Comprehensive analysis of the numbers,lengths and amino acid compositions of transmembrane helices in prokaryotic,eukaryotic and viral integral membrane proteins of high-resolution structure

We report a comprehensive analysis of the numbers, lengths and amino acid compositions of transmembrane helices in 235 high-resolution structures of integral membrane proteins. The properties of 1551 transmembrane helices in the structures were compared with those obtained by analysis of the same amino acid sequences using topology prediction tools. Explanations for the 81 (5.2%) missing or additional transmembrane helices in the prediction results were identified. Main reasons for missing transmembrane helices were mis-identification of N-terminal signal peptides, breaks in α-helix conformation or charged residues in the middle of transmembrane helices and transmembrane helices with unusual amino acid composition. The main reason for additional transmembrane helices was mis-identification of amphipathic helices, extramembrane helices or hairpin re-entrant loops. Transmembrane helix length had an overall median of 24 residues and an average of 24.9 ± 7.0 residues and the most common length was 23 residues. The overall content of residues in transmembrane helices as a percentage of the full proteins had a median of 56.8% and an average of 55.7 ± 16.0%. Amino acid composition was analysed for the full proteins, transmembrane helices and extramembrane regions. Individual proteins or types of proteins with transmembrane helices containing extremes in contents of individual amino acids or combinations of amino acids with similar physicochemical properties were identified and linked to structure and/or function. In addition to overall median and average values, all results were analysed for proteins originating from different types of organism (prokaryotic, eukaryotic, viral) and for subgroups of receptors, channels, transporters and others.     

Amino acid composition analysis of secondary transport proteins from Escherichia coli with relation to functional classification,ligand specificity and structure

We have performed an amino acid composition (AAC) analysis of the complete sequences for 235 secondary transport proteins from Escherichia coli, which have functions in the uptake and export of organic and inorganic metabolites, efflux of drugs and in controlling membrane potential. This revealed the trends in content for specific amino acid types and for combinations of amino acids with similar physicochemical properties. In certain proteins or groups of proteins, the so-called spikes of high content for a specific amino acid type or combination of amino acids were identified and confirmed statistically, which in some cases could be directly related to function and ligand specificity. This was prevalent in proteins with a function of multidrug or metal ion efflux. Any tool that can help in identifying bacterial multidrug efflux proteins is important for a better understanding of this mechanism of antibiotic resistance. Phylogenetic analysis based on sequence alignments and comparison of sequences at the N- and C-terminal ends confirmed transporter Family classification. Locations of specific amino acid types in some of the proteins that have crystal structures (EmrE, LacY, AcrB) were also considered to help link amino acid content with protein function. Though there are limitations, this work has demonstrated that a basic analysis of AAC is a useful tool to use in combination with other computational and experimental methods for classifying and investigating function and ligand specificity in a large group of transport or other membrane proteins, including those that are molecular targets for development of new drugs.     

Discontinuous membrane helices in transport proteins and their correlation with function

Alpha-helical bundles and beta-barrel proteins represent the two basic types of architecture known for integral membrane proteins. Irregular structural motifs have been revealed with the growing number of structures determined. "Discontinuous" helices are present in membrane proteins that actively transport ions. In the Ca(2+)-ATPase, a primary active transporter, and in the secondary transporters NhaA, LeuT(Aa), ClC H(+)/Cl(-) exchanger and Glt(Ph), the helical structure of two membrane segments is interrupted and the interjacent polypeptide chain forms an extended peptide. The discontinuous helices are integrated in the membrane either as transmembrane-spanning or hairpin-type segments. In addition, the secondary transporters have inverted internal duplication domains, which are only weakly correlated with their amino acid sequence. The symmetry comprises either parts of or the complete molecule, but always includes the discontinuous helices. The helix-peptide-helix motif is correlated with the ion translocation function. The extended peptides with their backbone atoms, the helix termini and the polar/charged amino acid residues in close vicinity provide the basis for ion recognition, binding and translocation.     

