Distinct protein interfaces in transmembrane domains suggest an in vivo folding model

Given the known high-resolution structures of alpha-helical transmembrane domains, we show that there are statistically distinct classes of transmembrane interfaces which relate to the folding and oligomerization of transmembrane domains. Distinct types of interfaces have been categorized and refer to those between: the same polypeptide chain, different polypeptide chains, helices that are sequential neighbors, and those that are nonsequential. These different interfaces may reflect different phases in the mechanism of transmembrane domain folding and are consistent with the current experimental evidence pertaining to the folding and oligomerization of transmembrane domains. The classes of helix-helix interfaces have been identified in terms of the numbers and different types of pairwise amino acid interactions. The specific measures used are interaction entropy, the information content of interacting partners compared to a random set of contacts, the amino acid composition of the classes and the abundances of specific amino acid pairs in close contact. Knowledge of the clear differences in the types of helix-helix contacts helps with the derivation of knowledge-based constraints which until now have focused on only the interiors of transmembrane domains as compared to the exterior. Taken together, an in vivo model for membrane protein folding is presented, which is distinct from the familiar two-stage model. The model takes into account the different interfaces of membrane helices defined herein, and the available data regarding folding in the translocation channel.     

Structure and mechanism of bacterial periplasmic transport systems

Bacterial periplasmic transport systems are complex, multicomponent permeases, present in Gram-negative bacteria. Many such permeases have been analyzed to various levels of detail. A generalized picture has emerged indicating that their overall structure consists of four proteins, one of which is a soluble periplasmic protein that binds the substrate and the other three are membrane bound. The liganded periplasmic protein interacts with the membrane components, which presumably form a complex, and which by a series of conformational changes allow the formation of an entry pathway for the substrate. The two extreme alternatives for such pathway involve either the formation of a nonspecific hydrophilic pore or the development of a ligand-binding site(s) on the membrane-bound complex. One of the membrane-bound components from each system constitutes a family of highly homologous proteins containing sequence domains characteristic of nucleotide-binding sites. Indeed, in several cases, they have been shown to bind ATP, which is thus postulated to be involved in the energy-coupling mechanism. Interestingly, eukaryotic proteins homologous to this family of proteins have been identified (mammalianmdr genes and Drosophilawhite locus), thus indicating that they perform a universal function, presumably related to energy coupling in membrane-related processes. The mechanism of energy coupling in periplasmic permeases is discussed.     

Sensory transducers of E. coli are composed of discrete structural and functional domains

The tar and tsr genes of E. coli encode functionally analogous transducer proteins that mediate two distinct classes of chemotactic response. The tap gene lies adjacent to tar, and is thought to encode another transducer protein. We present here the complete nucleotide sequence of the tar-tap region of the E. coli genome, together with a comparative analysis of the sequences of the Tar, Tap, and Tsr proteins. The proteins appear to have a simple transmembrane structure consisting of an extracytoplasmic amino-terminal domain, a membrane-spanning domain, and an intracellular carboxy-terminal domain. The carboxy-terminal domains of three proteins possess highly homologous sequences and contain sites of methylation involved in sensory adaptation, while the amino-terminal sequences are only distantly related to one another, consistent with their serving as chemoreceptor domains that have diverged functionally.     

Syndecans,signaling, and cell adhesion

Syndecans are transmembrane proteoglycans which can participate in diverse cell surface interactions, involving extracellular matrix macromolecules, growth factors, protease inhibitors, and even viral entry. Currently, all extracellular interactions are believed to be mediated by distinct structures within the heparan sulfate chains, leaving the roles of chondroitin sulfate chains and extracellular portion of the core proteins to be elucidated. Evidence that syndecans are a class of receptor involved in cell adhesion is mounting, and their small cytoplasmic domains may link with the microfilament cytoskeleton, thereby mediating signaling events. The molecular details are unknown, but the conservation of regions of syndecan cytoplasmic domains, and a strong tendency for homotypic association, support the idea that the ligand-induced clustering may be a discrete source of specific transmembrane signaling from matrix to cytoskeleton, as proposed for other classes of adhesion receptors. © 1996 Wiley-Liss, Inc.     

