A gamete-specific, sex-limited homeodomain protein in Chlamydomonas.

During fertilization in Chlamydomonas, gametes of opposite mating types interact with each other through sex-specific adhesion molecules on their flagellar surfaces. Flagellar adhesion brings the cell bodies of the gametes into close contact and initiates a signal transduction pathway in preparation for cell-cell fusion. We have identified a cDNA, gsp1, whose transcript levels are upregulated during flagellar adhesion. The GSP1 polypeptide is a novel, gamete-specific homeodomain protein, the first to be identified in an alga. Its homeodomain shows significant identity with several higher plant homeodomain proteins. Although encoded by a single copy gene present in cells of both mating types, immunoblot analysis showed that GSP1 was expressed in mating type (mt)+ gametes, but was not detectable in mt- gametes or in vegetative cells of either mating type. Moreover, GSP1 appeared late during gametogenesis, suggesting that it may function during adhesion with mt- gametes or after zygote formation. GSP1 is expressed in imp11, mt- mutant gametes, which have a lesion in the mid gene involved in sex determination and exhibit many phenotypic characteristics of mt+ gametes. Thus, gsp1 is negatively regulated by mid and is the first molecule to be identified in Chlamydomonas that shows sex-limited expression.

Accumulation of dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC transporters.

In healthy individuals, acute changes in cholesterol intake produce modest changes in plasma cholesterol levels. A striking exception occurs in sitosterolemia, an autosomal recessive disorder characterized by increased intestinal absorption and decreased biliary excretion of dietary sterols, hypercholesterolemia, and premature coronary atherosclerosis. We identified seven different mutations in two adjacent, oppositely oriented genes that encode new members of the adenosine triphosphate (ATP)-binding cassette (ABC) transporter family (six mutations in ABCG8 and one in ABCG5) in nine patients with sitosterolemia. The two genes are expressed at highest levels in liver and intestine and, in mice, cholesterol feeding up-regulates expressions of both genes. These data suggest that ABCG5 and ABCG8 normally cooperate to limit intestinal absorption and to promote biliary excretion of sterols, and that mutated forms of these transporters predispose to sterol accumulation and atherosclerosis.

Type II CAAX prenyl endopeptidases belong to a novel superfamily of putative membrane-bound metalloproteases.

In this article, a novel, large and diverse superfamily of putative membrane-bound proteins that includes the type II CAAX prenyl endopeptidases is described. The majority of the members of this superfamily are hypothetical proteins from bacteria and plants. Analysis of the conserved motifs, combined with available experimental data, suggests that these proteins are putative metal-dependent proteases that are potentially involved in protein and/or peptide modification and secretion.

KH domain: one motif, two folds.

The K homology (KH) module is a widespread RNA-binding motif that has been detected by sequence similarity searches in such proteins as heterogeneous nuclear ribonucleoprotein K (hnRNP K) and ribosomal protein S3. Analysis of spatial structures of KH domains in hnRNP K and S3 reveals that they are topologically dissimilar and thus belong to different protein folds. Thus KH motif proteins provide a rare example of protein domains that share significant sequence similarity in the motif regions but possess globally distinct structures. The two distinct topologies might have arisen from an ancestral KH motif protein by N- and C-terminal extensions, or one of the existing topologies may have evolved from the other by extension, displacement and deletion. C-terminal extension (deletion) requires ss-sheet rearrangement through the insertion (removal) of a ss-strand in a manner similar to that observed in serine protease inhibitors serpins. Current analysis offers a new look on how proteins can change fold in the course of evolution.

Two tricks in one bundle: helix-turn-helix gains enzymatic activity.

Many examples of enzymes that have lost their catalytic activity and perform other biological functions are known. The opposite situation is rare. A previously unnoticed structural similarity between the lambda integrase family (Int) proteins and the AraC family of transcriptional activators implies that the Int family evolved by duplication of an ancient DNA-binding homeodomain-like module, which acquired enzymatic activity. The two helix-turn-helix (HTH) motifs in Int proteins incorporate catalytic residues and participate in DNA binding. The active site of Int proteins, which include the type IB topoisomerases, is formed at the domain interface and the catalytic tyrosine residue is located in the second helix of the C-terminal HTH motif. Structural analysis of other 'tyrosine' DNA-breaking/rejoining enzymes with similar enzyme mechanisms, namely prokaryotic topoisomerase I, topoisomerase II and archaeal topoisomerase VI, reveals that the catalytic tyrosine is placed in a HTH domain as well. Surprisingly, the location of this tyrosine residue in the structure is not conserved, suggesting independent, parallel evolution leading to the same catalytic function by homologous HTH domains. The 'tyrosine' recombinases give a rare example of enzymes that evolved from ancient DNA-binding modules and present a unique case for homologous enzymatic domains with similar catalytic mechanisms but different locations of catalytic residues, which are placed at non-homologous sites.

