Convergent evolution of similar enzymatic function on different protein folds: the hexokinase, ribokinase, and galactokinase families of sugar kinases.

Kinases that catalyze phosphorylation of sugars, called here sugar kinases, can be divided into at least three distinct nonhomologous families. The first is the hexokinase family, which contains many prokaryotic and eukaryotic sugar kinases with diverse specificities, including a new member, rhamnokinase from Salmonella typhimurium. The three-dimensional structure of hexokinase is known and can be used to build models of functionally important regions of other kinases in this family. The second is the ribokinase family, of unknown three-dimensional structure, and comprises pro- and eukaryotic ribokinases, bacterial fructokinases, the minor 6-phosphofructokinase 2 from Escherichia coli, 6-phosphotagatokinase, 1-phosphofructokinase, and, possibly, inosine-guanosine kinase. The third family, also of unknown three-dimensional structure, contains several bacterial and yeast galactokinases and eukaryotic mevalonate and phosphomevalonate kinases and may have a substrate binding region in common with homoserine kinases. Each of the three families of sugar kinases appears to have a distinct three-dimensional fold, since conserved sequence patterns are strikingly different for the three families. Yet each catalyzes chemically equivalent reactions on similar or identical substrates. The enzymatic function of sugar phosphorylation appears to have evolved independently on the three distinct structural frameworks, by convergent evolution. In addition, evolutionary trees reveal that (1) fructokinase specificity has evolved independently in both the hexokinase and ribokinase families and (2) glucose specificity has evolved independently in different branches of the hexokinase family. These are examples of independent Darwinian adaptation of a structure to the same substrate at different evolutionary times. The flexible combination of active sites and three-dimensional folds observed in nature can be exploited by protein engineers in designing and optimizing enzymatic function.     

Operator recognition by the ROK transcription factor family members,NagC and Mlc

NagC and Mlc, paralogous members of the ROK family of proteins with almost identical helix-turn-helix DNA binding motifs, specifically regulate genes for transport and utilization of N-acetylglucosamine and glucose. We previously showed that two amino acids in a linker region outside the canonical helix-turn-helix motif are responsible for Mlc site specificity. In this work we identify four amino acids in the linker, which are required for recognition of NagC targets. These amino acids allow Mlc and NagC to distinguish between a C/G and an A/T bp at positions ±11 of the operators. One linker position, glycine in NagC and arginine in Mlc, corresponds to the major specificity determinant for the two proteins. In certain contexts it is possible to switch repression from Mlc-style to NagC-style, by interchanging this glycine and arginine. Secondary determinants are supplied by other linker positions or the helix-turn-helix motif. A wide genomic survey of unique ROK proteins shows that glycine- and arginine-rich sequences are present in the linkers of nearly all ROK family repressors. Conserved short sequence motifs, within the branches of the ROK evolutionary tree, suggest that these sequences could also be involved in operator recognition in other ROK family members.     

Partial protein domains: evolutionary insights and bioinformatics challenges

Protein domains are generally thought to correspond to units of evolution. New research raises questions about how such domains are defined with bioinformatics tools and sheds light on how evolution has enabled partial domains to be viable.With the rapid expansion in the number of determined protein sequences - over 92 million in UniProt in March 2015 - an ever-increasing number of biologists are using bioinformatics tools for annotation of these sequences. One widely used strategy is to identify occurrences of Pfam families within the sequence of interest [1]. A Pfam family is a multiple sequence alignment of the occurrences of a particular domain both in different species and in different regions of the same protein. The concept underpinning Pfam is that proteins typically comprise one or more domains (regions), each of which is an evolutionary unit that generally has a well-defined biological function. A significant sequence similarity between a query protein and a Pfam family provides the basis for annotations. Two recent articles [2,3] in Genome Biology evaluate the implications of having the query sequence only matching part of a Pfam family, which is an intriguing finding, given that a Pfam family is considered to be an evolutionary unit.     

A comparison of sequence and structure protein domain families as a basis for structural genomics.