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.     

Amino-acid solvation structure in transmembrane helices from molecular dynamics simulations

Understanding the solvation of amino acids in biomembranes is an important step to better explain membrane protein folding. Several experimental studies have shown that polar residues are both common and important in transmembrane segments, which means they have to be solvated in the hydrophobic membrane, at least until helices have aggregated to form integral proteins. In this work, we have used computer simulations to unravel these interactions on the atomic level, and classify intramembrane solvation properties of amino acids. Simulations have been performed for systematic mutations in poly-Leu helices, including not only each amino acid type, but also every z-position in a model helix. Interestingly, many polar or charged residues do not desolvate completely, but rather retain hydration by snorkeling or pulling in water/headgroups--even to the extent where many of them exist in a microscopic polar environment, with hydration levels corresponding well to experimental hydrophobicity scales. This suggests that even for polar/charged residues a large part of solvation cost is due to entropy, not enthalpy loss. Both hydration level and hydrogen bonding exhibit clear position-dependence. Basic side chains cause much less membrane distortion than acidic, since they are able to form hydrogen bonds with carbonyl groups instead of water or headgroups. This preference is supported by sequence statistics, where basic residues have increased relative occurrence at carbonyl z-coordinates. Snorkeling effects and N-/C-terminal orientation bias are directly observed, which significantly reduces the effective thickness of the hydrophobic core. Aromatic side chains intercalate efficiently with lipid chains (improving Trp/Tyr anchoring to the interface) and Ser/Thr residues are stabilized by hydroxyl groups sharing hydrogen bonds to backbone oxygens.     

Structure-based statistical analysis of transmembrane helices

Recent advances in determination of the high-resolution structure of membrane proteins now enable analysis of the main features of amino acids in transmembrane (TM) segments in comparison with amino acids in water-soluble helices. In this work, we conducted a large-scale analysis of the prevalent locations of amino acids by using a data set of 170 structures of integral membrane proteins obtained from the MPtopo database and 930 structures of water-soluble helical proteins obtained from the protein data bank. Large hydrophobic amino acids (Leu, Val, Ile, and Phe) plus Gly were clearly prevalent in TM helices whereas polar amino acids (Glu, Lys, Asp, Arg, and Gln) were less frequent in this type of helix. The distribution of amino acids along TM helices was also examined. As expected, hydrophobic and slightly polar amino acids are commonly found in the hydrophobic core of the membrane whereas aromatic (Trp and Tyr), Pro, and the hydrophilic amino acids (Asn, His, and Gln) occur more frequently in the interface regions. Charged amino acids are also statistically prevalent outside the hydrophobic core of the membrane, and whereas acidic amino acids are frequently found at both cytoplasmic and extra-cytoplasmic interfaces, basic amino acids cluster at the cytoplasmic interface. These results strongly support the experimentally demonstrated biased distribution of positively charged amino acids (that is, the so-called the positive-inside rule) with structural data.     

Anionic phospholipids are determinants of membrane protein topology.

The orientation of many membrane proteins is determined by the asymmetric distribution of positively charged amino acid residues in cytoplasmic and translocated loops. The positive-inside rule states that loops with large amounts of these residues tend to have cytoplasmic locations. Orientations of constructs derived from the inner membrane protein leader peptidase from Escherichia coli were found to depend on the anionic phospholipid content of the membrane. Lowering the contents of anionic phospholipids facilitated membrane passage of positively charged loops. On the other hand, elevated contents of acidic phospholipids in the membrane rendered translocation more sensitive to positively charged residues. The results demonstrate that anionic lipids are determinants of membrane protein topology and suggest that interactions between negatively charged phospholipids and positively charged amino acid residues contribute to the orientation of membrane proteins.     

