HMt, a histone-related protein from Methanobacterium thermoautotrophicum delta H.

HMt, a histone-related protein, has been isolated and characterized from Methanobacterium thermoautotrophicum delta H. HMt preparations contain two polypeptides designated HMt1 and HMt2, encoded by the hmtA and hmtB genes, respectively, that have been cloned, sequenced, and expressed in Escherichia coli. HMt1 and HMt2 are predicted to contain 68 and 67 amino acid residues, respectively, and have calculated molecular masses of 7,275 and 7,141 Da, respectively. Aligning the amino acid sequences of HMt1 and HMt2 with the sequences of HMf1 and HMf2, the subunit polypeptides of HMf, a histone-related protein from the hyperthermophile Methanothermus fervidus, revealed that 40 amino acid residues (approximately 60%) are conserved in all four polypeptides. In pairwise comparisons, these four polypeptides are 66 to 84% identical. The sequences and locations of the TATA box promoter elements and ribosome binding sites are very similar upstream of the hmtA and hmtB genes in M. thermoautotrophicum and upstream of the hmfA and hmfB genes in M. fervidus. HMt binding compacted linear pUC19 DNA molecules in vitro and therefore increased their electrophoretic mobilities through agarose gels. At protein/DNA mass ratios of < 0.2:1, HMt binding caused an increase in the overall negative superhelicity of relaxed, circular DNA molecules, but at HMt/DNA mass ratios of > 0.2:1, positive supercoils were introduced into these molecules. HMt and HMf are indistinguishable in terms of their abilities to compact and constrain DNA molecules in positive toroidal supercoils in vitro. Histone-related proteins with these properties are therefore not limited to reverse gyrase-containing hyperthermophilic species.     

Sequence landmark patterns identify and characterize protein families

The three-dimensional structures of homologous proteins are usually conserved during evolution, as are critical residues in a few short sequence motifs that often constitute the active site in enzymes. The precise spatial organization of such sites depends on the lengths and positions of the secondary structural elements connecting the motifs. We show how members of protein superfamilies, such as kinesins, myosins, and G(alpha) subunits of trimeric G proteins, are identified and classed by simply counting the number of amino acid residues between important sequence motifs in their nucleotide triphosphate-hydrolyzing domains. Subfamily-specific landmark patterns (motif to motif scores) are principally due to inserts and gaps in surface loops. Unusual protein sequences and possible sequence prediction errors are detected.     

Homology modelling and protein engineering strategy of subtilases, the family of subtilisin-like serine proteinases.

Subtilases are members of the family of subtilisin-like serine proteases. Presently, greater than 50 subtilases are known, greater than 40 of which with their complete amino acid sequences. We have compared these sequences and the available three-dimensional structures (subtilisin BPN', subtilisin Carlsberg, thermitase and proteinase K). The mature enzymes contain up to 1775 residues, with N-terminal catalytic domains ranging from 268 to 511 residues, and signal and/or activation-peptides ranging from 27 to 280 residues. Several members contain C-terminal extensions, relative to the subtilisins, which display additional properties such as sequence repeats, processing sites and membrane anchor segments. Multiple sequence alignment of the N-terminal catalytic domains allows the definition of two main classes of subtilases. A structurally conserved framework of 191 core residues has been defined from a comparison of the four known three-dimensional structures. Eighteen of these core residues are highly conserved, nine of which are glycines. While the alpha-helix and beta-sheet secondary structure elements show considerable sequence homology, this is less so for peptide loops that connect the core secondary structure elements. These loops can vary in length by greater than 150 residues. While the core three-dimensional structure is conserved, insertions and deletions are preferentially confined to surface loops. From the known three-dimensional structures various predictions are made for the other subtilases concerning essential conserved residues, allowable amino acid substitutions, disulphide bonds, Ca(2+)-binding sites, substrate-binding site residues, ionic and aromatic interactions, proteolytically susceptible surface loops, etc. These predictions form a basis for protein engineering of members of the subtilase family, for which no three-dimensional structure is known.     

Identification and nucleotide sequence of the glycoprotein gB gene of equine herpesvirus 4.