MOTIVATION: Protein families can be defined based on structure or sequence similarity. We wanted to compare two protein family databases, one based on structural and one on sequence similarity, to investigate to what extent they overlap, the similarity in definition of corresponding families, and to create a list of large protein families with unknown structure as a resource for structural genomics. We also wanted to increase the sensitivity of fold assignment by exploiting protein family HMMs. RESULTS: We compared Pfam, a protein family database based on sequence similarity, to Scop, which is based on structural similarity. We found that 70% of the Scop families exist in Pfam while 57% of the Pfam families exist in Scop. Most families that occur in both databases correspond well to each other, but in some cases they are different. Such cases highlight situations in which structure and sequence approaches differ significantly. The comparison enabled us to compile a list of the largest families that do not occur in Scop; these are suitable targets for structure prediction and determination, and may be useful to guide projects in structural genomics. It can be noted that 13 out of the 20 largest protein families without a known structure are likely transmembrane proteins. We also exploited Pfam to increase the sensitivity of detecting homologs of proteins with known structure, by comparing query sequences to Pfam HMMs that correspond to Scop families. For SWISSPROT+TREMBL, this yielded an increase in fold assignment from 31% to 42% compared to using FASTA only. This method assigned a structure to 22% of the proteins in Saccharomyces cerevisiae, 24% in Escherichia coli, and 16% in Methanococcus jannaschii.     

Mammalian hexokinase 1: Evolutionary conservation and structure to function analysis

We have amplified and sequenced the complete coding region of bovine hexokinase isoenzyme 1 (HK1) from brain RNA with PCR primers selected for sequence conservation. The sequence information was analyzed to evaluate the evolutionary and structure-function relationships among the mammalian and yeast HK isoenzymes. Structure to function analysis identified an unduplicated, invariant N-terminal domain involved in HK1 outer mitochondrial membrane targeting, as well as putative carbohydrate and nucleotide-binding sites in the regulatory and catalytic halves of HK1 essential to enzyme function. The ATP-binding site in the catalytic half of the HK1 protein resembles nucleotide-binding regions from protein kinases, with the single amino acid replacement (lysine to glutamate) in the ATP-binding site of the amino half explaining the loss of HK1 catalytic function in the regulatory domain. Sequence comparisons suggest that the 50-kDa mammalian and yeast glucokinases arose separately in evolution. In addition to providing valuable phylogenetic and structure-function insights, this work provides an efficient strategy for rapid cloning and sequencing of the coding regions for other HKs and related proteins.     

A new lectin family with structure similarity to actinoporins revealed by the crystal structure of Xerocomus chrysenteron lectin XCL

A newly defined family of fungal lectins displays no significant sequence similarity to any protein in the databases. These proteins, made of about 140 amino acid residues, have sequence identities ranging from 38% to 65% and share binding specificity to N-acetyl galactosamine. One member of this family, the lectin XCL from Xerocomus chrysenteron, induces drastic changes in the actin cytoskeleton after sugar binding at the cell surface and internalization, and has potent insecticidal activity. The crystal structure of XCL to 1.4 A resolution reveals the architecture of this new lectin family. The fold of the protein is not related to any of the several lectin folds documented so far. Unexpectedly, the structure similarity is significant with actinoporins, a family of pore-forming toxins. The specific structural features and sequence signatures in each protein family suggest a potential sugar binding site in XCL and a possible evolutionary relationship between these proteins. Finally, the tetrameric assembly of XCL reveals a complex network of protomer-protomer interfaces and generates a large, hydrated cavity of 1000 A3, which may become accessible to larger solutes after a small conformational change of the protein.     

Modeling bacterial UDP-HexNAc: polyprenol-P HexNAc-1-P transferases

Protein N-glycosylation in eukaryotes and peptidoglycan biosynthesis in bacteria are both initiated by the transfer of a D-N-acetylhexosamine 1-phosphate to a membrane-bound polyprenol phosphate. These reactions are catalyzed by a family of transmembrane proteins known as the UDP-D-N-acetylhexosamine: polyprenol phosphate D-N-acetylhexosamine 1-phosphate transferases. The sole eukaryotic member of this family, the d-N-acetylglucosamine 1-phosphate transferase (GPT), is specific for UDP-GlcNAc as the donor substrate and uses dolichol phosphate as the membrane-bound acceptor. The bacterial translocases, MraY, WecA, and WbpL, utilize undecaprenol phosphate as the acceptor substrate, but differ in their specificity for the UDP-sugar donor substrate. The structural basis of this sugar nucleotide specificity is uncertain. However, potential carbohydrate recognition (CR) domains have been identified within the C-terminal cytoplasmic loops of MraY, WecA, and WbpL that are highly conserved in family members with the same UDP-N-acetylhexosamine specificity. This review focuses on the catalytic mechanism and substrate specificity of these bacterial UDP-D-N-acetylhexosamine: polyprenol phosphate D-N-acetylhexosamine 1-P transferases and may provide insights for the development of selective inhibitors of cell wall biosynthesis.     