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

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

Interaction of 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes

We investigated the interaction of six 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes. The net charge of the peptides at neutral pH varies from −1 to +6. Circular dichroism spectra indicate that peptides with a high net positive charge tend to fold into a helical conformation in the presence of negatively charged lipid vesicles. In helical conformation, their average hydrophobic moment and hydrophobicity would render them surface-active. The composition of amino acids on the polar face of the helix in the peptides is considerably different. The peptides show variations in their ability to permeabilise zwitterionic and anionic lipid vesicles. Whereas increased net positive charge favours greater permeabilisation, the distribution of charged residues in the polar face also plays a role in determining membrane activity. The distribution of amino acids in the polar face of the helix in the peptides that were investigated do not fall into the canonical classes described. Amphipathic helices, which are part of proteins, with a pattern of amino acid distribution different from those observed in class L, A and others, could help in providing newer insights into peptide-membrane interactions.     

Melibiose carrier of Escherichia coli: use of cysteine mutagenesis to identify the amino acids on the hydrophilic face of transmembrane helix 2.

The melibiose carrier from Escherichia coli is a galactoside-cation symporter. Based on both experimental evidence and hydropathy analysis, 12 transmembrane helices have been assigned to this integral membrane protein. Transmembrane helix 2 contains several charged and polar amino acids that have been shown to be essential for the cation-coupled transport of melibiose. Starting with the cysteine-less melibiose carrier, we have individually substituted cysteine for amino acids 39-66, which includes the proposed transmembrane helix 2. In the resulting derivative carriers, we measured the transport of melibiose, determined the effect of the hydrophilic sulfhydryl reagent, p-chloromercuribenzenesulfonic acid (PCMBS), on transport in intact cells and inside out vesicles, and examined the ability of melibiose to protect the carrier from inactivation by the sulfhydryl reagent. We found a set of seven positions in which the reaction with the sulfhydryl reagent caused partial or complete loss of carrier function measured in intact cells or inside-out vesicles. The presence of melibiose protected five of these positions from reaction with PCMBS. The reaction of two additional positions with PCMBS resulted in the partial loss of transport function only in inside-out vesicles. Melibiose protected these two positions from reaction with the reagent. Together, the PCMBS-sensitive sites and charged residues assigned to helix 2 form a cluster of amino acids that map in three rows with each row comprised of every fourth residue. This is the pattern expected of residues that are part of an alpha-helical structure and thus the rows are tilted at an angle of 25 degrees to the helical axis. We suggest that these residues line the path of melibiose and its associated cation through the carrier.     

Helical integrity and microsolvation of transmembrane domains from Flaviviridae envelope glycoproteins

Charged and polar amino acids in the transmembrane domains of integral membrane proteins can be crucial for protein function and also promote helix-helix association or protein oligomerization. Yet, our current understanding is still limited on how these hydrophilic amino acids are efficiently translocated from the Sec61/SecY translocon into the cell membrane during the biogenesis of membrane proteins. In hepatitis C virus, the putative transmembrane segments of envelope glycoproteins E1 and E2 were suggested to heterodimerize via a Lys-Asp ion-pair in the host endoplasmic reticulum. Therefore in this work, we carried out molecular dynamic simulations in explicit lipid bilayer and solvent environment to explore the stability of all possible bridging ion-pairs using the model of H-segment helix dimers. We observed that, frequently, several water molecules penetrated from the interface into the membrane core to stabilize the charged and polar pairs. The hydration time and amount of water molecules in the membrane core depended on the position of the charged residues as well as on the type of ion-pairs. Similar microsolvation events were observed in simulations of the putative E1-E2 transmembrane helix dimers. Simulations of helix monomers from other members of the Flaviviridae family suggest that these systems show similar behaviors. Thus this study illustrates the important contribution of water microsolvation to overcome the unfavorable energetic cost of burying charged and polar amino acids in membrane lipid bilayers. Also, it emphasizes the novel role of bridging charged or polar interactions stabilized by water molecules in the hydrophobic lipid bilayer core that has an important biological function for helix dimerization in several envelope glycoproteins from the family of Flaviviridae viruses.     