Treble clef finger--a functionally diverse zinc-binding structural motif.

Detection of similarity is particularly difficult for small proteins and thus connections between many of them remain unnoticed. Structure and sequence analysis of several metal-binding proteins reveals unexpected similarities in structural domains classified as different protein folds in SCOP and suggests unification of seven folds that belong to two protein classes. The common motif, termed treble clef finger in this study, forms the protein structural core and is 25-45 residues long. The treble clef motif is assembled around the central zinc ion and consists of a zinc knuckle, loop, beta-hairpin and an alpha-helix. The knuckle and the first turn of the helix each incorporate two zinc ligands. Treble clef domains constitute the core of many structures such as ribosomal proteins L24E and S14, RING fingers, protein kinase cysteine-rich domains, nuclear receptor-like fingers, LIM domains, phosphatidylinositol-3-phosphate-binding domains and His-Me finger endonucleases. The treble clef finger is a uniquely versatile motif adaptable for various functions. This small domain with a 25 residue structural core can accommodate eight different metal-binding sites and can have many types of functions from binding of nucleic acids, proteins and small molecules, to catalysis of phosphodiester bond hydrolysis. Treble clef motifs are frequently incorporated in larger structures or occur in doublets. Present analysis suggests that the treble clef motif defines a distinct structural fold found in proteins with diverse functional properties and forms one of the major zinc finger groups.

Common fold in helix-hairpin-helix proteins.

Helix-hairpin-helix (HhH) is a widespread motif involved in non-sequence-specific DNA binding. The majority of HhH motifs function as DNA-binding modules, however, some of them are used to mediate protein-protein interactions or have acquired enzymatic activity by incorporating catalytic residues (DNA glycosylases). From sequence and structural analysis of HhH-containing proteins we conclude that most HhH motifs are integrated as a part of a five-helical domain, termed (HhH)(2) domain here. It typically consists of two consecutive HhH motifs that are linked by a connector helix and displays pseudo-2-fold symmetry. (HhH)(2) domains show clear structural integrity and a conserved hydrophobic core composed of seven residues, one residue from each alpha-helix and each hairpin, and deserves recognition as a distinct protein fold. In addition to known HhH in the structures of RuvA, RadA, MutY and DNA-polymerases, we have detected new HhH motifs in sterile alpha motif and barrier-to-autointegration factor domains, the alpha-subunit of Escherichia coli RNA-polymerase, DNA-helicase PcrA and DNA glycosylases. Statistically significant sequence similarity of HhH motifs and pronounced structural conservation argue for homology between (HhH)(2) domains in different protein families. Our analysis helps to clarify how non-symmetric protein motifs bind to the double helix of DNA through the formation of a pseudo-2-fold symmetric (HhH)(2) functional unit.

Structure and mechanism of homoserine kinase: prototype for the GHMP kinase superfamily.

BACKGROUND: Homoserine kinase (HSK) catalyzes an important step in the threonine biosynthesis pathway. It belongs to a large yet unique class of small metabolite kinases, the GHMP kinase superfamily. Members in the GHMP superfamily participate in several essential metabolic pathways, such as amino acid biosynthesis, galactose metabolism, and the mevalonate pathway. RESULTS: The crystal structure of HSK and its complex with ADP reveal a novel nucleotide binding fold. The N-terminal domain contains an unusual left-handed betaalphabeta unit, while the C-terminal domain has a central alpha-beta plait fold with an insertion of four helices. The phosphate binding loop in HSK is distinct from the classical P loops found in many ATP/GTP binding proteins. The bound ADP molecule adopts a rare syn conformation and is in the opposite orientation from those bound to the P loop-containing proteins. Inspection of the substrate binding cavity indicates several amino acid residues that are likely to be involved in substrate binding and catalysis. CONCLUSIONS: The crystal structure of HSK is the first representative in the GHMP superfamily to have determined structure. It provides insight into the structure and nucleotide binding mechanism of not only the HSK family but also a variety of enzymes in the GHMP superfamily. Such enzymes include galactokinases, mevalonate kinases, phosphomevalonate kinases, mevalonate pyrophosphate decarboxylases, and several proteins of yet unknown functions.

