Evolution and adaptation of single-pass transmembrane proteins

A comparative analysis of 6039 single-pass (bitopic) membrane proteins from six evolutionarily distant organisms was performed based on data from the Membranome database. The observed repertoire of bitopic proteins is significantly enlarged in eukaryotic cells and especially in multicellular organisms due to the diversification of enzymes, emergence of proteins involved in vesicular trafficking, and expansion of receptors, structural, and adhesion proteins. The majority of bitopic proteins in multicellular organisms are located in the plasma membrane (PM) and involved in cell communication. Bitopic proteins from different membranes significantly diverge in terms of their biological functions, size, topology, domain architecture, physical properties of transmembrane (TM) helices and propensity to form homodimers. Most proteins from eukaryotic PM and endoplasmic reticulum (ER) have the N-out topology. The predicted lengths of TM helices and hydrophobic thicknesses, stabilities and hydrophobicities of TM α-helices are the highest for proteins from eukaryotic PM, intermediate for proteins from prokaryotic cells, ER and Golgi apparatus, and lowest for proteins from mitochondria, chloroplasts, and peroxisomes. Tyr and Phe residues accumulate at the cytoplasmic leaflet of PM and at the outer leaflet of membranes of bacteria, Golgi apparatus, and nucleus. The propensity for dimerization increases from unicellular to multicellular eukaryotes, from enzymes to receptors, and from intracellular membrane proteins to PM proteins. More than half of PM proteins form homodimers with a 2:1 ratio of right-handed to left-handed helix packing arrangements. The inverse ratio (1:2) was observed for dimers from the ER, Golgi and vesicles.     

Phenylalanine promotes interaction of transmembrane domains via GxxxG motifs

Interactions of transmembrane helices play a crucial role in the folding and oligomerisation of integral membrane proteins. In order to uncover novel sequence motifs mediating these interactions, we randomised one face of a transmembrane helix with a set of non-polar or moderately polar amino acids. Those sequences capable of self-interaction upon integration into bacterial inner membranes were selected by means of the ToxR/POSSYCCAT system. A comparison between low/medium-affinity and high-affinity sequences reveals that high-affinity sequences are strongly enriched in phenylalanine residues that are frequently observed at the − 3 position of GxxxG motifs, thus yielding FxxGxxxG motifs. Mutation of Phe or GxxxG in selected sequences significantly reduces self-interaction of the transmembrane domains without affecting their efficiency of membrane integration. Conversely, grafting FxxGxxxG onto unrelated transmembrane domains strongly enhances their interaction. Further, we find that FxxGxxxG is significantly over-represented in transmembrane domains of bitopic membrane proteins. The same motif contributes to self-interaction of the vesicular stomatitis virus G protein transmembrane domain. We conclude that Phe stabilises membrane-spanning GxxxG motifs. This is one example of how the role of certain side-chains in helix-helix interfaces is modulated by sequence context.     

Ryanodine receptor regulation by intramolecular interaction between cytoplasmic and transmembrane domains

Ryanodine receptors (RyR) function as Ca(2+) channels that regulate Ca(2+) release from intracellular stores to control a diverse array of cellular processes. The massive cytoplasmic domain of RyR is believed to be responsible for regulating channel function. We investigated interaction between the transmembrane Ca(2+)-releasing pore and a panel of cytoplasmic domains of the human cardiac RyR in living cells. Expression of eGFP-tagged RyR constructs encoding distinct transmembrane topological models profoundly altered intracellular Ca(2+) handling and was refractory to modulation by ryanodine, FKBP12.6 and caffeine. The impact of coexpressing dsRed-tagged cytoplasmic domains of RyR2 on intracellular Ca(2+) phenotype was assessed using confocal microscopy coupled with parallel determination of in situ protein: protein interaction using fluorescence resonance energy transfer (FRET). Dynamic interactions between RyR cytoplasmic and transmembrane domains were mediated by amino acids 3722-4610 (Interacting or "I"-domain) which critically modulated intracellular Ca(2+) handling and restored RyR sensitivity to caffeine activation. These results provide compelling evidence that specific interaction between cytoplasmic and transmembrane domains is an important mechanism in the intrinsic modulation of RyR Ca(2+) release channels.     

