Entire Superfamily Research Articles

Topological Predictions for Integral Membrane Permeases of the Phosphoenolpyruvate:Sugar Phosphotransferase System

We report bioinformatic analyses of the largest superfamily of integral membrane permeases of the bacterial phosphotransferase system (PTS), the Enzyme IIC constituents of the Glc superfamily. Phylogenetic analyses reveal that this superfamily consists of five equally distant families, the Glucose (Glc), β-Glucoside (Bgl), Fructose (Fru), Mannitol (Mtl) and Lactose (Lac) families. Average hydropathy, amphipathicity and similarity plots were generated for these five families as well as for the entire superfamily. Charged residue distribution was analyzed, and the most conserved sequence motif, common to all five families, was identified. The results show that the members of all five families exhibit similar average hydropathy plots with regions of average amphipathicity and relative conservation also being similar. Evidence is presented suggesting that the Glucitol (Gut) family of Enzyme IIC constituents is a distant member of the Glc superfamily. Based on our analyses we offer a topological model that resembles, but differs in detail from the two previously proposed models.

A Unified View of the Role of Electrostatic Interactions in Modulating the Gating of Cys Loop Receptors

In the Cys loop superfamily of ligand-gated ion channels, a global conformational change, initiated by agonist binding, results in channel opening and the passage of ions across the cell membrane. The detailed mechanism of channel gating is a subject that has lent itself to both structural and electrophysiological studies. Here we defined a gating interface that incorporates elements from the ligand binding domain and transmembrane domain previously reported as integral to proper channel gating. An overall analysis of charged residues within the gating interface across the entire superfamily showed a conserved charging pattern, although no specific interacting ion pairs were conserved. We utilized a combination of conventional mutagenesis and the high precision methodology of unnatural amino acid incorporation to study extensively the gating interface of the mouse muscle nicotinic acetylcholine receptor. We found that charge reversal, charge neutralization, and charge introduction at the gating interface are often well tolerated. Furthermore, based on our data and a reexamination of previously reported data on gamma-aminobutyric acid, type A, and glycine receptors, we concluded that the overall charging pattern of the gating interface, and not any specific pairwise electrostatic interactions, controls the gating process in the Cys loop superfamily.

Signature of the oligomeric behaviour of nuclear receptors at the sequence and structural level.

Nuclear receptors (NRs) are ligand-dependent transcription factors that control a large number of physiological events through the regulation of gene transcription. NRs function either as homodimers or as heterodimers with retinoid X receptor/ultraspiracle protein (RXR/USP). A structure-based sequence analysis aimed at discovering the molecular mechanism that controls the dimeric association of the ligand-binding domain reveals two sets of differentially conserved residues, which partition the entire NR superfamily into two classes related to their oligomeric behaviour. Site-directed mutagenesis confirms the functional importance of these residues for the dimerization process and/or transcriptional activity. All homodimers belong to class I, in which the related residues contribute a communication pathway of two salt bridges linking helix 1 on the cofactor-binding site to the dimer interface. A salt bridge involving a differentially conserved arginine residue in loop H8-H9 defines the signature motif of heterodimers. RXR/USP and all Caenorhabditis elegans NRs belong to class I, supporting the hypothesis of an earlier emergence of this class.

Gapped alignment of protein sequence motifs through Monte Carlo optimization of a hidden Markov model

