces of the pore-forming helices. This general observation adds considerable support to hypotheses concerning the structure of the ion-channels formed by alamethicin and melittin. As proline kinks helices, our observation suggests that the pores formed in ion-channel proteins tend to be funnel-shaped having a constriction near their center. Such a structure can aid in the capture of ions by the channel (an entropic effect) and should help in the gating mechanism of the channel. The observation will aid identification of putative transmembrane helices of ion-channels.     

Comprehensive analysis of the helix-X-helix motif in soluble proteins

Alpha-helices are the most common secondary structures found in globular proteins. In this report, we analyze the stereochemical and sequence properties of helix-X-helix (HXH) motifs in which two alpha-helices are linked by a single residue, in search of characteristic structures and sequence signals. The analysis is carried out on a database of 837 nonredundant HXH motifs. The kinks are characterized by the bend angle between the axes of the N-terminal and C-terminal helices and the wobble angle corresponding to the rotation of C-terminal helix axis on the plane perpendicular to the N-terminal one. The phi-psi dihedral angles of the linker residue are clustered in six distinct areas of the Ramachandran plot: two areas are located in the additional allowed alpha region (alpha(1) and alpha(2)), two areas are in the additional allowed beta region (beta(1) and beta(2)) and two areas have positive phi values (alpha(L) and beta(M)). Each phi/psi region corresponds to characteristic bend and wobble angles and amino acid distributions. Bend angles can vary from 0 degrees to 160 degrees. Most wobble angles correspond to a counter-clockwise rotation of the C-terminal helix. Proline residues are rigorously excluded from the linker position X but have a high propensity at position X+1 of the beta(1) and beta(2) motifs (12 and 7, respectively) and at position X+3 of the alpha(1) motifs (9). Glycine linkers are located either in the alpha(L) region (20%) or in the beta(M) region (80%). This latter conformation is characterized by a marked bend angle (124 degrees +/- 18 degrees) and a clockwise wobble. Among other amino acids, Asn is remarkable for its high propensity (>3) at the linker position of the alpha(2), beta(1), and beta(2) motifs. Stabilization of HXH motifs by H-bonds between polar side chains of the linker and polar groups of the backbone is determined. A method based on position-specific scoring matrices is developed for conformational prediction. The accuracy of the predictions reaches 80% when the method is applied to proline-induced kinks or to kinks with bend angles in the 50 degrees-100 degrees range.     

Crystal structure of human Ankyrin G death domain

Ankyrins (Ank) are a ubiquitously expressed family of multifunctional membrane adapter proteins. Ankyrin G (AnkG) is critical for assembling and maintenance of the axon initial segment. Here we present the 2.1 Å crystal structure of human AnkG death domain (hAnkG‐DD). The core death domain is composed of six α‐helices and three 3₁₀‐helices. It forms a hydrophobic pocket on the surface of the molecule. The C‐terminal tail of the hAnkG‐DD curves back to have the aromatic ring of a phenylalanine residue, Phe100 insert into this pocket, which anchors the flexible tail onto the core domain. Related DDs were selected for structure comparison. The major variations are at the C‐terminal region, including the α6 and the long C‐terminal extension. The results of size exclusion chromatography and analytical ultracentrifugation suggest that hAnkG‐DD exists as monomer in solution. Our work should help for the future investigation of the structure–function of AnkG. Proteins 2014; 82:3476–3482. © 2014 Wiley Periodicals, Inc.     

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.     

Simultaneous prediction of protein secondary structure and transmembrane spans

Prediction of transmembrane spans and secondary structure from the protein sequence is generally the first step in the structural characterization of (membrane) proteins. Preference of a stretch of amino acids in a protein to form secondary structure and being placed in the membrane are correlated. Nevertheless, current methods predict either secondary structure or individual transmembrane states. We introduce a method that simultaneously predicts the secondary structure and transmembrane spans from the protein sequence. This approach not only eliminates the necessity to create a consensus prediction from possibly contradicting outputs of several predictors but bears the potential to predict conformational switches, i.e., sequence regions that have a high probability to change for example from a coil conformation in solution to an α‐helical transmembrane state. An artificial neural network was trained on databases of 177 membrane proteins and 6048 soluble proteins. The output is a 3 × 3 dimensional probability matrix for each residue in the sequence that combines three secondary structure types (helix, strand, coil) and three environment types (membrane core, interface, solution). The prediction accuracies are 70.3% for nine possible states, 73.2% for three‐state secondary structure prediction, and 94.8% for three‐state transmembrane span prediction. These accuracies are comparable to state‐of‐the‐art predictors of secondary structure (e.g., Psipred) or transmembrane placement (e.g., OCTOPUS). The method is available as web server and for download at www.meilerlab.org . Proteins 2013; 81:1127–1140. © 2013 Wiley Periodicals, Inc.     

