Buried water molecules in helical transmembrane proteins

Buried water molecules (having no contact with bulk solvent) in 30 helical transmembrane (TM) protein structures were identified. The average amount of buried water in helical TM proteins is about the same as for all water-soluble (WS) proteins, but it is greater than the average for helical WS proteins. Buried waters in TM proteins make more polar contacts, and are more frequently found contacting helices than in WS proteins. The distribution of the buried water binding sites across the membrane profile shows that the sites to some extent reflect protein function. There is also evidence for asymmetry of the sites, with more in the extracellular half of the membrane. Many of the buried water contact sites are conserved across families of proteins, including family members having different functions. This suggests that at least some buried waters play a role in structural stabilization. Disease-causing mutations, which are known to result in misfolded TM proteins, occur at buried water contact sites at a higher than random frequency, which also supports a stabilizing role for buried water molecules.     

How strongly do sequence conservation patterns and empirical scales correlate with exposure patterns of transmembrane helices of membrane proteins?

Given the difficulty in determining high-resolution structures of helical membrane proteins, sequence-based prediction methods can be useful in elucidating diverse physiological processes mediated by this important class of proteins. Predicting the angular orientations of transmembrane (TM) helices about the helix axes, based on the helix parameters from electron microscopy data, is a classical problem in this regard. This problem has triggered the development of a number of different empirical scales. Recently, sequence conservation patterns were also made use of for improved predictions. Empirical scales and sequence conservation patterns (collectively termed as "prediction scales") have also found frequent applications in other research areas of membrane proteins: for example, in structure modeling and in prediction of buried TM helices. This trend is expected to grow in the near future unless there are revolutionary developments in the experimental characterization of membrane proteins. Thus, it is timely and imperative to carry out a comprehensive benchmark test over the prediction scales proposed so far to determine their pros and cons. In the current analysis, we use exposure patterns of TM helices as a golden standard, because if one develops a prediction scale that correlates perfectly with exposure patterns of TM helices, it will enable one to predict buried residues (or buried faces) of TM helices with an accuracy of 100%. Our analysis reveals several important points. (1) It demonstrates that sequence conservation patterns are much more strongly correlated with exposure patterns of TM helices than empirical scales. (2) Scales that were specifically parameterized using structure data (structure-based scales) display stronger correlation than hydrophobicity-based scales, as expected. (3) A nonnegligible difference is observed among the structure-based scales in their correlational property, suggesting that not every learning algorithm is equally effective. (4) A straightforward framework of optimally combining sequence conservation patterns and empirical scales is proposed, which reveals that improvements gained from combining the two sources of information are not dramatic in almost all cases. In turn, this calls for the development of fundamentally different scales that capture the essentials of membrane protein folding for substantial improvements.     

Coils in the membrane core are conserved and functionally important

With the increasing number of available α-helical transmembrane (TM) protein structures, the traditional picture of membrane proteins has been challenged. For example, reentrant regions, which enter and exit the membrane at the same side, and interface helices, which lie parallel with the membrane in the membrane-water interface, are common. Furthermore, TM helices are frequently kinked, and their length and tilt angle vary. Here, we systematically analyze 7% of all residues within the deep membrane core that are in coil state. These coils can be found in TM-helix kinks as major breaks in TM helices and as parts of reentrant regions.Coil residues are significantly more conserved than other residues. Due to the polar character of the coil backbone, they are either buried or located near aqueous channels. Coil residues are frequently found within channels and transporters, where they introduce the flexibility and polarity required for transport across the membrane. Therefore, we believe that coil residues in the membrane core, while constituting a structural anomaly, are essential for the function of proteins.     

The Effect of Loops on the Structural Organization of α-Helical Membrane Proteins

Loops connecting the transmembrane (TM) α-helices in membrane proteins are expected to affect the structural organization of the thereby connected helices and the helical bundles as a whole. This effect, which has been largely ignored previously, is studied here by analyzing the x-ray structures of 41 α-helical membrane proteins. First we define the loop flexibility ratio, R, and find that 53% of the loops are stretched, where a stretched loop constrains the distance between the two connected helices. The significance of this constraining effect is supported by experiments carried out with bacteriorhodopsin and rhodopsin, in which cutting or eliminating their (predominately stretched) loops has led to a decrease in protein stability, and for rhodopsin, in most cases, also to the destruction of the structure. We show that for nonstretched loops in the extramembranous regions, the fraction of hydrophobic residues is comparable to that for soluble proteins; furthermore (as is also the case for soluble proteins), the hydrophobic residues in these regions are preferentially buried. This is expected to lead to the compact structural organization of the loops, which is transferred to the TM helices, causing them to assemble. We argue that a soluble protein complexed with a membrane protein similarly promotes compactness; other properties of such complexes are also studied. We calculate complementary attractive interactions between helices, including hydrogen bonds and van der Waals interactions of sequential motifs, such as GXXXG. The relative and combined effects of all these factors on the association of the TM helices are discussed and protein structures with only a few of these factors are analyzed. Our study emphasizes the need for classifying membrane proteins into groups according to structural organization. This classification should be considered when procedures for structural analysis or prediction are developed and applied. Detailed analysis of each structure is provided at http://flan.blm.cs.cmu.edu/memloop/     