BackgroundCertain protein families are highly conserved across distantly related organisms and belong to large and functionally diverse superfamilies. The patterns of conservation present in these protein sequences presumably are due to selective constraints maintaining important but unknown structural mechanisms with some constraints specific to each family and others shared by a larger subset or by the entire superfamily. To exploit these patterns as a source of functional information, we recently devised a statistically based approach called contrast hierarchical alignment and interaction network (CHAIN) analysis, which infers the strengths of various categories of selective constraints from co-conserved patterns in a multiple alignment. The power of this approach strongly depends on the quality of the multiple alignments, which thus motivated development of theoretical concepts and strategies to improve alignment of conserved motifs within large sets of distantly related sequences.ResultsHere we describe a hidden Markov model (HMM), an algebraic system, and Markov chain Monte Carlo (MCMC) sampling strategies for alignment of multiple sequence motifs. The MCMC sampling strategies are useful both for alignment optimization and for adjusting position specific background amino acid frequencies for alignment uncertainties. Associated statistical formulations provide an objective measure of alignment quality as well as automatic gap penalty optimization. Improved alignments obtained in this way are compared with PSI-BLAST based alignments within the context of CHAIN analysis of three protein families: Giα subunits, prolyl oligopeptidases, and transitional endoplasmic reticulum (p97) AAA+ ATPases.ConclusionWhile not entirely replacing PSI-BLAST based alignments, which likewise may be optimized for CHAIN analysis using this approach, these motif-based methods often more accurately align very distantly related sequences and thus can provide a better measure of selective constraints. In some instances, these new approaches also provide a better understanding of family-specific constraints, as we illustrate for p97 ATPases. Programs implementing these procedures and supplementary information are available from the authors.

The SUPERFAMILY database in 2004: additions and improvements.

The SUPERFAMILY database provides structural assignments to protein sequences and a framework for analysis of the results. At the core of the database is a library of profile Hidden Markov Models that represent all proteins of known structure. The library is based on the SCOP classification of proteins: each model corresponds to a SCOP domain and aims to represent an entire superfamily. We have applied the library to predicted proteins from all completely sequenced genomes (currently 154), the Swiss-Prot and TrEMBL databases and other sequence collections. Close to 60% of all proteins have at least one match, and one half of all residues are covered by assignments. All models and full results are available for download and online browsing at http://supfam.org. Users can study the distribution of their superfamily of interest across all completely sequenced genomes, investigate with which other superfamilies it combines and retrieve proteins in which it occurs. Alternatively, concentrating on a particular genome as a whole, it is possible first, to find out its superfamily composition, and secondly, to compare it with that of other genomes to detect superfamilies that are over- or under-represented. In addition, the webserver provides the following standard services: sequence search; keyword search for genomes, superfamilies and sequence identifiers; and multiple alignment of genomic, PDB and custom sequences.

Identification of the prokaryotic ligand-gated ion channels and their implications for the mechanisms and origins of animal Cys-loop ion channels

Acetylcholine receptor type ligand-gated ion channels are well known in animals. Homologs are identified in prokaryotes that may act as chemotactic receptors.

Homo- and hetero-oligomerization of G protein-coupled receptors

G protein-coupled receptors (GPCRs) form homo-oligomeric and hetero-oligomeric complexes. This understanding has prompted a re-evaluation of many aspects of GPCR biology, however the concept of receptor complexes has not been fully integrated into the current thinking about GPCR structure and function. Nevertheless, receptor oligomerization is a pivotal aspect of the structure and function of GPCRs that has been shown to have implications for receptor trafficking, signaling, and pharmacology and more intricate models for understanding the physiological roles of these receptors are emerging. Here, we summarize some of the advances made in understanding the structural basis and the functional roles of homo- and hetero- oligomerization in this important group of receptors. Although this discussion focuses primarily on the dopamine receptors, particularly the D2 dopamine receptor, and the opioid and serotonin receptors, we discuss the principles governing the oligomerization of all rhodopsin-like GPCRs and potentially of the entire superfamily of these receptors.

Evolutionary analysis reveals collective properties and specificity in the C-type lectin and lectin-like domain superfamily.

Members of the C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily share a common fold and are involved in a variety of functions, such as generalized defense mechanisms against foreign agents, discrimination between healthy and pathogen-infected cells, and endocytosis and blood coagulation. In this work we used ConSurf, a computer program recently developed in our lab, to perform an evolutionary analysis of this superfamily in order to further identify characteristics of all or part of its members. Given a set of homologous proteins in the form of multiple sequence alignment (MSA) and an inferred phylogenetic tree, ConSurf calculates the conservation score in every alignment position, taking into account the relationships between the sequences and the physicochemical similarity between the amino acids. The scores are then color-coded onto the three-dimensional structure of one of the homologous proteins. We provide here and at http://ashtoret.tau.ac.il/ approximately sharon a detailed analysis of the conservation pattern obtained for the entire superfamily and for two subgroups of proteins: (a) 21 CTLs and (b) 11 heterodimeric CTLD toxins. We show that, in general, proteins of the superfamily have one face that is constructed mostly of conserved residues and another that is not, and we suggest that the former face is involved in binding to other proteins or domains. In the CTLs examined we detected a region of highly conserved residues, corresponding to the known calcium- and carbohydrate-binding site of the family, which is not conserved throughout the entire superfamily, and in the CTLD toxins we found a patch of highly conserved residues, corresponding to the known dimerization region of these proteins. Our analysis also detected patches of conserved residues with yet unknown function(s).