A survey of left-handed polyproline II helices

Left-handed polyproline II helices (PPII) are contiguous elements of protein secondary structure in which the phi and psi angles of constituent residues are restricted to around -75 degrees and 145 degrees, respectively. They are important in structural proteins, in unfolded states and as ligands for signaling proteins. Here, we present a survey of 274 nonhomologous polypeptide chains from proteins of known structure for regions that form these structures. Such regions are rare, but the majority of proteins contain at least one PPII helix. Most PPII helices are shorter than five residues, although the longest found contained 12 amino acids. Proline predominates in PPII, but Gln and positively charged residues are also favored. The basis of Gln's prevalence is its ability to form an i, i + 1 side-chain to main-chain hydrogen bond with the backbone carbonyl oxygen of the proceeding residue; this helps to fix the psi angle of the Gln and the phi and psi of the proceeding residue in PPII conformations and explains why Gln is favored at the first position in a PPII helix. PPII helices are highly solvent exposed, which explains why apolar amino acids are disfavored despite preferring this region of phi/psi space when in isolation. PPII helices have perfect threefold rotational symmetry and within these structures we find significant correlation between the hydrophobicity of residues at i and i + 3; thus, PPII helices in globular proteins can be considered to be amphipathic.     

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.     

A study of the membrane-water interface region of membrane proteins

The most conspicuous structural characteristic of the alpha-helical membrane proteins is their long transmembrane alpha-helices. However, other structural elements, as yet largely ignored in statistical studies of membrane protein structure, are found in those parts of the protein that are located in the membrane-water interface region. Here, we show that this region is enriched in irregular structure and in interfacial helices running roughly parallel with the membrane surface, while beta-strands are extremely rare. The average amino acid composition is different between the interfacial helices, the parts of the transmembrane helices located in the interface region, and the irregular structures. In this region, hydrophobic and aromatic residues tend to point toward the membrane and charged/polar residues tend to point away from the membrane. The interface region thus imposes different constraints on protein structure than do the central hydrocarbon core of the membrane and the surrounding aqueous phase.     

TMKink: a method to predict transmembrane helix kinks

A hallmark of membrane protein structure is the large number of distorted transmembrane helices. Because of the prevalence of bends, it is important to not only understand how they are generated but also to learn how to predict their occurrence. Here, we find that there are local sequence preferences in kinked helices, most notably a higher abundance of proline, which can be exploited to identify bends from local sequence information. A neural network predictor identifies over two-thirds of all bends (sensitivity 0.70) with high reliability (specificity 0.89). It is likely that more structural data will allow for better helix distortion predictors with increased coverage in the future. The kink predictor, TMKink, is available at http://tmkinkpredictor.mbi.ucla.edu/.     

Side-chain contributions to membrane protein structure and stability

The molecular forces that stabilize membrane protein structure are poorly understood. To investigate these forces we introduced alanine substitutions at 24 positions in the B helix of bacteriorhodopsin and examined their effects on structure and stability. Although most of the results can be rationalized in terms of the folded structure, there are a number of surprises. (1) We find a remarkably high frequency of stabilizing mutations (17%), indicating that membrane proteins are not highly optimized for stability. (2) Helix B is kinked, with the kink centered around Pro50. The P50A mutation has no effect on stability, however, and a crystal structure reveals that the helix remains bent, indicating that tertiary contacts dominate in the distortion of this helix. (3) We find that the protein is stabilized by about 1kcal/mol for every 38A(2) of surface area buried, which is quite similar to soluble proteins in spite of their dramatically different environments. (4) We find little energetic difference, on average, in the burial of apolar surface or polar surface area, implying that van der Waals packing is the dominant force that drives membrane protein folding.     

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.