TMPDB: a database of experimentally-characterized transmembrane topologies

TMPDB is a database of experimentally-characterized transmembrane (TM) topologies. TMPDB release 6.2 contains a total of 302 TM protein sequences, in which 276 are alpha-helical sequences, 17 beta-stranded, and 9 alpha-helical sequences with short pore-forming helices buried in the membrane. The TM topologies in TMPDB were determined experimentally by means of X-ray crystallography, NMR, gene fusion technique, substituted cysteine accessibility method, N-linked glycosylation experiment and other biochemical methods. TMPDB would be useful as a test and/or training dataset in improving the proposed TM topology prediction methods or developing novel methods with higher performance, and as a guide for both the bioinformaticians and biologists to better understand TM proteins. TMPDB and its subsets are freely available at the following web site: http://bioinfo.si.hirosaki-u.ac.jp/~TMPDB/.     

Assembly of transmembrane helices of simple polytopic membrane proteins from sequence conservation patterns

The transmembrane (TM) domains of most membrane proteins consist of helix bundles. The seemingly simple task of TM helix bundle assembly has turned out to be extremely difficult. This is true even for simple TM helix bundle proteins, i.e., those that have the simple form of compact TM helix bundles. Herein, we present a computational method that is capable of generating native-like structural models for simple TM helix bundle proteins having modest numbers of TM helices based on sequence conservation patterns. Thus, the only requirement for our method is the presence of more than 30 homologous sequences for an accurate extraction of sequence conservation patterns. The prediction method first computes a number of representative well-packed conformations for each pair of contacting TM helices, and then a library of tertiary folds is generated by overlaying overlapping TM helices of the representative conformations. This library is scored using sequence conservation patterns, and a subsequent clustering analysis yields five final models. Assuming that neighboring TM helices in the sequence contact each other (but not that TM helices A and G contact each other), the method produced structural models of Calpha atom root-mean-square deviation (CA RMSD) of 3-5 A from corresponding crystal structures for bacteriorhodopsin, halorhodopsin, sensory rhodopsin II, and rhodopsin. In blind predictions, this type of contact knowledge is not available. Mimicking this, predictions were made for the rotor of the V-type Na(+)-adenosine triphosphatase without such knowledge. The CA RMSD between the best model and its crystal structure is only 3.4 A, and its contact accuracy reaches 55%. Furthermore, the model correctly identifies the binding pocket for sodium ion. These results demonstrate that the method can be readily applied to ab initio structure prediction of simple TM helix bundle proteins having modest numbers of TM helices.     

kPROT: a knowledge-based scale for the propensity of residue orientation in transmembrane segments. Application to membrane protein structure prediction

Modeling of integral membrane proteins and the prediction of their functional sites requires the identification of transmembrane (TM) segments and the determination of their angular orientations. Hydrophobicity scales predict accurately the location of TM helices, but are less accurate in computing angular disposition. Estimating lipid-exposure propensities of the residues from statistics of solved membrane protein structures has the disadvantage of relying on relatively few proteins. As an alternative, we propose here a scale of knowledge-based Propensities for Residue Orientation in Transmembrane segments (kPROT), derived from the analysis of more than 5000 non-redundant protein sequences. We assume that residues that tend to be exposed to the membrane are more frequent in TM segments of single-span proteins, while residues that prefer to be buried in the transmembrane bundle interior are present mainly in multi-span TMs. The kPROT value for each residue is thus defined as the logarithm of the ratio of its proportions in single and multiple TM spans. The scale is refined further by defining it for three discrete sections of the TM segment; namely, extracellular, central, and intracellular. The capacity of the kPROT scale to predict angular helical orientation was compared to that of alternative methods in a benchmark test, using a diversity of multi-span alpha-helical transmembrane proteins with a solved 3D structure. kPROT yielded an average angular error of 41 degrees, significantly lower than that of alternative scales (62 degrees -68 degrees ). The new scale thus provides a useful general tool for modeling and prediction of functional residues in membrane proteins. A WWW server (http://bioinfo.weizmann.ac.il/kPROT) is available for automatic helix orientation prediction with kPROT.     

