Transmembrane Helix Region Research Articles

Genomic analysis of membrane protein families: abundance and conserved motifs.

Polytopic membrane proteins can be related to each other on the basis of the number of transmembrane helices and sequence similarities. Building on the Pfam classification of protein domain families, and using transmembrane-helix prediction and sequence-similarity searching, we identified a total of 526 well-characterized membrane protein families in 26 recently sequenced genomes. To this we added a clustering of a number of predicted but unclassified membrane proteins, resulting in a total of 637 membrane protein families. Analysis of the occurrence and composition of these families revealed several interesting trends. The number of assigned membrane protein domains has an approximately linear relationship to the total number of open reading frames (ORFs) in 26 genomes studied. Caenorhabditis elegans is an apparent outlier, because of its high representation of seven-span transmembrane (7-TM) chemoreceptor families. In all genomes, including that of C. elegans, the number of distinct membrane protein families has a logarithmic relation to the number of ORFs. Glycine, proline, and tyrosine locations tend to be conserved in transmembrane regions within families, whereas isoleucine, valine, and methionine locations are relatively mutable. Analysis of motifs in putative transmembrane helices reveals that GxxxG and GxxxxxxG (which can be written GG4 and GG7, respectively; see Materials and methods) are among the most prevalent. This was noted in earlier studies; we now find these motifs are particularly well conserved in families, however, especially those corresponding to transporters, symporters, and channels. We carried out a genome-wide analysis on patterns of the classified polytopic membrane protein families and analyzed the distribution of conserved amino acids and motifs in the transmembrane helix regions in these families.

Molecular mechanism of the P-type ATPases.

The recent determination of the structure of the Ca2+-ATPase of sarcoplasmic reticulum to atomic resolution in the Ca2+-bound state and to near atomic resolution in the Ca2+-free, decavanadate-bound state has paved the way for an ultimate complete understanding of the molecular mechanism of the P-type ATPases. Analysis of this new structure information together with the large amount of biochemical information about these enzymes that preceded it has produced important new revelations about how the P-type ATPases work. Most models propose that these transporters operate by a strictly conformational energy coupling mechanism in which conformational changes in the large cytoplasmic head region mechanically drive the ions to be transported from their binding sites in the transmembrane helix region 50 A away. However, while these enzymes do indeed undergo profound conformational changes, the available evidence suggests that they do not mechanically transduce the chemical energy of ATP hydrolysis into transmembrane ion gradients via these conformational changes. As an alternative, it is proposed that the effects of the chemical events that occur at the phosphorylation/dephosphorylation site in the cytoplasmic region are exerted on the ion-binding sites via two well-defined charge transfer pathways that electronically connect the chemical reaction site with the site of ion binding. The recognition of these charge transfer pathways provides rational explanations of all of the key biochemical features of the P-type ATPase catalytic cycle. Thus, although a few details await elucidation, a nearly complete understanding of the P-type ATPase reaction mechanism may be at hand.

Cloning and characterization of a cDNA encoding human differentiation antigen 5D4

A 1 846 bp cDNA is isolated from a human tonsil cell lambda gt 11 cDNA library (ATCC No. 37546) with mAb 5D4 reactive strongly with human B cell line 3D5, but weakly with human B cell line Daudi and human T cell line Jurkat as a probe. RT-PCR also shows a strong reaction in 3D5 cell and a weak reaction in Daudi and Jurkat cell for 5D4 mRNA. There is an open reading frame from 88 to 1 209 bp in 5D4 cDNA encoding a 374 AA protein. Both the Northern blot analysis and the two consecutive stop codens before start coden demonstrate that the cDNA is a full-length cDNA. Secondary structure prediction suggests that there are a region from 295 to 334 AA in the protein with strong hydrophobicity and a transmembrane helix region with high score from 313 to 334 AA with an orientation from the inside to the outside of the cell.