Quantitative real-time PCR protocol for analysis of nuclear receptor signaling pathways

A major goal of the Nuclear Receptor Signaling Atlas (NURSA) is to elucidate the biochemical and physiological roles of nuclear receptors in vivo. Characterizing the tissue expression pattern of individual receptors and their target genes in whole animals under various pharmacological conditions and genotypes is an essential component of this aim. Here we describe a high-throughput quantitative, real-time, reverse-transcription PCR (QPCR) method for the measurement of both the relative level of expression of a particular transcript in a given tissue or cell type, and the relative change in expression of a particular transcript after pharmacologic or genotypic manipulation. This method is provided as a standardized protocol for those in the nuclear receptor field. It is meant to be a simplified, easy to use protocol for the rapid, high-throughput measurement of transcript levels in a large number of samples. A subsequent report will provide validated primer and probe sequence information for the entire mouse and human nuclear receptor superfamily.

Inhibitor scaffolds as new allele specific kinase substrates.

The elucidation of protein kinase signaling networks is challenging due to the large size of the protein kinase superfamily (>500 human kinases). Here we describe a new class of orthogonal triphosphate substrate analogues for the direct labeling of analogue-specific kinase protein targets. These analogues were constructed as derivatives of the Src family kinase inhibitor PP1 and were designed based on the crystal structures of PP1 bound to HCK and N(6)-(benzyl)-ADP bound to c-Src (T338G). 3-Benzylpyrazolopyrimidine triphosphate (3-benzyl-PPTP) proved to be a substrate for a mutant of the MAP kinase p38 (p38-T106G/A157L/L167A). 3-Benzyl-PPTP was preferred by v-Src (T338G) (k(cat)/K(M) = 3.2 x 10(6) min(-)(1) M(-)(1)) over ATP or the previously described ATP analogue, N(6) (benzyl) ATP. For the kinase CDK2 (F80G)/cyclin E, 3-benzyl-PPTP demonstrated catalytic efficiency (k(cat)/K(M) = 2.6 x 10(4) min(-)(1) M(-)(1)) comparable to ATP (k(cat)/K(M) = 5.0 x 10(4) min(-)(1) M(-)(1)) largely due to a significantly better K(M) (6.4 microM vs 530 microM). In kinase protein substrate labeling experiments both 3-benzyl-PPTP and 3-phenyl-PPTP prove to be over 4 times more orthogonal than N(6)-(benzyl)-ATP with respect to the wild-type kinases found in murine spleenocyte cell lysates. These experiments also demonstrate that [gamma-(32)P]-3-benzyl-PPTP is an excellent phosphodonor for labeling the direct protein substrates of CDK2 (F80G)/E in murine spleenocyte cell lysates, even while competing with cellular levels (4 mM) of unlabeled ATP. The fact that this new more highly orthogonal nucleotide is accepted by three widely divergent kinases studied here suggests that it is likely to be generalizable across the entire kinase superfamily.