The nucleotide sequence of the glycoprotein gB gene of equine herpesvirus 4 (EHV-4) was determined. The gene was located within a BamHI genomic library by a combination of Southern and dot-blot hybridization with probes derived from the herpes simplex virus type 1 (HSV-1) gB DNA sequence. The predominant portion of the coding sequences was mapped to a 2.95-kilobase BamHI-EcoRI subfragment at the left-hand end of BamHI-C. Potential TATA box, CAT box, and mRNA start site sequences and the translational initiation codon were located in the BamHI M fragment of the virus, which is located immediately to the left of BamHI-C. A polyadenylation signal, AATAAA, occurs nine nucleotides past the chain termination codon. Translation of these sequences would give a 110-kilodalton protein possessing a 5' hydrophobic signal sequence, a hydrophilic surface domain containing 11 potential N-linked glycosylation sites, a hydrophobic transmembrane domain, and a 3' highly charged cytoplasmic domain. A potential internal proteolytic cleavage site, Arg-Arg/Ser, was identified at residues 459 to 461. Analysis of this protein revealed amino acid sequence homologies of 47% with HSV-1 gB, 54% with pseudorabies virus gpII, 51% with varicella-zoster virus gpII, 29% with human cytomegalovirus gB, and 30% with Epstein-Barr virus gB. Alignment of EHV-4 gB with HSV-1 (KOS) gB further revealed that four potential N-linked glycosylation sites and all 10 cysteine residues on the external surface of the molecules are perfectly conserved, suggesting that the proteins possess similar secondary and tertiary structures. Thus, we showed that EHV-4 gB is highly conserved with the gB and gpII glycoproteins of other herpesviruses, suggesting that this glycoprotein has a similar overall function in each virus.     

Molecular cloning, characterization, and expression of a redox-responsive cutinase from Monilinia fructicola (Wint.) Honey

cDNA clones encoding a cutinase expressed in cutin-induced cultures of the plant pathogen Monilinia fructicola were isolated using a protein-based strategy. The largest cDNA (Mfcut1) was found to contain an open reading frame of 603 bp that predicted a 20.2-kDa protein of 201 amino acids with a 20-amino-acid secretory signal peptide and a pI of 8.4. The predicted protein contained cutinase/lipase consensus sequences with active site serines and potential protein kinase phosphorylation sites. Comparison of the deduced amino sequence from Mfcut1 with other fungal cutinase sequences revealed new features, which include conserved cysteines, C-terminal aromatic residues, and a novel histidine substitution in the D-H active site motif. The presence in the growth medium of antioxidants, such as caffeic acid, suppressed mRNA accumulation and enzyme activity of a cutinase from M. fructicola. MFCUT1 was expressed at high levels as a His-tagged fusion protein in Pichia pastoris and purified to apparent homogeneity in a single step by Ni(2+)-nitrilotriacetic acid affinity chromatography. Analysis of variant MFCUT1 mutants in which the novel serine and histidine residues were replaced by site-directed mutagenesis indicated that these residues had an important effect on enzyme activity.     

A synthetic arabinose-inducible promoter confers high levels of recombinant protein expression in hyperthermophilic archaeon Sulfolobus islandicus

Despite major progresses in genetic studies of hyperthermophilic archaea, recombinant protein production in these organisms always suffers from low yields and a robust expression system is still in great demand. Here we report a versatile vector that confers high levels of protein expression in Sulfolobus islandicus, a hyperthermophilic crenarchaeon. Two expression vectors, pSeSD and pEXA, harboring 11 unique restriction sites were constructed. They contain coding sequences of two hexahistidine (6×His) peptide tags and those coding for two protease sites, the latter of which make it possible to remove the peptide tags from expressed recombinant proteins. While pEXA employed an araS promoter for protein expression, pSeSD utilized P(araS-SD), an araS derivative promoter carrying an engineered ribosome-binding site (RBS; a Shine-Dalgarno [SD] sequence). We found that P(araS-SD) directed high levels of target gene expression. More strikingly, N-terminal amino acid sequencing of recombinant proteins unraveled that the protein synthesized from pEXA-N-lacS lacked the designed 6×His tag and that translation initiation did not start at the ATG codon of the fusion gene. Instead, it started at multiple sites downstream of the 6×His codons. Intriguingly, inserting an RBS site upstream of the ATG codon regained the expression of the 6×His tag, as shown with pSeSD-N-lacS. These results have yielded novel insight into the archaeal translation mechanism. The crenarchaeon Sulfolobus can utilize N-terminal coding sequences of proteins to specify translation initiation in the absence of an RBS site.     

Nucleotide and predicted amino acid sequence analysis of the fusion protein and hemagglutinin-neuraminidase protein genes among Newcastle disease virus isolates. Phylogenetic relationships among the Paramyxovirinae based on attachment glycoprotein sequences