C-terminal domains of Escherichia coli topoisomerase I belong to the zinc-ribbon superfamily.

Detection of remote evolutionary connections is increasingly difficult with sequence and structural divergence. A combination of sequence and structural analysis, in which statistically supported sequence similarity had a crucial impact, revealed that Escherichia coli topoisomerase I C-terminal fragment is evolutionarily related to the three tetracysteine zinc-binding domains of the enzyme. Spatial structure analysis of this C-terminal fragment indicates that it consists of two structurally similar domains and suggests homology between them. Sequence similarity between the zinc-binding domains of type Ia topoisomerases and transcription regulators of known spatial structure helps to conclude that E. coli topo I contains five copies of a zinc ribbon domain at the C terminus. Two of these domains, corresponding to the C-terminal fragment, lost their cysteine residues and are probably not able to bind zinc. Present analyses lead to the classification of the C-terminal fragment of E. coli topoisomerase I as a member of zinc ribbon superfamily, despite the absence of zinc-binding sites.

Phosphatidylinositol phosphate kinase: a link between protein kinase and glutathione synthase folds.

Comparisons of serine/threonine protein kinase (PK) and type IIbeta phosphatidylinositol phosphate kinase (PIPK) structures with each other and also with other proteins reveal structural and functional similarity between the two kinases and proteins of the glutathione synthase fold (ATP-grasp). This suggests that these enzymes are evolutionarily related. The structure of PIPK, which clearly resembles both PK and ATP-grasp, provides a link between the two proteins and establishes that the C-terminal domains of PK, PIPK and ATP-grasp share the same fold. The functional implications of the proposed homology are discussed.

Mh1 domain of Smad is a degraded homing endonuclease.

Smad proteins are eukarytic transcription regulators in the TGF-beta signaling cascade. Using a combination of sequence and structure-based analyses, we argue that MH1 domain of Smad is homologous to the diverse His-Me finger endonuclease family enzymes. The similarity is particularly extensive with the I-PpoI endonuclease. In addition to the global fold similarities, both proteins possess a conserved motif of three cysteine residues and one histidine residue which form a zinc-binding site in I-PpoI. Sequence and structure conservation in the motif region strongly suggest that MH1 domain may also incorporate a metal ion in its structural core. MH1 of Smad3 and I-PpoI exhibit similar nucleic acid binding mode and interact with DNA major groove through an antiparallel beta-sheet. MH1 is an example of transcription regulator derived from the ancient enzymatic domain that lost its catalytic activity but retained DNA-binding sites.

The Zn-peptidase superfamily: functional convergence after evolutionary divergence.

Zn-dependent carboxypeptidases (ZnCP) cleave off the C-terminal amino acid residues from proteins and peptides. Here we describe a superfamily that unites classical ZnCP with other enzymes, most of which are known (or likely) to participate in metal-dependent peptide bond cleavage, but not necessarily in polypeptide substrates. It is demonstrated that aspartoacylase (ASP gene) and succinylglutamate desuccinylase (ASTE gene) are members of the ZnCP family. The Zn-binding site along with the structural core of the protein is shown to be conserved between ZnCP and another large family of hydrolases that includes mostly aminopeptidases (ZnAP). Both families (ZnCP and ZnAP) include not only proteases but also enzymes that perform N-deacylation, and enzymes that catalyze N-desuccinylation of amino acids. This is a result of functional convergence that apparently occurred after the divergence of the two families.

Homology between O-linked GlcNAc transferases and proteins of the glycogen phosphorylase superfamily.