Subunit IV of Cytochrome bc1 Complex from Rhodobacter sphaeroides: LOCALIZATION OF REGIONS ESSENTIAL FOR INTERACTION WITH THE THREE-SUBUNIT CORE COMPLEX

Recombinant subunit IV mutants which identify the regions essential for restoration of bc(1) activity to the three-subunit core complex of Rhodobacter sphaeroides were generated and characterized. Four C-terminal truncated mutants: IV(1-109), IV(1-85), IV(1-76), and IV(1-40) had 100, 0, 0, and 0% of reconstitutive activity of the wild-type IV, indicating that residues 86-109 are essential. IV(1-109) is associated with the core complex in the same manner as the wild-type IV while mutants IV(1-85), IV(1-76), and IV(1-40) do not associate with the core complex, indicating that subunit IV requires its transmembrane helix region (residues 86-109) for assembly into the bc(1) complex. Since GST-IV(86-109) fusion protein has little reconstitutive activity, some region(s) in residues 1-85 are required for bc(1) activity restoration after subunit IV is incorporated into the complex through the transmembrane helix, presumably by interaction with cytochrome b in the core complex. The interacting regions are identified as residues 41-53 and 77-85, since mutants IV(21-109), IV(41-109), IV(54-109), and IV(77-109) had 95, 98, 53, and 53% of the reconstitutive activity of the wild-type IV. These two interacting regions are on the cytoplasmic side of the chromatophore membrane and closed to the DE loop and helix G of cytochrome b, respectively.

Crystallization, structure and dynamics of the proton-translocating P-type ATPase.

Large single three-dimensional crystals of the dodecylmaltoside complex of the Neurospora crassa plasma membrane H(+)-ATPase (H(+) P-ATPase) can be grown in polyethylene-glycol-containing solutions optimized for moderate supersaturation of both the protein surfaces and detergent micellar region. Large two-dimensional H(+) P-ATPase crystals also grow on the surface of such mixtures and on carbon films located at such surfaces. Electron crystallographic analysis of the two-dimensional crystals grown on carbon films has recently elucidated the structure of the H(+) P-ATPase at a resolution of 0.8 nm in the membrane plane. The two-dimensional crystals comprise two offset layers of ring-shaped ATPase hexamers with their exocytoplasmic surfaces face to face. Side-to-side interactions between the cytoplasmic regions of the hexamers in each layer can be seen, and an interaction between identical exocytoplasmic loops in opposing hexamer layers holds the two layers together. Detergent rings around the membrane-embedded region of the hexamers are clearly visible, and detergent-detergent interactions between the rings are also apparent. The crystal packing forces thus comprise both protein-protein and detergent-detergent interactions, supporting the validity of the original crystallization strategy. Ten transmembrane helices in each ATPase monomer are well-defined in the structure map. They are all relatively straight, closely packed, moderately tilted at various angles with respect to a plane normal to the membrane surface and average approximately 3.5 nm in length. The transmembrane helix region is connected in at least three places to the larger cytoplasmic region, which comprises several discrete domains separated by relatively wide, deep clefts. Previous work has shown that the H(+) P-ATPase undergoes substantial conformational changes during its catalytic cycle that are not changes in secondary structure. Importantly, the results of hydrogen/deuterium exchange experiments indicate that these conformational changes are probably rigid-body interdomain movements that lead to cleft closure. When interpreted within the framework of established principles of enzyme catalysis, this information on the structure and dynamics of the H(+) P-ATPase molecule provides the basis of a rational model for the sequence of events that occurs as the ATPase proceeds through its transport cycle. The forces that drive the sequence can also be clearly stipulated. However, an understanding of the molecular mechanism of ion transport catalyzed by the H(+) P-ATPase awaits an atomic resolution structure.

Characterization of a region of the lutropin receptor extracellular domain near transmembrane helix 1 that is important in ligand-mediated signaling.