Proline localized to the interaction interface can mediate self-association of transmembrane domains

Assembly of transmembrane domains (TMDs) is a critical step in the function of membrane proteins. In recent years, the role of specific amino acids in TMD–TMD interactions has been better characterized, with more emphasis on polar and aromatic residues. Despite the high abundance of proline residues in TMDs, contribution of proline to TMD–TMD association has not been intensively studied. Here, we evaluated statistically the frequency of appearance, and experimentally the contribution of proline, compared to other hydrophobic amino acids (Gly, Ala, Val, Leu, Ile, and Met), with regard to TMD–TMD self-assembly. Our model system is the assembly motif (²²QxxS²⁵) found previously in TMDs of the Escherichia coli aspartate receptor (Tar-1). Statistically, our data revealed that all different motifs, except PxxS (P/S), have frequencies similar to their theoretical random expectancy within a database of 41916 sequences of TMDs, while PxxS motif is underrepresented. Experimentally, using the ToxR assembly system, the SDS-gel running pattern of biotin-conjugated TMD peptides, and FRET experiments between fluorescence-labeled peptides, we found that only the P/S motif preserves the dimerization ability of wild-type Tar-1 TMD. Although proline is known as a helix breaker in solution, Circular Dichroism spectroscopy revealed that the secondary structure of the P/S and the wild-type peptides are similar. All together, these data suggest that proline can stabilize TM self-assembly when localized to the interaction interface of a transmembrane oligomer. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

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.     

Membrane anchoring and interaction between transmembrane domains are crucial for K+ channel function

The small viral channel Kcv is a Kir-like K(+) channel of only 94 amino acids. With this simple structure, the tetramer of Kcv represents the pore module of all complex K(+) channels. To examine the structural contribution of the transmembrane domains (TMDs) to channel function, we performed Ala scanning mutagenesis of the two domains and tested the functionality of the mutants in a yeast complementation assay. The data reveal, in combination with computational models, that the upper halves of both TMDs, which face toward the external medium, are rather rigid, whereas the inner parts are more flexible. The rigidity of the outer TMD is conferred by a number of essential aromatic amino acids that face the membrane and probably anchor this domain in the bilayer. The inner TMD is intimately connected with the rigid part of the outer TMD via π···π interactions between a pair of aromatic amino acids. This structural principle is conserved within the viral K(+) channels and also present in Kir2.2, implying a general importance of this architecture for K(+) channel function.     

Site-directed mutagenesis reveals putative regions of protein interaction within the transmembrane domains of connexins

Through cysteine-scanning mutagenesis, the authors have compared sites within the transmembrane domains of two connexins, one from the alpha-class (Cx50) and one from the beta-class (Cx32), where amino acid substitution disrupts the function of gap junction channels. In Cx32, 11 sites resulted in no channel function, or an aberrant voltage gating phenotype referred to as "reverse gating," whereas in Cx50, 7 such sites were identified. In both connexins, the sites lie along specific faces of transmembrane helices, suggesting that these may be sites of transmembrane domain interactions. In Cx32, one broad face of the M1 transmembrane domain and a narrower, polar face of M3 were identified, including one site that was shown to come into close apposition with M4 in the closed state. In Cx50, the same face of M3 was identified, but sensitive sites in M1 differed from Cx32. Many fewer sites in M1 disrupted channel function in Cx50, and those that did were on a different helical face to the sensitive sites in Cx32. A more in depth study of two sites in M1 and M2 of Cx32 showed that side-chain length or branching are important for maintenance of normal channel behavior, consistent with this being a site of transmembrane domain interaction.     

A stable interaction between syntaxin 1a and synaptobrevin 2 mediated by their transmembrane domains

The proteins synaptobrevin (VAMP), SNAP-25 and syntaxin 1 are essential for neuronal exocytosis. They assemble into a stable ternary complex which is thought to initiate membrane fusion. In vitro, the transmembrane domains of syntaxin and synaptobrevin are not required for association. Here we report a novel interaction between synaptobrevin and syntaxin that requires the presence of the transmembrane domains. When co-reconstituted into liposomes, the proteins form a stable binary complex that cannot be disassembled by NSF and that is resistant to denaturation by SDS. Cleavage of synaptobrevin with tetanus toxin does not affect the interaction. Furthermore, the complex is formed when a truncated version of syntaxin is used that contains only 12 additional amino acid residues outside the membrane anchor. We conclude that the interaction is mediated by the transmembrane domains.     