Identification and characterization of subfamily-specific signatures in a large protein superfamily by a hidden Markov model approach

BackgroundMost profile and motif databases strive to classify protein sequences into a broad spectrum of protein families. The next step of such database studies should include the development of classification systems capable of distinguishing between subfamilies within a structurally and functionally diverse superfamily. This would be helpful in elucidating sequence-structure-function relationships of proteins.ResultsHere, we present a method to diagnose sequences into subfamilies by employing hidden Markov models (HMMs) to find windows of residues that are distinct among subfamilies (called signatures). The method starts with a multiple sequence alignment (MSA) of the subfamily. Then, we build a HMM database representing all sliding windows of the MSA of a fixed size. Finally, we construct a HMM histogram of the matches of each sliding window in the entire superfamily. To illustrate the efficacy of the method, we have applied the analysis to find subfamily signatures in two well-studied superfamilies: the cadherin and the EF-hand protein superfamilies. As a corollary, the HMM histograms of the analyzed subfamilies revealed information about their Ca2+ binding sites and loops.ConclusionsThe method is used to create HMM databases to diagnose subfamilies of protein superfamilies that complement broad profile and motif databases such as BLOCKS, PROSITE, Pfam, SMART, PRINTS and InterPro.

Taxonomy of Campylobacter, Arcobacter, Helicobacter and related bacteria: current status, future prospects and immediate concerns.

The taxonomy of the genus Campylobacter has changed dramatically since its inception in 1963. At that time the genus comprised just two species. At present, taxa that were once assigned to Campylobacter may belong to one of over 50 species distributed among six genera. Most of these taxa belong to a phylogenetically distinct group referred to as either ribosomal RNA (rRNA) superfamily VI or the epsilon division of the class Proteobacteria. The taxonomic diversity of the group is matched by the diverse habitats in which they may be found, and by the wide range of diseases that they are associated with. Recognition of their clinical and economic importance has resulted in intense interest in the group, and the application of increasingly sophisticated isolation, detection and chemotaxonomic methods continues to elucidate new aspects of their biodiversity. However, despite the advances in new bacterial systematics, there remain a number of important issues concerning the classification of various campylobacterial taxa that require careful consideration. Ultimately, these issues are relevant to many working in the field of applied microbiology, including clinicians, veterinarians, epidemiologists and taxonomists. The purpose of this article is briefly to review the major developments in the taxonomy of Campylobacter from its inception to the present day; summarize the most recent changes in the field; analyse current topical issues of special relevance to applied microbiologists, including identification of the bacteria; and speculate on future prospects for campylobacterial taxonomy.

The neuronal MAP kinase cascade: a biochemical signal integration system subserving synaptic plasticity and memory.

The mitogen-activated protein kinase (MAP kinase, MAPK) cascade, as the name implies, was originally discovered as a critical regulator of cell division and differentiation. As further details of this signaling cascade were worked out, it became clear that the MAPK cascade is in fact a prototype for a family of signaling cascades that share the motif of three serially linked kinases regulating each other by sequential phosphorylation. Thus, a revised nomenclature arose that uses the term MAPK to refer to the entire superfamily of signaling cascades (comprising the erks, the JNKs and the p38 stress activated protein kinases), and specifies the prototype MAPK as the extracellular signal-regulated kinase (erk). The two erk MAPK isoforms, p44 MAPK and p42 MAPK, are referred to as erk1 and erk2, respectively. The erks are abundantly expressed in neurons in the mature central nervous system, raising the question of why the prototype molecular regulators of cell division and differentiation are present in these non-dividing, terminally differentiated neurons. This review will describe the beginnings of an answer to this question. Interestingly, the general model has begun to emerge that the erk signaling system has been co-opted in mature neurons to function in synaptic plasticity and memory. Moreover, recent insights have led to the intriguing prospect that these molecules serve as biochemical signal integrators and molecular coincidence detectors for coordinating responses to extracellular signals in neurons. In this review I will first outline the essential components of this signal transduction cascade, and briefly describe recent results implicating the erks in mammalian synaptic plasticity and learning. I will then proceed to outline recent results implicating the erks as molecular signal integrators and, potentially, coincidence detectors. Finally, I will speculate on what the critical downstream effectors of the erks are in neurons, and how they might provide a readout of the integrated signal.