Molecular evolution and structural analysis of the Ca(2+) release-activated Ca(2+) channel subunit, Orai

Depletion of intracellular Ca(2+) stores evokes Ca(2+) entry across the plasma membrane by inducing Ca(2+) release-activated Ca(2+) (CRAC) currents in many cell types. Recently, Orai and STIM proteins were identified as the molecular identities of the CRAC channel subunit and the endoplasmic reticulum Ca(2+) sensor, respectively. Here, extensive database searching and phylogenetic analysis revealed several lineage-specific duplication events in the Orai protein family, which may account for the evolutionary origins of distinct functional properties among mammalian Orai proteins. Based on similarity to key structural domains and essential residues for channel functions in Orai proteins, database searching also identifies a putative primordial Orai sequence in hyperthermophilic archaeons. Furthermore, modern Orai appears to acquire new structural domains as early as Urochodata, before divergence into vertebrates. The evolutionary patterns of structural domains might be related to distinct functional properties of Drosophila and mammalian CRAC currents. Interestingly, Orai proteins display two conserved internal repeats located at transmembrane segments 1 and 3, both of which contain key amino acids essential for channel function. These findings demonstrate biochemical and physiological relevance of Orai proteins in light of different evolutionary origins and will provide novel insights into future structural and functional studies of Orai proteins.     

Common structural requirements for heptahelical domain function in class A and class C G protein-coupled receptors

G protein-coupled receptors (GPCRs) are key players in cell communication. Several classes of such receptors have been identified. Although all GPCRs possess a heptahelical domain directly activating G proteins, important structural and sequence differences within receptors from different classes suggested distinct activation mechanisms. Here we show that highly conserved charged residues likely involved in an interaction network between transmembrane domains (TM) 3 and 6 at the cytoplasmic side of class C GPCRs are critical for activation of the gamma-aminobutyric acid type B receptor. Indeed, the loss of function resulting from the mutation of the conserved lysine residue into aspartate or glutamate in the TM3 of gamma-aminobutyric acid type B(2) can be partly rescued by mutating the conserved acidic residue of TM6 into either lysine or arginine. In addition, mutation of the conserved lysine into an acidic residue leads to a nonfunctional receptor that displays a high agonist affinity. This is reminiscent of a similar ionic network that constitutes a lock stabilizing the inactive state of many class A rhodopsin-like GPCRs. These data reveal that despite their original structure, class C GPCRs share with class A receptors at least some common structural feature controlling G protein activation.     

Genomic and functional characterization of the diverse immunoglobulin domain-containing protein (DICP) family

A heretofore-unrecognized multigene family encoding diverse immunoglobulin (Ig) domain-containing proteins (DICPs) was identified in the zebrafish genome. Twenty-nine distinct loci mapping to three chromosomal regions encode receptor-type structures possessing two classes of Ig ectodomains (D1 and D2). The sequence and number of Ig domains, transmembrane regions and signaling motifs vary between DICPs. Interindividual polymorphism and alternative RNA processing contribute to DICP diversity. Molecular models indicate that most D1 domains are of the variable (V) type; D2 domains are Ig-like. Sequence differences between D1 domains are concentrated in hypervariable regions on the front sheet strands of the Ig fold. Recombinant DICP Ig domains bind lipids, a property shared by mammalian CD300 and TREM family members. These findings suggest that novel multigene families encoding diversified immune receptors have arisen in different vertebrate lineages and affect parallel patterns of ligand recognition that potentially impact species-specific advantages.     