Transmembrane helix dimerization: beyond the search for sequence motifs

Studies of the dimerization of transmembrane (TM) helices have been ongoing for many years now, and have provided clues to the fundamental principles behind membrane protein (MP) folding. Our understanding of TM helix dimerization has been dominated by the idea that sequence motifs, simple recognizable amino acid sequences that drive lateral interaction, can be used to explain and predict the lateral interactions between TM helices in membrane proteins. But as more and more unique interacting helices are characterized, it is becoming clear that the sequence motif paradigm is incomplete. Experimental evidence suggests that the search for sequence motifs, as mediators of TM helix dimerization, cannot solve the membrane protein folding problem alone. Here we review the current understanding in the field, as it has evolved from the paradigm of sequence motifs into a view in which the interactions between TM helices are much more complex. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Interhelical hydrogen bonds and spatial motifs in membrane proteins: polar clamps and serine zippers

Polar and ionizable amino acid residues are frequently found in the transmembrane (TM) regions of membrane proteins. In this study, we show that they help to form extensive hydrogen bond connections between TM helices. We find that almost all TM helices have interhelical hydrogen bonding. In addition, we find that a pair of contacting TM helices is packed tighter when there are interhelical hydrogen bonds between them. We further describe several spatial motifs in the TM regions, including "Polar Clamp" and "Serine Zipper," where conserved Ser residues coincide with tightly packed locations in the TM region. With the examples of halorhodopsin, calcium-transporting ATPase, and bovine cytochrome c oxidase, we discuss the roles of hydrogen bonds in stabilizing helical bundles in polytopic membrane proteins and in protein functions.     

A computational analysis of non-genomic plasma membrane progestin binding proteins: Signaling through ion channel-linked cell surface receptors

A number of plasma membrane progestin receptors linked to non-genomic events have been identified. These include: (1) α1-subunit of the Na⁺/K⁺-ATPase (ATP1A1), (2) progestin binding PAQR proteins, (3) membrane progestin receptor alpha (mPRα), (4) progesterone receptor MAPR proteins and (5) the association of nuclear receptor (PRB) with the plasma membrane. This study compares: the pore-lining regions (ion channels), transmembrane (TM) helices, caveolin binding (CB) motifs and leucine-rich repeats (LRRs) of putative progesterone receptors. ATP1A1 contains 10 TM helices (TM-2, 4, 5, 6 and 8 are pores) and 4 CB motifs; whereas PAQR5, PAQR6, PAQR7, PAQRB8 and fish mPRα each contain 8 TM helices (TM-3 is a pore) and 2–4 CB motifs. MAPR proteins contain a single TM helix but lack pore-lining regions and CB motifs. PRB contains one or more TM helices in the steroid binding region, one of which is a pore. ATP1A1, PAQR5/7/8, mPRα, and MAPR-1 contain highly conserved leucine-rich repeats (LRR, common to plant membrane proteins) that are ligand binding sites for ouabain-like steroids associated with LRR kinases. LRR domains are within or overlap TM helices predicted to be ion channels (pore-lining regions), with the variable LRR sequence either at the C-terminus (PAQR and MAPR-1) or within an external loop (ATP1A1). Since ouabain-like steroids are produced by animal cells, our findings suggest that ATP1A1, PAQR5/7/8 and mPRα represent ion channel-linked receptors that respond physiologically to ouabain-like steroids (not progestin) similar to those known to regulate developmental and defense-related processes in plants.     

GALLEX,a measurement of heterologous association of transmembrane helices in a biological membrane

Whereas a variety of two-hybrid systems are available to measure the interaction of soluble proteins, related methods are significantly less developed for the measurement of membrane protein interactions. Here we present a two-hybrid system to follow the heterodimerization of membrane proteins in the Escherichia coli inner membrane. The method is based on the repression of a reporter gene activity by two LexA DNA binding domains with different DNA binding specificities. When coupled to transmembrane domains, heterodimeric association is reported by repression of beta-galactosidase synthesis. The LexA-transmembrane chimeric proteins were found to correctly insert into the membrane, and reproducible signals were obtained measuring the homodimerization as well as heterodimerization of wild-type and mutant glycophorin A transmembrane helices. The GALLEX data were compared with data recently gained by other methods and discussed in the general context of heteroassociation of single TM helices. Additionally, the formation of heterodimers between the TM domains of the alpha(4) and the beta(7) integrin subunits were tested. The results show that both homo- and heterodimerization of membrane proteins can be measured accurately using the GALLEX system.     