Highly virulent Newcastle disease virus (NDV) isolates are List A pathogens for commercial poultry, and reports of their isolation among member nations must be made to the Office of International Epizootes (OIE). The virus is classified as a member of the order Mononegavirales in the family Paramyxoviridae of the subfamily Paramyxovirinae. Two interactive surface glycoproteins, the fusion (F) and hemagglutinin-neuraminidase (HN) proteins, play essential roles in NDV attachment and fusion of cells during infection. Antibodies to the F or HN proteins are capable of virus neutralization; however, no full-length sequences are available for these genes from recently obtained virulent isolates. Therefore, nucleotide and predicted amino acid sequences of the F and HN protein genes from 16 NDV isolates representing highly virulent viruses from worldwide sources were obtained for comparison to older virulent isolates and vaccine strains. The F protein amino acid sequence was relatively conserved among isolates maintaining potential glycosylation sites and C residues for disulfide bonds. A dibasic amino acid motif was present at the cleavage site among more virulent isolates, while the low virulence viruses did not have this sequence. However, a Eurasian collared dove virus had a K114Q substitution at the F cleavage site unique among NDV isolates. The HN protein among NDV isolates maintained predicted catalytic and active site residues necessary for neuraminidase activity and hemagglutination. Length of the HN for the Eurasian collared dove isolate and a previously reported heat resistant virulent isolate were longer relative to other more recent virulent isolates. Phylogenetically NDV isolates separated into four groups with more recent virulent isolates forming a diverse branch, while all the avian paramyxoviruses formed their own clade distinct from other members of the Paramyxoviridae.     

RNA-dependent activation of primer RNA production by influenza virus polymerase: different regions of the same protein subunit constitute the two required RNA-binding sites.

The capped RNA primers required for the initiation of influenza virus mRNA synthesis are produced by the viral polymerase itself, which consists of three proteins PB1, PB2 and PA. Production of primers is activated only when the 5'- and 3'-terminal sequences of virion RNA (vRNA) bind sequentially to the polymerase, indicating that vRNA molecules function not only as templates for mRNA synthesis but also as essential cofactors which activate catalytic functions. Using thio U-substituted RNA and UV crosslinking, we demonstrate that the 5' and 3' sequences of vRNA bind to different amino acid sequences in the same protein subunit, the PB1 protein. Mutagenesis experiments proved that these two amino acid sequences constitute the functional RNA-binding sites. The 5' sequence of vRNA binds to an amino acid sequence centered around two arginine residues at positions 571 and 572, causing an allosteric alteration which activates two new functions of the polymerase complex. In addition to the PB2 protein subunit acquiring the ability to bind 5'-capped ends of RNAs, the PB1 protein itself acquires the ability to bind the 3' sequence of vRNA, via a ribonucleoprotein 1 (RNP1)-like motif, amino acids 249-256, which contains two phenylalanine residues required for binding. Binding to this site induces a second allosteric alteration which results in the activation of the endonuclease that produces the capped RNA primers needed for mRNA synthesis. Hence, the PB1 protein plays a central role in the catalytic activity of the viral polymerase, not only in the catalysis of RNA-chain elongation but also in the activation of the enzyme activities that produce capped RNA primers.     

Functional control of the binuclear metal site in the metallo-beta-lactamase-like fold by subtle amino acid replacements

At present there are three protein families that share a common structural domain, the alphabeta/betaalpha fold of class B beta-lactamases: zinc beta-lactamases, glyoxalases II, and A-type flavoproteins. A detailed inspection of their superimposed structures was undertaken and showed that although these proteins contain binuclear metal sites in spatially equivalent positions, there are some subtle differences within the first ligand sphere that determine a distinct composition of metals. Although zinc beta-lactamases contain either a mono or a di-zinc center, the catalytically active form of glyoxalase II contains a mixed iron-zinc binuclear center, whereas A-type flavoproteins contain a di-iron site. These variations on the type of metal site found within a common fold are correlated with the subtle variations in the nature of the ligating amino acid residues and are discussed in terms of the different reactions catalyzed by each of the protein families. Correlation of these observations with sequence data results in the definition of a sequence motif that comprises the possible binuclear metal site ligands in this broad family. The evolution of the proteins sharing this common fold and factors modulating reactivity are also discussed.     

Pyrimidine-specific ribonucleoside hydrolase from the archaeon Sulfolobus solfataricus--biochemical characterization and homology modeling

We report the characterization of the pyrimidine-specific ribonucleoside hydrolase from the hyperthermophilic archaeon Sulfolobus solfataricus (SsCU-NH). The gene SSO0505 encoding SsCU-NH was cloned and expressed in Escherichia coli and the recombinant protein was purified to homogeneity. SsCU-NH is a homotetramer of 140 kDa that recognizes uridine and cytidine as substrates. SsCU-NH shares 34% sequence identity with pyrimidine-specific nucleoside hydrolase from E. coli YeiK. The alignment of the amino acid sequences of SsCU-NH with nucleoside hydrolases whose 3D structures have been solved indicates that the amino acid residues involved in the calcium- and ribose-binding sites are preserved. SsCU-NH is highly thermophilic with an optimum temperature of 100 degrees C and is characterized by extreme thermodynamic stability (T(m) = 106 degrees C) and kinetic stability (100% residual activity after 1 h incubation at 90 degrees C). Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is necessary for the integrity of the active site. The structure of the enzyme determined by homology modeling provides insight into the proteolytic analyses as well as into mechanisms of thermal stability. This is the first nucleoside hydrolase from Archaea.