Marginally hydrophobic transmembrane α‐helices shaping membrane protein folding

Cells have developed an incredible machinery to facilitate the insertion of membrane proteins into the membrane. While we have a fairly good understanding of the mechanism and determinants of membrane integration, more data is needed to understand the insertion of membrane proteins with more complex insertion and folding pathways. This review will focus on marginally hydrophobic transmembrane helices and their influence on membrane protein folding. These weakly hydrophobic transmembrane segments are by themselves not recognized by the translocon and therefore rely on local sequence context for membrane integration. How can such segments reside within the membrane? We will discuss this in the light of features found in the protein itself as well as the environment it resides in. Several characteristics in proteins have been described to influence the insertion of marginally hydrophobic helices. Additionally, the influence of biological membranes is significant. To begin with, the actual cost for having polar groups within the membrane may not be as high as expected; the presence of proteins in the membrane as well as characteristics of some amino acids may enable a transmembrane helix to harbor a charged residue. The lipid environment has also been shown to directly influence the topology as well as membrane boundaries of transmembrane helices—implying a dynamic relationship between membrane proteins and their environment.     

How proteins adapt to a membrane-water interface

Membrane proteins present a hydrophobic surface to the surrounding lipid, whereas portions protruding into the aqueous milieu expose a polar surface. But how have proteins evolved to deal with the complex environment at the membrane-water interface? Some insights have been provided by high-resolution structures of membrane proteins, and recent studies of the role of individual amino acids in mediating protein-lipid contacts have shed further light on this issue. It now appears clear that the polar-aromatic residues Trp and Tyr have a specific affinity for a region near the lipid carbonyls, whereas positively charged residues extend into the lipid phosphate region.     

Molecular dynamics simulation of the unfolding of individual bacteriorhodopsin helices in sodium dodecyl sulfate micelles

We report molecular dynamics simulations of the trends in the changes in secondary structure of the seven individual helices of bacteriorhodopsin when inserted into sodium dodecyl sulfate (SDS) micelles, and their dependence on the amino acid sequence. The results indicate that the partitioning of the helices in the micelles and their stability are dependent on the hydrophobicity of the transmembrane segments. Helices A, B, and E are stable and retain their initial secondary structure throughout the 100 ns simulation time. In contrast, helices C, D, F, and G show structural perturbations within the first 10 ns. The instabilities are localized near charged residues within the transmembrane segments. The overall structural instability of the helix is correlated with its partitioning to the surface of the micelle and its interaction with polar groups there. The in silico experiments were performed to complement the in vitro experiments that examined the partial denaturation of bacteriorhodopsin in SDS described in the preceding article (DOI 10.1021/bi201769z ). The simulations are consistent with the trends revealed by the experimental results but strongly underestimate the extent of helix to extended coil transformation. The reason may be either that the sampling time was not sufficiently long or, more interestingly, that interhelix residue interactions play a role in the unfolding of the helices.     

Membrane topology of the ZntB efflux system of Salmonella enterica serovar Typhimurium

The membrane topology of the ZntB Zn(2+) transport protein of Salmonella enterica serovar Typhimurium was determined by constructing deletion derivatives of the protein and genetically fusing them to blaM or lacZ cassettes. The enzymatic activities of the hybrid proteins indicate that ZntB is a bitopic integral membrane protein consisting largely of two independent domains. The first 266 amino acids form a large, highly charged domain within the cytoplasm, while the remaining 61 residues form a small membrane domain containing two membrane-spanning segments. The overall orientation towards the cytoplasm is consistent with the ability of ZntB to facilitate zinc efflux.     