Self-complementary motifs (SCM) in α-crystallin small heat shock proteins

Small heat shock proteins (sHsp) have been implicated in many cell processes involving the dynamics of protein–protein interactions. Two unusual sequences containing self-complementary motifs (SCM) have been identified within the conserved α-crystallin domain of sHsps. When two SCMs are aligned in an anti-parallel direction (N to C and C to N), the charged or polar residues form either salt bridges or hydrogen bonds while the non-polar residues participate in hydrophobic interactions. When aligned in reverse order, the residues of these motifs in α-crystallin subunits form either hydrophobic and/or polar interactions. Homology based molecular modeling of the C-terminal domain of α-crystallin subunits using the crystal structure of MjHSP16.5 suggests that SCM1 and 2 participate in stabilizing secondary structure and subunit interactions. Also there is overwhelming evidence that these motifs are important in the chaperone-like activity of α-crystallin subunits. These sequences are conserved and appear to be characteristic of the entire sHsp superfamily. Similar motifs are also present in the Hsp70 family and the immunoglobulin superfamily.

The alpha-mannosidases: phylogeny and adaptive diversification.

alpha-Mannosidase enzymes comprise a class of gylcoside hydrolases involved in the maturation and degradaton of glycoprotein-linked oligosaccharides. Various alpha-mannosidase enzymatic activities are encoded by an ancient and ubiquitous gene superfamily. A comparative sequence analysis was employed here to characterize the evolutionary relationships and dynamics of the alpha-mannosidase superfamily. A series of lineage-specific BLAST searches recovered the first ever recognized archaean and eubacterial alpha-mannosidase sequences, in addition to numerous eukaryotic sequences. Motif-based alignment and subsequent phylogenetic analysis of the entire superfamily revealed the presence of three well-supported monophyletic clades that represent discrete alpha-mannosidase families. The comparative method was used to evaluate the phylogenetic distribution of alpha-mannosidase functional variants within families. Results of this analysis demonstrate a pattern of functional diversification of alpha-mannosidase paralogs followed by conservation of function among orthologs. Nucleotide polymorphism among the most closely related pair of duplicated genes was analyzed to evaluate the role of natural selection in the functional diversification of alpha-mannosidase paralogs. Ratios of nonsynonymous and synonymous variation show an increase in the rate of nonsynonymous change after duplication and a relative excess of fixed nonsynonymous changes between the two groups of paralogs. These data point to a possible role for positive Darwinian selection in the evolution of alpha-mannosidase functional diversification following gene duplication.

The C-terminal domain of HPII catalase is a member of the type I glutamine amidotransferase superfamily

Discovering distant evolutionary relationships between proteins requires detecting subtle similarities. Here we use a combination of sequence and structure analysis to show that the C-terminal domain of Escherichia coli HPII catalase with available spatial structure is a divergent member of the type I glutamine amidotransferase (GAT) superfamily. GAT-containing proteins include many biosynthetic enzymes such as E. coli carbamoyl phosphate synthetase and anthranilate synthase. Typical GAT domains have Rossmann fold-like topology and possess a catalytic triad similar to that of proteases. The C-terminal domain of HPII catalase has the GAT Rossmann fold but lacks the triad and therefore loses enzymatic activity. In addition, we detect significant sequence similarity between thiJ domains, some of which are known to have protease activity, and typical GAT proteins. Evolutionary tree analysis of the entire GAT superfamily indicates that the HPII catalase is more closely related to thiJ domains than to classical GAT domains and is likely to have evolved from a thiJ-like protein. This work illustrates the strength of sequence-based profile analysis techniques coupled with structural superpositions in developing an evolutionarily relevant classification of protein structures. Proteins 2001;42:230-236.

Carnitine Transporter OCTN2 Mutations in Systemic Primary Carnitine Deficiency: A Novel Arg169Gln Mutation and a Recurrent Arg282ter Mutation Associated with an Unconventional Splicing Abnormality

Systemic primary carnitine deficiency (CDSP, MIM 212140) is a disorder of fatty acid oxidation manifesting in acute metabolic decompensation or in progressive cardiomyopathy and muscle weakness. Mutations in the plasmalemmal organic cation/carnitine transporter OCTN2 were recently identified in CDSP patients of diverse ethnic backgrounds. We have performed OCTN2 mutation analysis in two unrelated German patients with primary carnitine deficiency and identified three molecular abnormalities. On one of the four chromosomes analyzed, we detected an Arg169Gln missense mutation that affects an arginine residue absolutely conserved in the entire transporter superfamily to which OCTN2 belongs. On the three other chromosomes, we found an Arg282ter nonsense mutation in exon 5. This mutation is embedded into different haplotypes of closely spaced intragenic dimorphisms in our two patients and was recently described in a patient of Asiatic Indian background, so it appears to be a recurrent or ancient founder mutation that may account for more CDSP cases. Finally, we found that the Arg282ter nonsense mutation is associated with a splicing abnormality at the intron 6/exon 7 junction. However, no mutations are present in exon 6, intron 6, or exon 7, suggesting that defective splicing of exon 7 on the Arg282ter allele is due to an unconventional, long-distance mechanism.