The O-linked GlcNAc transferases (OGTs) are a recently characterized group of largely eukaryotic enzymes that add a single beta-N-acetylglucosamine moiety to specific serine or threonine hydroxyls. In humans, this process may be part of a sugar regulation mechanism or cellular signaling pathway that is involved in many important diseases, such as diabetes, cancer, and neurodegeneration. However, no structural information about the human OGT exists, except for the identification of tetratricopeptide repeats (TPR) at the N terminus. The locations of substrate binding sites are unknown and the structural basis for this enzyme's function is not clear. Here, remote homology is reported between the OGTs and a large group of diverse sugar processing enzymes, including proteins with known structure such as glycogen phosphorylase, UDP-GlcNAc 2-epimerase, and the glycosyl transferase MurG. This relationship, in conjunction with amino acid similarity spanning the entire length of the sequence, implies that the fold of the human OGT consists of two Rossmann-like domains C-terminal to the TPR region. A conserved motif in the second Rossmann domain points to the UDP-GlcNAc donor binding site. This conclusion is supported by a combination of statistically significant PSI-BLAST hits, consensus secondary structure predictions, and a fold recognition hit to MurG. Additionally, iterative PSI-BLAST database searches reveal that proteins homologous to the OGTs form a large and diverse superfamily that is termed GPGTF (glycogen phosphorylase/glycosyl transferase). Up to one-third of the 51 functional families in the CAZY database, a glycosyl transferase classification scheme based on catalytic residue and sequence homology considerations, can be unified through this common predicted fold. GPGTF homologs constitute a substantial fraction of known proteins: 0.4% of all non-redundant sequences and about 1% of proteins in the Escherichia coli genome are found to belong to the GPGTF superfamily.

Estimating the number of protein folds and families from complete genome data.

Using the data on proteins encoded in complete genomes, combined with a rigorous theory of the sampling process, we estimate the total number of protein folds and families, as well as the number of folds and families in each genome. The total number of folds in globular, water- soluble proteins is estimated at about 1000, with structural information currently available for about one-third of the number. The sequenced genomes of unicellular organisms encode from approximately 25%, for the minimal genomes of the Mycoplasmas, to 70-80% for larger genomes, such as Escherichia coli and yeast, of the total number of folds. The number of protein families with significant sequence conservation was estimated to be between 4000 and 7000, with structures available for about 20% of these.

Crystal structure of human ornithine decarboxylase at 2.1 A resolution: structural insights to antizyme binding.

The polyamines spermidine and spermine are ubiquitous and required for cell growth and differentiation in eukaryotes. Ornithine decarboxylase (ODC, EC 4.1.1.17) performs the first step in polyamine biosynthesis, the decarboxylation of ornithine to putrescine. Elevated polyamine levels can lead to down-regulation of ODC activity by enhancing the translation of antizyme mRNA, resulting in subsequent binding of antizyme to ODC monomers which targets ODC for proteolysis by the 26S proteasome. The crystal structure of ornithine decarboxylase from human liver has been determined to 2.1 A resolution by molecular replacement using truncated mouse ODC (Delta425-461) as the search model and refined to a crystallographic R-factor of 21.2% and an R-free value of 28.8%. The human ODC model includes several regions that are disordered in the mouse ODC crystal structure, including one of two C-terminal basal degradation elements that have been demonstrated to independently collaborate with antizyme binding to target ODC for degradation by the 26S proteasome. The crystal structure of human ODC suggests that the C terminus, which contains basal degradation elements necessary for antizyme-induced proteolysis, is not buried by the structural core of homodimeric ODC as previously proposed. Analysis of the solvent-accessible surface area, surface electrostatic potential, and the conservation of primary sequence between human ODC and Trypanosoma brucei ODC provides clues to the identity of potential protein-binding-determinants in the putative antizyme binding element in human ODC.

The subunit interfaces of oligomeric enzymes are conserved to a similar extent to the overall protein sequences.

It is well established that, within families of homologous enzymes, amino acid residues that are involved in the chemistry of the reaction are highly conserved. To determine if residues at the subunit interface of oligomeric enzymes with shared active sites are also conserved, comparative analysis of five enzyme families was undertaken. For the chosen enzyme families, sequence data were available for a large number of proteins and a three-dimensional structure was known for at least two members of each family. The analysis indicates that the subunit interface and the hydrophobic core of proteins from all five families have diverged to a similar extent to the overall protein sequences.

The alpha-subunit of protein prenyltransferases is a member of the tetratricopeptide repeat family.

Lipidation catalyzed by protein prenyltransferases is essential for the biological function of a number of eukaryotic proteins, many of which are involved in signal transduction and vesicular traffic regulation. Sequence similarity searches reveal that the alpha-subunit of protein prenyltransferases (PTalpha) is a member of the tetratricopeptide repeat (TPR) superfamily. This finding makes the three-dimensional structure of the rat protein farnesyltransferase the first structural model of a TPR protein interacting with its protein partner. Structural comparison of the two TPR domains in protein farnesyltransferase and protein phosphatase 5 indicates that variation in TPR consensus residues may affect protein binding specificity through altering the overall shape of the TPR superhelix. A general approach to evolutionary analysis of proteins with repetitive sequence motifs has been developed and applied to the protein prenyltransferases and other TPR proteins. The results suggest that all members in PTalpha family originated from a common multirepeat ancestor, while the common ancestor of PTalpha and other members of TPR superfamily is likely to be a single repeat protein.