Combining hydrophobicity and helicity: a novel approach to membrane protein structure prediction

In spite of the overwhelming numbers and critical biological functions of membrane proteins, only a few have been characterized by high-resolution structural techniques. From the structures that are known, it is seen that their transmembrane (TM) segments tend to fold most often into alpha-helices. To evaluate systematically the features of these TM segments, we have taken two approaches: (1) using the experimentally-measured residence behavior of specifically designed hydrophobic peptides in RP-HPLC, a scale was derived based directly on the properties of individual amino acids incorporated into membrane-interactive helices: and (2) the relative alpha-helical propensity of each of the 20 amino acids was measured in the organic non-polar environment of n-butanol. By combining the resulting hydrophobicity and helical propensity data, in conjunction with consideration of the 'threshold hydrophobicity' required for spontaneous membrane integration of protein segments, an approach was developed for prediction of TM segments wherein each must fulfill the dual requirements of hydrophobicity and helicity. Evaluated against the available high-resolution structural data on membrane proteins, the present combining method is shown to provide accurate predictions for the locations of TM helices. In contrast, no segment in soluble proteins was predicted as a 'TM helix'.     

An automatic method for predicting transmembrane protein structures using cryo-EM and evolutionary data

The transmembrane (TM) domains of many integral membrane proteins are composed of alpha-helix bundles. Structure determination at high resolution (<4 A) of TM domains is still exceedingly difficult experimentally. Hence, some TM-protein structures have only been solved at intermediate (5-10 A) or low (>10 A) resolutions using, for example, cryo-electron microscopy (cryo-EM). These structures reveal the packing arrangement of the TM domain, but cannot be used to determine the positions of individual amino acids. The observation that typically, the lipid-exposed faces of TM proteins are evolutionarily more variable and less charged than their core provides a simple rule for orienting their constituent helices. Based on this rule, we developed score functions and automated methods for orienting TM helices, for which locations and tilt angles have been determined using, e.g., cryo-EM data. The method was parameterized with the aim of retrieving the native structure of bacteriorhodopsin among near- and far-from-native templates. It was then tested on proteins that differ from bacteriorhodopsin in their sequences, architectures, and functions, such as the acetylcholine receptor and rhodopsin. The predicted structures were within 1.5-3.5 A from the native state in all cases. We conclude that the computational method can be used in conjunction with cryo-EM data to obtain approximate model structures of TM domains of proteins for which a sufficiently heterogeneous set of homologs is available. We also show that in those proteins in which relatively short loops connect neighboring helices, the scoring functions can discriminate between near- and far-from-native conformations even without the constraints imposed on helix locations and tilt angles that are derived from cryo-EM.     

Energetics, stability, and prediction of transmembrane helices

We show that the peptide backbone of an alpha-helix places a severe thermodynamic constraint on transmembrane (TM) stability. Neglect of this constraint by commonly used hydrophobicity scales underlies the notorious uncertainty of TM helix prediction by sliding-window hydropathy plots of membrane protein (MP) amino acid sequences. We find that an experiment-based whole-residue hydropathy scale (WW scale), which includes the backbone constraint, identifies TM helices of membrane proteins with an accuracy greater than 99 %. Furthermore, it correctly predicts the minimum hydrophobicity required for stable single-helix TM insertion observed in Escherichia coli. In order to improve membrane protein topology prediction further, we introduce the augmented WW (aWW) scale, which accounts for the energetics of salt-bridge formation. An important issue for genomic analysis is the ability of the hydropathy plot method to distinguish membrane from soluble proteins. We find that the method falsely predicts 17 to 43 % of a set of soluble proteins to be MPs, depending upon the hydropathy scale used.     

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.     

Empirical lipid propensities of amino acid residues in multispan alpha helical membrane proteins