Filopodia formation driven by membrane glycoprotein M6a depends on the interaction of its transmembrane domains

Membrane glycoprotein M6a, which belongs to the tetraspan proteolipid protein family, promotes structural plasticity in neurons and cell lines by unknown mechanisms. This glycoprotein is encoded by Gpm6a, a stress‐regulated gene. The hippocampus of animals chronically stressed by either psychosocial or physical stressors shows decreased M6a expression. Stressed Gpm6a‐null mice develop a claustrophobia‐like phenotype. In humans, de novo duplication of GPM6A results in learning/behavioral abnormalities, and two single‐nucleotide polymorphisms (SNPs) in the non‐coding region are linked to mood disorders. Here, we studied M6a dimerization in neuronal membranes and its functional relevance. We showed that the self‐interaction of M6a transmembrane domains (TMDs) might be driving M6a dimerization, which is required to induce filopodia formation. Glycine mutants located in TMD2 and TMD4 of M6a affected its dimerization, thus preventing M6a‐induced filopodia formation in neurons. In silico analysis of three non‐synonymous SNPs located in the coding region of TMDs suggested that these mutations induce protein instability. Indeed, these SNPs prevented M6a from being functional in neurons, owing to decreased stability, dimerization or improper folding. Interestingly, SNP3 (W141R), which caused endoplasmic reticulum retention, is equivalent to that mutated in PLP1, W161L, which causes demyelinating Pelizaeus–Merzbacher disease.

     

Families of membranous proteins can be characterized by the amino acid composition of their transmembrane domains

SUMMARY: In eukaryotes, membranous proteins account for 20-30% of the proteome. Most of these proteins contain one or more transmembrane (TM) domains. These are short segments that transverse the bilayer lipid membrane. Various properties of the TM domains, such as their number, their topology and their arrangement within the membrane, are closely related to the protein's cellular functions. The properties of the TM domains also determine the cellular targeting and localization of these proteins. It is not known, however, whether the information encoded by TM domains suffices for the purpose of classifying proteins into their functional families. This is the question we address here. We introduce an algorithm that creates a profile of each functional family of membranous proteins based only on the amino acid composition of their TM domains. This is complemented by a classifier program for each such family (to determine whether a given protein belongs to it or not). We find that in most instances TM domains contain enough information to allow an accurate discrimination of approximately 80% sensitivity and approximately 90% specificity among unrelated polytopic functional families with the same number of TM domains. SUPPLEMENTARY INFORMATION: Available at www.protonet.cs.huji.ac.il/TM/     

Reliable expression and purification of highly insoluble transmembrane domains

A general procedure for the reliable preparation of insoluble transmembrane domains has been developed. Improved expression schemes were developed by expressing the transmembrane domains of caveolin proteins 1, 2, and 3 as a fusion to the Trp leader protein. This construct readily formed inclusion bodies during overexpression, allowing high levels of protein to be achieved. Cleavage of the transmembrane domain away from the Trp leader carrier protein was performed with cyanogen bromide. The transmembrane domains were then purified using reverse-phase high-performance liquid chromatography with a C4 column and were eluted with a mixture of 1-butanol and acetic acid. Using this method, the 39-42 amino acid transmembrane domains from caveolin proteins 1, 2, and 3 were successfully purified to homogeneity. Further verification of this method was successfully done with Rfbp(18-51), another insoluble transmembrane domain.     

Structural biology of transmembrane domains: efficient production and characterization of transmembrane peptides by NMR

Structural characterization of transmembrane peptides (TMPs) is justified because transmembrane domains of membrane proteins appear to often function independently of the rest of the protein. However, the challenge in obtaining milligrams of isotopically labeled TMPs to study these highly hydrophobic peptides by nuclear magnetic resonance (NMR) is significant. In the present work, a protocol is developed to produce, isotopically label, and purify TMPs in high yield as well as to initially characterize the TMPs with CD and both solution and solid-state NMR. Six TMPs from three integral membrane proteins, CorA, M2, and KdpF, were studied. CorA and KdpF are from Mycobacterium tuberculosis, while M2 is from influenza A virus. Several milligrams of each of these TMPs ranging from 25 to 89 residues were obtained per liter of M9 culture. The initial structural characterization results showed that these peptides were well folded in both detergent micelles and lipid bilayer preparations. The high yield, the simplicity of purification, and the convenient protocol represents a suitable approach for NMR studies and a starting point for characterizing the transmembrane domains of membrane proteins.     