Bacterial periplasmic permeases belong to a family of transport proteins operating from Escherichia coli to human: Traffic ATPases

Bacterial periplasmic transport systems are complex permeases composed of a soluble substrate-binding receptor and a membrane-bound complex containing 2-4 proteins. Recent developments have clearly demonstrated that these permeases are energized by the hydrolysis of ATP. Several in vitro systems have allowed a detailed study of the essential parameters functioning in these permeases. Several of the component proteins have been shown to interact with each other and the actual substrate for the transport process has been shown to be the liganded soluble receptor. The affinity of this substrate for the membrane complex is approximately 10 microM. The involvement of ATP in energy coupling is mediated by one of the proteins in the membrane complex. For each specific permease, this protein is a member of a family of conserved proteins which bind ATP. The similarity between the members of this family is high and extends itself beyond the consensus motifs for ATP binding. Interestingly, over the last few years, several eukaryotic membrane-bound proteins have been discovered which bear a high level of homology to the family of the conserved components of bacterial periplasmic permeases. Most of these proteins are known to, or can be inferred to participate in a transport process, such as in the case of the multidrug resistance protein (MDR), the STE6 gene product of yeast, and possibly the cystic fibrosis protein. This homology suggests a similarity in the mechanism of action and possibly a common evolutionary origin. This exciting development will stimulate progress in both the prokaryotic and eukaryotic areas of research by the use of overlapping procedures and model building. We propose that this universal class of permeases be called 'Traffic ATPases' to distinguish them from other types of transport systems, and to highlight their involvement in the transport of a vast variety of substrates in either direction relative to the cell interior and their use of ATP as energy source.     

Clustering of MAL genes in Hansenula polymorpha: cloning of the maltose permease gene and expression from the divergent intergenic region between the maltose permease and maltase genes

Hansenula polymorpha uses maltase to grow on maltose and sucrose. Inspection of genomic clones of H. polymorpha showed that the maltase gene HPMAL1 is clustered with genes corresponding to Saccharomyces cerevisiae maltose permeases and MAL activator genes orthologues. We sequenced the H. polymorpha maltose permease gene HPMAL2 of the cluster. The protein (582 amino acids) deduced from the HPMAL2 gene is predicted to have eleven transmembrane domains and shows 39-57% identity with yeast maltose permeases. The identity of the protein is highest with maltose permeases of Debaryomyces hansenii and Candida albicans. Expression of the HPMAL2 in a S. cerevisiae maltose permease-negative mutant CMY1050 proved functionality of the permease protein encoded by the gene. HPMAL1 and HPMAL2 genes are divergently positioned similarly to maltase and maltose permease genes in many yeasts. A two-reporter assay of the expression from the HPMAL1-HPMAL2 intergenic region showed that expression of both genes is coordinately regulated, repressed by glucose, induced by maltose, and that basal expression is higher in the direction of the permease gene.     

Molecular analysis of the myosin gene family in Arabidopsis thaliana

Myosin is believed to act as the molecular motor for many actin-based motility processes in eukaryotes. It is becoming apparent that a single species may possess multiple myosin isoforms, and at least seven distinct classes of myosin have been identified from studies of animals, fungi, and protozoans. The complexity of the myosin heavy-chain gene family in higher plants was investigated by isolating and characterizing myosin genomic and cDNA clones from Arabidopsis thaliana. Six myosin-like genes were identified from three polymerase chain reaction (PCR) products (PCR1, PCR11, PCR43) and three cDNA clones (ATM2, MYA2, MYA3). Sequence comparisons of the deduced head domains suggest that these myosins are members of two major classes. Analysis of the overall structure of the ATM2 and MYA2 myosins shows that they are similar to the previously-identified ATM1 and MYA1 myosins, respectively. The MYA3 appears to possess a novel tail domain, with five IQ repeats, a six-member imperfect repeat, and a segment of unique sequence. Northern blot analyses indicate that some of the Arabidopsis myosin genes are preferentially expressed in different plant organs. Combined with previous studies, these results show that the Arabidopsis genome contains at least eight myosin-like genes representing two distinct classes.     