Comparison of fragments comprising the first two helices of the human y4 and the yeast ste2p g-protein-coupled receptors

Solution NMR techniques are used to determine the structure and the topology of micelle integration of a large fragment of the Y4 receptor, a human G-protein-coupled receptor, that contains the entire N-terminal domain plus the first two transmembrane (TM) segments. The structure calculations reveal that the putative TM helices are indeed helical to a large extent, but that interruptions of secondary structure occur close to internal polar or charged residues. This view is supported by ¹⁵N relaxation data, amide-water exchange rates, and attenuations from micelle-integrating spin labels. No contacts between different helices are observed. This is in contrast to a similar TM1-TM2 fragment from the yeast Ste2p receptor for which locations of the secondary and the tertiary structure agreed well with the predictions from a homology model. The difference in structure is discussed in terms of principal biophysical properties of residues within central regions of the putative TM helices. Overall, using the biophysical scale of Wimley and White the TM regions of Ste2p display much more favorable free energies for membrane integration. Accordingly, the full secondary structure and the tertiary structure in TM1-TM2 of the Y4 receptor is likely to be formed only when tertiary contacts with other TM segments are created during folding of the receptor.     

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.     

Generalized transposon shuttle mutagenesis in Neisseria gonorrhoeae: a method for isolating epithelial cell invasion-defective mutants

Hybrid genes were constructed to express bifunctional hybrid proteins in which staphyloccal nuclease A with or without an amino-terminai OmpA signal sequence was fused with TEM β-lactamase (at the carboxyl terminal side) using the signal peptide of the major outer membrane lipoprotein of Escherichia coli as an internal linker. The hybrid proteins were found to be inserted in the membrane. Orientation of the hybrid protein with the OmpA signal peptide showed that the nuclease was translocated into the periplasm and the β-lactamase remained in the cytoplasm. This indicates that the cleavable OmpA signal peptide served as a secretory signal for nuclease and the internal lipoprotein signal served as the transmembrane anchor, in the absence of the OmpA signal sequence the topology of the hybrid protein was reversed indicating that the internal lipoprotein signal peptide initially served as the signal peptide for the secretion of the carboxy terminal β-lactamase domain across the membrane and subsequently as a membrane anchoring signal. The role of charged amino acids in the translocation and transmembrane orientation of membrane proteins was also analysed by introducing charged amino acids to either or both sides of the internal lipoprotein signal sequence in the bifunctional hybrid proteins in the absence of the amino-terminal signal sequence. Introduction of two lysine residues at the carboxy-terminal side of the internal signal sequence reversed the topology of the transmembrane protein by translocating the aminoterminal nuclease domain across the membrane, leaving the carboxyl terminal β-actamase domain in the cytoplasm. When three more lysine residues were added to the amino-terminal side of the internal signal sequence of the same construct the membrane topology flipped back to the original orientation. A similar reversion of the topology could be obtained by introducing negatively charged residues at the amino-terminal side of the internal signal sequence. Present results demonstrate for the first time that a bifunctional transmembrane protein can be engineered to assume either of the two opposite orientations and that charge balance around the transmembrane domain is a major factor in controlling the topology of a transmembrane protein.     

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

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

Intra-Helical Salt Bridge Contribution to Membrane Protein Insertion

Salt bridges between negatively (D, E) and positively charged (K, R, H) amino acids play an important role in protein stabilization. This has a more prevalent effect in membrane proteins where polar amino acids are exposed to a hydrophobic environment. In transmembrane (TM) helices the presence of charged residues can hinder the insertion of the helices into the membrane. It is possible that the formation of salt bridges could decrease the cost of membrane integration. However, the presence of intra-helical salt bridges in TM domains and their effect on insertion has not been properly studied yet. In this work, we show that potentially salt-bridge forming pairs are statistically over-represented in TM-helices. We then selected some candidates to experimentally determine the contribution of these electrostatic interactions to the translocon-assisted membrane insertion process. Using both in vitro and whole cell systems, we confirm the presence of intra-helical salt bridges in TM segments during biogenesis and determined that they contribute ～0.5 kcal/mol to the apparent free energy of membrane insertion (ΔG_app). Our observations suggest that salt bridge interactions can be stabilized during translocon-mediated insertion and thus could be relevant to consider for the future development of membrane protein prediction software.