SUPFAM—a database of potential protein superfamily relationships derived by comparing sequence-based and structure-based families: implications for structural genomics and function annotation in genomes

Members of a superfamily of proteins could result from divergent evolution of homologues with insignificant similarity in the amino acid sequences. A superfamily relationship is detected commonly after the three-dimensional structures of the proteins are determined using X-ray analysis or NMR. The SUPFAM database described here relates two homologous protein families in a multiple sequence alignment database of either known or unknown structure. The present release (1.1), which is the first version of the SUPFAM database, has been derived by analysing Pfam, which is one of the commonly used databases of multiple sequence alignments of homologous proteins. The first step in establishing SUPFAM is to relate Pfam families with the families in PALI, which is an alignment database of homologous proteins of known structure that is derived largely from SCOP. The second step involves relating Pfam families which could not be associated reliably with a protein superfamily of known structure. The profile matching procedure, IMPALA, has been used in these steps. The first step resulted in identification of 1280 Pfam families (out of 2697, i.e. 47%) which are related, either by close homologous connection to a SCOP family or by distant relationship to a SCOP family, potentially forming new superfamily connections. Using the profiles of 1417 Pfam families with apparently no structural information, an all-against-all comparison involving a sequence-profile match using IMPALA resulted in clustering of 67 homologous protein families of Pfam into 28 potential new superfamilies. Expansion of groups of related proteins of yet unknown structural information, as proposed in SUPFAM, should help in identifying ‘priority proteins’ for structure determination in structural genomics initiatives to expand the coverage of structural information in the protein sequence space. For example, we could assign 858 distinct Pfam domains in 2203 of the gene products in the genome of Mycobacterium tubercolosis. Fifty-one of these Pfam families of unknown structure could be clustered into 17 potentially new superfamilies forming good targets for structural genomics. SUPFAM database can be accessed at http://pauling.mbu.iisc.ernet.in/~supfam.     

Bioinformatic prediction and analysis of eukaryotic protein kinases in the rat genome

Eukaryotic protein kinases, containing a conserved catalytic domain, represent one of the largest superfamilies of the eukaryotic proteins and play distinct roles in cell signaling and diseases. Near completion of rat genome sequencing project enables the evaluation of a near complete set of rat protein kinases. Publicly accessible genetic sequence databases were searched for rat protein kinases, and 515 eukaryotic protein kinases, 40 atypical protein kinases and 45 kinase pseudogenes were identified. The rat has 509 putative protein kinases orthologous to human kinases. Unlike microtubule affinity-regulating kinases, the rat has a few more kinases, in addition to the orthologous pairs of mouse kinases. The comparison of 11 different eukaryotic species revealed the evolutionary conservation of this diverse family of proteins. The evolutionary rate studies of human disease and non-disease associated kinases suggested that relatively uniform selective pressures have been applied to these kinase classes. This bioinformatic study of the rat protein kinases provides a suitable framework for further characterization of the functional and structural properties of these protein kinases.     

Modulation of substrate specificity of the DnaK chaperone by alteration of a hydrophobic arch

Hsp70 chaperones assist protein folding by reversible interaction with extended hydrophobic segments of substrate polypeptides. We investigated the contribution of three structural elements of the substrate- binding cavity of the Escherichia coli homologue, DnaK, to substrate specificity by investigating mutant DnaK proteins for binding to cellulose-bound peptides. Deletion of the C-terminal subdomain (Delta539-638) and blockage of the access to the hydrophobic pocket in the substrate-binding cavity (V436F) did not change the specificity, although the latter exchange reduced the affinity to all peptides investigated. Mutations (A429W, M404A/A429W) that affect the formation of a hydrophobic arch spanning over the bound substrate disfavored DnaK binding, especially to peptides with short stretches of consecutive hydrophobic residues flanked by acidic residues, while binding to most other peptides remained unchanged. The arch thus contributes to the substrate specificity of DnaK. This finding is of particular interest, since of all the residues of the substrate-binding cavity that contact bound substrate, only the arch-forming residues show significant variation within the Hsp70 family.     