P-glycoprotein structure and evolutionary homologies

Analysis of multidrug resistant cell lines has led to the identification of the P-glycoprotein multigene family. Two of the three classes of mammalian P-glycoproteins have the ability to confer cellular resistance to a broad range of structurally and functionally diverse cytotoxic agents. P-glycoproteins are integral membrane glycoproteins comprised of two similar halves, each consisting of six membrane spanning domains followed by a cytoplasmic domain which includes a nucleotide binding fold. The P-glycoprotein is a member of a large superfamily of transport proteins which utilize ATP to translocate a wide range of substrates across biological membranes. This superfamily includes transport complexes comprised of multicomponent systems, half P-glycoproteins and P-glycoprotein-like homologs which appear to require approximately 12 α-helical transmembrane domains and two nucleotide binding folds for substrate transport. P-glycoprotein homologs have been isolated and characterized from a wide range of species. Amino acid sequences, the similarities between the halves and intron/exon boundaries have been compared to understand the evolutionary origins of the P-glycoprotein. This revised version was published online in August 2006 with corrections to the Cover Date.     

Calcium transport across the sarcoplasmic reticulum: structure and function of Ca2+-ATPase and the ryanodine receptor.

Contraction of striated muscle results from a rise in cytoplasmic calcium concentration in a process termed excitation/contraction coupling. Most of this calcium moves back and forth across the sarcoplasmic-reticulum membrane in cycles of contraction and relaxation. The channel responsible for release from the sarcoplasmic reticulum is the ryanodine receptor, whereas Ca2+-ATPase effects reuptake in an ATP-dependent manner. The structures of these two molecules have been studied by cryoelectron microscopy, with helical crystals in the case of Ca2+-ATPase and as isolated tetramers in the case of ryanodine receptor. Structures of Ca2+-ATPase at 8-A resolution reveal the packing of transmembrane helices and have allowed fitting of a putative ATP-binding domain among the cytoplasmic densities. Comparison of ATPases in different conformations gives hints about the conformational changes that accompany the reaction cycle. Structures of ryanodine receptor at 30-A resolution reveal a multitude of isolated domains in the cytoplasmic portion, as well as a distinct transmembrane assembly. Binding sites for various protein ligands have been determined and conformational changes induced by ATP, calcium and ryanodine have been characterized. Both molecules appear to use large conformational changes to couple interactions in their cytoplasmic domains with calcium transport through their membrane domains, and future studies at higher resolution will focus on the mechanisms for this coupling.     

The major facilitator superfamily (MFS) revisited

The major facilitator superfamily (MFS) is the largest known superfamily of secondary carriers found in the biosphere. It is ubiquitously distributed throughout virtually all currently recognized organismal phyla. This superfamily currently (2012) consists of 74 families, each of which is usually concerned with the transport of a certain type of substrate. Many of these families, defined phylogenetically, do not include even a single member that is functionally characterized. In this article, we probe the evolutionary origins of these transporters, providing evidence that they arose from a single 2-transmembrane segment (TMS) hairpin structure that triplicated to give a 6-TMS unit that duplicated to a 12-TMS protein, the most frequent topological type of these permeases. We globally examine MFS protein topologies, focusing on exceptional proteins that deviate from the norm. Nine distantly related families appear to have members with 14?TMSs in which the extra two are usually centrally localized between the two 6-TMS repeat units. They probably have arisen by intragenic duplication of an adjacent hairpin. This alternative topology probably arose multiple times during MFS evolution. Convincing evidence for MFS permeases with fewer than 12?TMSs was not forthcoming, leading to the suggestion that all 12?TMSs are required for optimal function. Some homologs appear to have 13, 14, 15 or 16 TMSs, and the probable locations of the extra TMSs were identified. A few MFS permeases are fused to other functional domains or are fully duplicated to give 24-TMS proteins with dual functions. Finally, the MFS families with no known function were subjected to genomic context analyses leading to functional predictions.     