The lutropin receptor (LHR), a member of the G protein-coupled receptor family, contains a relatively large N-terminal extracellular domain, accounting for about half of the receptor and responsible for high affinity ligand binding, and a standard heptahelical portion with connecting loops and a C-terminal tail. LHR and the other two glycoprotein hormone receptors, i.e. the follitropin and TSH receptors, contain an invariant 10-amino acid residue sequence, FNPCEDIMGY (residues 328-337 in rat LHR), in the extracellular domain separated by only a few amino acid residues from the beginning of transmembrane helix 1. In view of the invariant nature of this region in the three glycoprotein hormone receptors and preliminary data in the literature on the importance of Glu332 and Asp333 in signal transduction, we undertook a systematic investigation of all 10 amino acid residues because this region may function as a switch or trigger for communicating ligand binding to the extracellular domain with a conformational change of the membrane-embedded C-terminal half of the receptor to activate G proteins, particularly Gs. A total of 36 single, double, and multiple replacements, as well as two deletions, of LHR were prepared and characterized in transiently transfected COS-7 cells. Of these mutants LHRs, 26 expressed on the cell surface in sufficient numbers that quantitative assessments could be made of human choriogonadotropin binding and ligand-mediated cAMP production. Replacements of Cys331 abolished ligand binding to intact cells, although binding could be detected after solubilization of the cells. Replacements of the other nine amino acid residues that did not interfere with receptor folding or trafficking had no significant effect on ligand binding affinity; however, replacements of Pro330, Glu332, and Asp333 resulted in diminished signaling, especially for the two acidic residues. An interesting observation was made in which replacement of Tyr337 with Ala or Asp, while having no profound change on receptor function, could overcome to some extent limited expression of replacements at positions 332 and/or 333, thus permitting a more definitive analysis of signaling. Replacement of the decapeptide sequence with Gly10 prevents expression, whereas deletion of all 10 residues and deletion of Glu332-Asp333 prevents functional expression at the cell surface. Thus, this invariant sequence in the glycoprotein hormones is required for proper folding, trafficking, and ligand-mediated signaling, but not ligand binding, in LHR. Amino acid residues, Glu332, Asp333, and to a limited extent, Pro330, are important in ligand-mediated signaling but not ligand binding.

A novel electron transfer mechanism suggested by crystallographic studies of mitochondrial cytochrome bc1 complex

The crystal structure of bovine mitochondrial cytochrome bc1 complex, an integral membrane protein complex of 11 different subunits with a total molecular mass of 242 kDa, demonstrated a tightly associated dimer consisting of three major regions: a matrix region primarily made of subunits core1, core2, 6, and 9; a transmembrane-helix region of 26 helices in the dimer contributed by cytochrome b, cytochrome c1, the Rieske iron-sulfur protein (ISP), subunits 7, 10, and 11; and an intermembrane-space region composed of extramembrane domains of ISP, cytochrome c1, and subunit 8. The structure also revealed the positions of and distances between irons of prosthetic groups, and two symmetry related cavities in the transmembrane-helix region upon dimerization of the bc1 complex. Extensive crystallographic studies on crystals of bc1 complexed with inhibitors of electron transfer identified binding pockets for both Qo and Qi site inhibitors. Discrete binding sites for subtypes of Qo site inhibitors have been mapped onto the Qo binding pocket, and bindings of different subtypes of Qo site inhibitors are capable of inducing dramatic conformational changes in the extramembrane domain of ISP. A novel electron transfer mechanism for the bc1 complex consistent with crystallographic observations is discussed.Key words: bc1 complex, electron transfer, structure, conformational change, inhibitor.

A novel electron transfer mechanism suggested by crystallographic studies of mitochondrial cytochrome bc1 complex.

The crystal structure of bovine mitochondrial cytochrome bc1 complex, an integral membrane protein complex of 11 different subunits with a total molecular mass of 242 kDa, demonstrated a tightly associated dimer consisting of three major regions: a matrix region primarily made of subunits core1, core2, 6, and 9; a transmembrane-helix region of 26 helices in the dimer contributed by cytochrome b, cytochrome c1, the Rieske iron-sulfur protein (ISP), subunits 7, 10, and 11; and an intermembrane-space region composed of extramembrane domains of ISP, cytochrome c1, and subunit 8. The structure also revealed the positions of and distances between irons of prosthetic groups, and two symmetry related cavities in the transmembrane-helix region upon dimerization of the bc1 complex. Extensive crystallographic studies on crystals of bc1 complexed with inhibitors of electron transfer identified binding pockets for both Qo and Qi site inhibitors. Discrete binding sites for subtypes of Qo site inhibitors have been mapped onto the Qo binding pocket, and bindings of different subtypes of Qo site inhibitors are capable of inducing dramatic conformational changes in the extramembrane domain of ISP. A novel electron transfer mechanism for the bc1 complex consistent with crystallographic observations is discussed.