Pfam 3.1: 1313 multiple alignments and profile HMMs match the majority of proteins.

Pfam is a collection of multiple alignments and profile hidden Markov models of protein domain families. Release 3.1 is a major update of the Pfam database and contains 1313 families which are available on the World Wide Web in Europe at http://www.sanger.ac.uk/Software/Pfam/ and http://www.cgr.ki.se/Pfam/, and in the US at http://pfam.wustl.edu/. Over 54% of proteins in SWISS-PROT-35 and SP-TrEMBL-5 match a Pfam family. The primary changes of Pfam since release 2.1 are that we now use the more advanced version 2 of the HMMER software, which is more sensitive and provides expectation values for matches, and that it now includes proteins from both SP-TrEMBL and SWISS-PROT.     

Circular Permutation Provides an Evolutionary Link between Two Families of Calcium-dependent Carbohydrate Binding Modules

The microbial deconstruction of the plant cell wall is a critical biological process, which also provides important substrates for environmentally sustainable industries. Enzymes that hydrolyze the plant cell wall generally contain non-catalytic carbohydrate binding modules (CBMs) that contribute to plant cell wall degradation. Here we report the biochemical properties and crystal structure of a family of CBMs (CBM60) that are located in xylanases. Uniquely, the proteins display broad ligand specificity, targeting xylans, galactans, and cellulose. Some of the CBM60s display enhanced affinity for their ligands through avidity effects mediated by protein dimerization. The crystal structure of vCBM60, displays a β-sandwich with the ligand binding site comprising a broad cleft formed by the loops connecting the two β-sheets. Ligand recognition at site 1 is, exclusively, through hydrophobic interactions, whereas binding at site 2 is conferred by polar interactions between a protein-bound calcium and the O2 and O3 of the sugar. The observation, that ligand recognition at site 2 requires only a β-linked sugar that contains equatorial hydroxyls at C2 and C3, explains the broad ligand specificity displayed by vCBM60. The ligand-binding apparatus of vCBM60 displays remarkable structural conservation with a family 36 CBM (CBM36); however, the residues that contribute to carbohydrate recognition are derived from different regions of the two proteins. Three-dimensional structure-based sequence alignments reveal that CBM36 and CBM60 are related by circular permutation. The biological and evolutionary significance of the mechanism of ligand recognition displayed by family 60 CBMs is discussed.     

Nuclear protein kinase activities during the cell cycle of HeLa S3 cells

To ascertain the activity and substrate specificity of nuclear protein kinases during various stages of the cell cycle of HeLa S3 cells, a nuclear phospho-protein-enriched sample was extracted from synchronised cells and assayed in vitro in the presence of homologous substrates. The nuclear protein kinases increased in activity during S and G2 phase to a level that was twice that of kinases from early S phase cells. The activity was reduced during mitosis but increased again in G1 phase. When the phosphoproteins were separated into five fractions by cellulose-phosphate chromatography each fraction, though not homogenous, exhibited differences in activity. Variations in the activity of the protein kinase fractions were observed during the cell cycle, similar to those observed for the unfractionated kinases. Sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis of the proteins phosphorylated by each of the five kinase fractions demonstrated a substrate specificity. The fractions also exhibited some cell cycle stage-specific preference for substrates; kinases from G1 cells phosphorylated mainly high molecular weight polypeptides, whereas lower molecular weight species were phosphorylated by kinases from the S, G2 and mitotic stages of the cell cycle. Inhibition of DNA and histone synthesis by cytosine arabinoside had no effect on the activity or substrate specificity of S phase kinases. Some kinase fractions phosphorylated histones as well as non-histone chromosomal proteins and this phosphorylation was also cell cycle stage dependent. The presence of histones in the in vitro assay influenced the ability of some fractions to phosphorylate particular non-histone polypeptides; non-histone proteins also appeared to affect the in vitro phosphorylation of histones.     