Crystal structure of a scavenger receptor cysteine-rich domain sheds light on an ancient superfamily.

Scavenger receptor cysteine-rich (SRCR) domains are found widely in cell surface molecules and in some secreted proteins, where they are thought to mediate ligand binding. We have determined the crystal structure at 2.0 A resolution of the SRCR domain of Mac-2 binding protein (M2BP), a tumor-associated antigen and matrix protein. The structure reveals a curved six-stranded beta-sheet cradling an alpha-helix. Structure-based sequence alignment demonstrates that the M2BP SRCR domain is a valid template for the entire SRCR protein superfamily. This allows an interpretation of previous mutagenesis data on ligand binding to the lymphocyte receptor CD6.

Multiple activation steps of the N-formyl peptide receptor.

The human N-formyl peptide receptor (FPR) is representative of a growing family of G protein-coupled receptors (GPCR) that respond to chemokines and chemoattractants. Despite the importance of this receptor class to immune function, relatively little is known about the molecular mechanisms involved in their activation. To reveal steps required for the activation of GPCR receptors, we utilized mutants of the FPR which have previously been shown to be incapable of binding and activating G proteins. For this study, the FPR mutants were expressed in human myeloid U937 cells and characterized for functions in addition to G protein coupling, such as receptor phosphorylation and ligand-induced receptor internalization. The results demonstrated that one of the mutants, R123G, though being unable to activate G protein, was capable of undergoing ligand-induced phosphorylation as well as internalization. Receptor internalization was monitored by following the fate of the ligand as well as by directly monitoring the fate of the receptor. The results with the R123G mutant were in contrast to those obtained for mutants D71A and R309G/E310A/R311G which, though being expressed at the cell surface and binding ligand, were incapable of being phosphorylated or internalized upon agonist stimulation. These results suggest that following ligand binding at least two "steps" are required for full activation of the wild-type FPR. That these observations may be of more general importance in GPCR-mediated signaling is suggested by the highly conserved nature of the mutants studied: D71, R123, and the site represented by amino acids 309-311 are very highly conserved throughout the entire superfamily of G protein-coupled receptors. Models of receptor activation based on the observed results are discussed.

Interdomain communication regulating ligand binding by PPAR-gamma.

Binding to receptors in the cell nucleus is crucial for the action of lipophilic hormones and ligands. PPAR-gamma (for peroxisome proliferator-activated receptor) is a nuclear hormone receptor that mediates adipocyte differentiation and modulates insulin sensitivity, cell proliferation and inflammatory processes. PPAR-gamma ligands have been implicated in the development of atherogenic foam cells and as potential cancer treatments. Transcriptional activity of PPAR-gamma is induced by binding diverse ligands, including natural fatty acid derivatives, antidiabetic thiazolidinediones, and non-steroidal anti-inflammatory drugs. Ligand binding by PPAR-gamma, as well as by the entire nuclear-receptor superfamily, is an independent property of the carboxy-terminal ligand-binding domain (LBD) of the receptor. Here we show that ligand binding by PPAR-gamma is regulated by intramolecular communication between its amino-terminal A/B domain and its carboxy-terminal LBD. Modification of the A/B domain, for example by physiological phosphorylation by MAP kinase, reduces ligand-binding affinity, thus negatively regulating the transcriptional and biological functions of PPAR-gamma. The ability of the A/B domain to regulate ligand binding has important implications for the evaluation and mechanism of action of potentially therapeutic ligands that bind PPAR-gamma and that are likely to extend to other members of the nuclear-receptor superfamily.