Lymphocyte transformation and transmembrane potential depressors

Potassium and rubidium at high concentrations, as well as valinomycin, gramicidin, and ouabain, inhibit the PHA-induced synthesis of DNA. They act primarily at an early step in the G 1 period. All these inhibitors have in common a tendency to depress the transmembrane potential suggesting a relationship between the potential and lymphocyte transformation.     

Single-spanning transmembrane domains in cell growth and cell-cell interactions

As a whole, integral membrane proteins represent about one third of sequenced genomes, and more than 50% of currently available drugs target membrane proteins, often cell surface receptors. Some membrane protein classes, with a defined number of transmembrane (TM) helices, are receiving much attention because of their great functional and pharmacological importance, such as G protein-coupled receptors possessing 7 TM segments. Although they represent roughly half of all membrane proteins, bitopic proteins (with only 1 TM helix) have so far been less well characterized. Though they include many essential families of receptors, such as adhesion molecules and receptor tyrosine kinases, many of which are excellent targets for biopharmaceuticals (peptides, antibodies, et al.). A growing body of evidence suggests a major role for interactions between TM domains of these receptors in signaling, through homo and heteromeric associations, conformational changes, assembly of signaling platforms, etc. Significantly, mutations within single domains are frequent in human disease, such as cancer or developmental disorders. This review attempts to give an overview of current knowledge about these interactions, from structural data to therapeutic perspectives, focusing on bitopic proteins involved in cell signaling.     

L-selectin transmembrane and cytoplasmic domains are monomeric in membranes

A recombinant protein termed CLS, which corresponds to the C-terminal portion of human L-selectin and contains its entire transmembrane and cytoplasmic domains (residues Ser473-Arg542), has been produced and its oligomeric state in detergents characterized. CLS migrates in the SDS polyacrylamide gel at a pace that is typically expected from a complex twice of its molecular weight. Additional studies revealed, however, that this is due to residues in the cytoplasmic domain, as mutations in this region or its deletion significantly increased the electrophoretic rate of CLS. Analytical ultracentrifugation and fluorescence resonance energy transfer studies indicated that CLS reconstituted in dodecylphosphocholine detergent micelles is monomeric. When the transmembrane domain of L-selectin is inserted into the inner membrane of Escherichia coli as a part of a chimeric protein in the TOXCAT assay, little oligomerization of the chimeric protein is observed. Overall, these results suggest that transmembrane and cytoplasmic domains of L-selectin lack the propensity to self-associate in membranes, in contrast to the previously documented dimerization of the transmembrane domain of closely related P-selectin. This study will provide constraints for future investigations on the interaction of L-selectin and its associating proteins.     

Tryptophan supports interaction of transmembrane helices

Interactions of transmembrane helices play an important role in folding and oligomerization of integral membrane proteins. The interfacial residues of these helices frequently correspond to heptad repeat motifs. In order to uncover novel mechanisms underlying these interactions, we randomised a heptad repeat pattern with a complete set of amino acids. Those sequences that were capable of high-affinity self-interaction upon integration into bacterial inner membranes were selected by means of the POSSYCCAT system. A comparison between selected and non-selected sequences reveals that high-affinity sequences were strongly enriched in tryptophan residues that accumulated at specific positions of the heptad motif. Mutation of Trp in selected clones significantly reduced self-interaction of the transmembrane segments without affecting their efficiency of membrane integration. Conversely, grafting Trp onto artificial transmembrane segments strongly enhanced their interaction. We conclude that tryptophan supports interaction of transmembrane segments.     

Structural organization and interactions of transmembrane domains in tetraspanin proteins

Background  

Proteins of the tetraspanin family contain four transmembrane domains (TM1-4) linked by two extracellular loops and a short intracellular loop, and have short intracellular N- and C-termini. While structure and function analysis of the larger extracellular loop has been performed, the organization and role of transmembrane domains have not been systematically assessed.