Unstructured conformations are a substrate requirement for the Sir2 family of NAD-dependent protein deacetylases

The regulation of protein function is often achieved through post-translational modifications including phosphorylation, methylation, ubiquitination, and acetylation. The role of acetylation has been most extensively studied in the context of histones, but it is becoming increasingly evident that this modification now includes other proteins. The Sir2 family of NAD-dependent deacetylases was initially recognized as mediating gene silencing through histone deacetylation, but several family members display non-nuclear sub-cellular localization and deacetylate non-histone protein substrates. Although many structural and enzymatic studies of Sir2 proteins have been reported, how substrate recognition is achieved by this family of enzymes is unknown. Here we use in vitro deacetylase assays and a variety of potential substrates to examine the substrate specificity of yeast homologue Hst2. We show that Hst2 is specific for acetyl-lysine within proteins; it does not deacetylate small polycations such as acetyl-spermine or acetylated amino ter-mini of proteins. Furthermore we have found that Hst2 displays conformational rather than sequence specificity, preferentially deacetylating acetyl-lysine within unstructured regions of proteins. Our results suggest that this conformational requirement may be a general feature for substrate recognition in the Sir2 family.     

Architectures of the unique domains associated with the DEAD-box helicase motif

Helicases are motor proteins of biological system, which catalyze the opening of energetically stable duplex nucleic acids in an ATP-dependent manner and thereby are involved in almost all aspects of nucleic acid metabolism including cell cycle progression. They contain several conserved domains including the DEAD-box and also several unique domains associated with these. The Pfam database (http://pfam.janelia.org/) is a large collection of protein families, each represented by multiple sequence alignments and hidden Markov models (HMMs). A diverse range of proteins are found in nature, and the functional specificity to each protein, to a greater extent, is imparted by its domain architecture. To this extent, a DEAD-box ATP-dependent RNA helicase (LOC_Os01g36890; Genomic sequence length: 6284 nucleotides; CDS length: 1299 nucleotides; Protein length: 432 amino acids) was studied. The protein sequence was imported for domain search on Pfam. This particular Pfam entry after covering a large proportion of the sequences in the underlying database has generated a more comprehensive coverage across a wide range of phyla of the known domains that are associated with the typical DEAD-box helicase motif. A total of 362 domain architectures were recollected from the Pfam database for the Family: DEAD (PF00270). We have therefore systematically analyzed the domains closely associated with DEAD-motif, which occur in a variety of proteins and can provide insights into their function.     

Modelling and mutational evidence identify the substrate binding site and functional elements in APC amino acid transporters

The Amino acid-Polyamine-Organocation (APC) superfamily is the main family of amino acid transporters found in all domains of life and one of the largest families of secondary transporters. Here, using a sensitive homology threading approach and modelling we show that the predicted structure of APC members is extremely similar to the crystal structures of several prokaryotic transporters belonging to evolutionary distinct protein families with different substrate specificities. All of these proteins, despite having no primary amino acid sequence similarity, share a similar structural core, consisting of two V-shaped domains of five transmembrane domains each, intertwined in an antiparallel topology. Based on this model, we reviewed available data on functional mutations in bacterial, fungal and mammalian APCs and obtained novel mutational data, which provide compelling evidence that the amino acid binding pocket is located in the vicinity of the unwound part of two broken helices, in a nearly identical position to the structures of similar transporters. Our analysis is fully supported by the evolutionary conservation and specific amino acid substitutions in the proposed substrate binding domains. Furthermore, it allows predictions concerning residues that might be crucial in determining the specificity profile of APC members. Finally, we show that two cytoplasmic loops constitute important functional elements in APCs. Our work along with different kinetic and specificity profiles of APC members in easily manipulated bacterial and fungal model systems could form a unique framework for combining genetic, in-silico and structural studies, for understanding the function of one of the most important transporter families.     

The Pfam Protein Families Database

Pfam is a large collection of protein multiple sequence alignments and profile hidden Markov models. Pfam is available on the World Wide Web in the UK at http://www.sanger.ac.uk/Software/Pfam/, in Sweden at http://www.cgb.ki.se/Pfam/, in France at http://pfam.jouy.inra.fr/ and in the US at http://pfam.wustl.edu/. The latest version (6.6) of Pfam contains 3071 families, which match 69% of proteins in SWISS-PROT 39 and TrEMBL 14. Structural data, where available, have been utilised to ensure that Pfam families correspond with structural domains, and to improve domain-based annotation. Predictions of non-domain regions are now also included. In addition to secondary structure, Pfam multiple sequence alignments now contain active site residue mark-up. New search tools, including taxonomy search and domain query, greatly add to the functionality and usability of the Pfam resource.