Thermolysin and mitochondrial processing peptidase: how far structure-functional convergence goes.

The structure-functional convergence between two Zn-dependent proteases, namely thermolysin and mitochondrial processing peptidase (MPP), is described. These two families of nonhomologous enzymes show not only functional convergence of several active site residues as in chymotrypsin and subtilisin, but also structural convergence of overall molecular architectures including the beta-sheet arrangement and packing of the surrounding alpha-helices. The major functionally important structural elements are present in both enzymes with different topological connections and often in reverse main-chain orientation, but display similar packing. The structural comparison helps to rationalize sequence "inversion" of the HEXXH thermolysin consensus present as HXXEH in MPP. The described structural convergence may be due to a limited number of alternatives to build a Zn-protease that utilizes hydrogen bonding between a substrate main chain and the enzyme beta-sheet for substrate binding.

Modeling of the spatial structure of eukaryotic ornithine decarboxylases.

We used sequence and structural comparisons to determine the fold for eukaryotic ornithine decarboxylase, which we found is related to alanine racemase. These enzymes have no detectable sequence identity with any protein of known structure, including three pyridoxal phosphate-utilizing enzymes. Our studies suggest that the N-terminal domain of ornithine decarboxylase folds into a beta/alpha-barrel. Through the analysis of known barrel structures we developed a topographic model of the pyridoxal phosphate-binding domain of ornithine decarboxylase, which predicts that the Schiff base lysine and a conserved glycine-rich sequence both map to the C-termini of the beta-strands. Other residues in this domain that are likely to have essential roles in catalysis, substrate, and cofactor binding were also identified, suggesting that this model will be a suitable guide to mutagenic analysis of the enzyme mechanism.

Genome trees constructed using five different approaches suggest new major bacterial clades.

BACKGROUND: The availability of multiple complete genome sequences from diverse taxa prompts the development of new phylogenetic approaches, which attempt to incorporate information derived from comparative analysis of complete gene sets or large subsets thereof. Such attempts are particularly relevant because of the major role of horizontal gene transfer and lineage-specific gene loss, at least in the evolution of prokaryotes. RESULTS: Five largely independent approaches were employed to construct trees for completely sequenced bacterial and archaeal genomes: i) presence-absence of genomes in clusters of orthologous genes; ii) conservation of local gene order (gene pairs) among prokaryotic genomes; iii) parameters of identity distribution for probable orthologs; iv) analysis of concatenated alignments of ribosomal proteins; v) comparison of trees constructed for multiple protein families. All constructed trees support the separation of the two primary prokaryotic domains, bacteria and archaea, as well as some terminal bifurcations within the bacterial and archaeal domains. Beyond these obvious groupings, the trees made with different methods appeared to differ substantially in terms of the relative contributions of phylogenetic relationships and similarities in gene repertoires caused by similar life styles and horizontal gene transfer to the tree topology. The trees based on presence-absence of genomes in orthologous clusters and the trees based on conserved gene pairs appear to be strongly affected by gene loss and horizontal gene transfer. The trees based on identity distributions for orthologs and particularly the tree made of concatenated ribosomal protein sequences seemed to carry a stronger phylogenetic signal. The latter tree supported three potential high-level bacterial clades,: i) Chlamydia-Spirochetes, ii) Thermotogales-Aquificales (bacterial hyperthermophiles), and ii) Actinomycetes-Deinococcales-Cyanobacteria. The latter group also appeared to join the low-GC Gram-positive bacteria at a deeper tree node. These new groupings of bacteria were supported by the analysis of alternative topologies in the concatenated ribosomal protein tree using the Kishino-Hasegawa test and by a census of the topologies of 132 individual groups of orthologous proteins. Additionally, the results of this analysis put into question the sister-group relationship between the two major archaeal groups, Euryarchaeota and Crenarchaeota, and suggest instead that Euryarchaeota might be a paraphyletic group with respect to Crenarchaeota. CONCLUSIONS: We conclude that, the extensive horizontal gene flow and lineage-specific gene loss notwithstanding, extension of phylogenetic analysis to the genome scale has the potential of uncovering deep evolutionary relationships between prokaryotic lineages.