Characterizing the interactions between amino acid residues and lipid molecules is important for understanding the assembly of transmembrane helices and for studying membrane protein folding. In this study we develop TMLIP (TransMembrane helix-LIPid), an empirically derived propensity of individual residue types to face lipid membrane based on statistical analysis of high-resolution structures of membrane proteins. Lipid accessibilities of amino acid residues within the transmembrane (TM) region of 29 structures of helical membrane proteins are studied with a spherical probe of radius of 1.9 A. Our results show that there are characteristic preferences for residues to face the headgroup region and the hydrocarbon core region of lipid membrane. Amino acid residues Lys, Arg, Trp, Phe, and Leu are often found exposed at the headgroup regions of the membrane, where they have high propensity to face phospholipid headgroups and glycerol backbones. In the hydrocarbon core region, the strongest preference for interacting with lipids is observed for Ile, Leu, Phe and Val. Small and polar amino acid residues are usually buried inside helical bundles and are strongly lipophobic. There is a strong correlation between various hydrophobicity scales and the propensity of a given residue to face the lipids in the hydrocarbon region of the bilayer. Our data suggest a possibly significant contribution of the lipophobic effect to the folding of membrane proteins. This study shows that membrane proteins have exceedingly apolar exteriors rather than highly polar interiors. Prediction of lipid-facing surfaces of boundary helices using TMLIP1 results in a 54% accuracy, which is significantly better than random (25% accuracy). We also compare performance of TMLIP with another lipid propensity scale, kPROT, and with several hydrophobicity scales using hydrophobic moment analysis.     

Biophysical approaches to membrane protein structure determination.

Recently, there have been several technical advances in the use of solution and solid-state NMR spectroscopy to determine the structures of membrane proteins. The structures of several isolated transmembrane (TM) helices and pairs of TM helices have been solved by solution NMR methods. Similarly, the complete folds of two TM beta-barrel proteins with molecular weights of 16 and 19 kDa have been determined by solution NMR in detergent micelles. Solution NMR has also provided a first glimpse at the dynamics of an integral membrane protein. Structures of individual TM helices have also been determined by solid-state NMR. A combination of NMR with site-directed spin-label electron paramagnetic resonance or Fourier transform IR spectroscopy allows one to assemble quite detailed protein structures in the membrane.     

Converting a marginally hydrophobic soluble protein into a membrane protein

δ-Helices are marginally hydrophobic α-helical segments in soluble proteins that exhibit certain sequence characteristics of transmembrane (TM) helices [Cunningham, F., Rath, A., Johnson, R. M. & Deber, C. M. (2009). Distinctions between hydrophobic helices in globular proteins and TM segments as factors in protein sorting. J. Biol. Chem., 284, 5395-402]. In order to better understand the difference between δ-helices and TM helices, we have studied the insertion of five TM-like δ-helices into dog pancreas microsomal membranes. Using model constructs in which an isolated δ-helix is engineered into a bona fide membrane protein, we find that, for two δ-helices originating from secreted proteins, at least three single-nucleotide mutations are necessary to obtain efficient membrane insertion, whereas one mutation is sufficient in a δ-helix from the cytosolic protein P450BM-3. We further find that only when the entire upstream region of the mutated δ-helix in the intact cytochrome P450BM-3 is deleted does a small fraction of the truncated protein insert into microsomes. Our results suggest that upstream portions of the polypeptide, as well as embedded charged residues, protect δ-helices in globular proteins from being recognized by the signal recognition particle-Sec61 endoplasmic-reticulum-targeting machinery and that δ-helices in secreted proteins are mutationally more distant from TM helices than δ-helices in cytosolic proteins.     

Transmembrane helices before, during, and after insertion

The transmembrane (TM) helix is the fundamental structural unit of helix-bundle membrane proteins. Recent biophysical studies provide new insights into the interactions of TM helices with each other and with membrane lipid bilayers. The biological process of helix insertion is carried out by translocon complexes acting in concert with ribosomes. An electron cryo-microscopic reconstruction of these complexes reveals their architecture in new detail, and shows that the complex is constructed from four SecY/Sec61 heterotrimers and two TRAP complexes. A disulfide bridge study shows that elongating polypeptide chains pass through the pore previously identified in the X-ray structure of an archaeal SecY heterotrimer. The fundamental code used by the translocon to select polypeptide segments for insertion as TM helices has been broken. A detailed analysis of the TM amino acid distributions of helix-bundle membrane proteins of known structure recapitulates this code.     

Voltage-dependent energetics of alamethicin monomers in the membrane

The implicit membrane model IMM1 is extended to include the effect of transmembrane potential and used to investigate the optimal membrane binding configurations and energies for alamethicin helices. In the absence of voltage, the lowest energy configuration is on the membrane surface with a tilt allowing the N terminus to be fully buried. Slightly higher in energy is an also tilted configuration with the N terminus deeper in the membrane and almost crossing the membrane. In 26A membranes and in the presence of 0.1V voltage, the TM orientation becomes lower in energy. This is consistent with the assumption that voltage induces a transition from the interfacial to the inserted (TM) orientation. This effect of voltage is smaller in thicker membranes. The results are compared to previous experimental and theoretical studies and the findings are discussed in relation to the mechanism of channel formation by alamethicin.