Energy coupling in bacterial periplasmic transport systems. Studies in intact Escherichia coli cells

Periplasmic permeases are composed of four proteins, one of which has an ATP-binding site that has been postulated to be involved in energy coupling. Previous data suggested that these permeases derive energy from substrate level phosphorylation (Berger, E. A. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 1514-1518); however, conflicting results later cast doubt upon this hypothesis. Here, we make use of two well characterized periplasmic permeases and of a well characterized unc mutant (ATPase-) to examine this energetics problem in depth. We have utilized the histidine and maltose periplasmic permeases in Escherichia coli as model systems. Isogenic unc strains were used in order to study separately the effect of the proton-motive force and of ATP on transport. These parameters were analyzed concomitantly with transport assays. Starvation experiments indicate that both histidine and maltose transport require ATP generation and that a normal level of delta psi is not sufficient. Uncouplers such as carbonyl cyanide-m-chlorophenylhydrazone and 2,4-dinitrophenol dissipated the delta psi without decreasing the ATP level and without significant effect on these permeases, showing that delta psi is not needed. Inhibition of ATP synthesis by arsenate eliminates transport through both permeases, confirming the need for ATP. In agreement with previous results with the glutamine permease (Plate, C. A. (1979) J. Bacteriol. 137, 221-225), valinomycin plus K+ dissipates delta psi without affecting ATP levels and inhibits histidine transport; however, maltose transport is not inhibited under these conditions. This result is discussed in terms of the artefactual side effects caused by valinomycin/K+ treatment on some periplasmic permeases. Histidine transport is also shown to be sensitive to changes in the cytoplasmic pH. It is concluded that periplasmic permeases indeed have an obligatory requirement for ATP (or a closely related molecule), whereas the proton-motive force is neither sufficient nor essential.     

Distinct membrane localization and kinase association of the two isoforms of CD58

The adhesion molecule CD58 is involved in intercellular adhesion and in signal transduction. It is natively expressed in both a transmembrane form and a glycosylphosphatidylinositol (GPI)-anchored form, and hence provides a model for the study of two distinct membrane-anchored forms of the same protein in the same cell. We demonstrate here that the two isoforms of CD58 are localized in distinct membrane compartments. The GPI-anchored form localizes in lipid rafts, while the transmembrane form resides in nonraft domains. In addition to distinct membrane localization, the two isoforms of CD58 differ in their association with protein kinases. GPI-anchored CD58, residing in raft domains, is constitutively associated with protein kinases. However, cross-linking mediates a substantial increase in kinase activity which is predominantly associated with the transmembrane CD58 in nonraft membrane domains. The extensive inducible kinase activity, associated with transmembrane CD58, is demonstrated in wild-type cells as well as in GPI-deficient variant cells. Thus, although the transmembrane CD58 is excluded from rafts, it may trigger signaling independently of the GPI-linked isoform.     

Persistent interactions between the two transmembrane clusters dictate the targeting and functional assembly of adenylyl cyclase

Adenylyl cyclases possess complex structures like those of the ATP binding cassette (ABC) transporter family, which includes the cystic fibrosis transmembrane regulator, the P-glycoprotein, and ATP-sensitive K(+) channels [1-4]. These structures comprise a cytosolic N terminus followed by two tandem six-transmembrane cassettes, each associated with a highly homologous (ATP binding) cytosolic loop [5-8]. The catalytic domains, which are located in the two large cytoplasmic loops, are highly conserved and well studied. The crystal structure of these domains has even been described recently [9, 10]. However, nothing is known of the function or organization of the 12 transmembrane segments. In the present study we adopted a range of strategies including live-cell fluorescence resonance energy transfer (FRET) microscopy, coimmunoprecipitation, and functional assays of various truncated and substituted, fluorescently-tagged molecules to analyze the trafficking and activity of this molecule. When expressed as individual peptides, the two transmembrane domains - largely independently of any cytosolic region - formed a tight complex that was delivered to the plasma membrane. This cooperation between the two intact transmembrane domains was essential and sufficient to target the enzyme to the plasma membrane of the cell. The extracellular loop between the ninth and tenth transmembrane segments, which contains an N-glycosylation site, was also necessary. Furthermore, the interaction between the two transmembrane clusters played a critical role in bringing together the cytosolic catalytic domains to express functional adenylyl cyclase activity in the intact cell.