Structure of nitrilotriacetate monooxygenase component B from Mycobacterium thermoresistibile.

Mycobacterium tuberculosis belongs to a large family of soil bacteria which can degrade a remarkably broad range of organic compounds and utilize them as carbon, nitrogen and energy sources. It has been proposed that a variety of mycobacteria can subsist on alternative carbon sources during latency within an infected human host, with the help of enzymes such as nitrilotriacetate monooxygenase (NTA-Mo). NTA-Mo is a member of a class of enzymes which consist of two components: A and B. While component A has monooxygenase activity and is responsible for the oxidation of the substrate, component B consumes cofactor to generate reduced flavin mononucleotide, which is required for component A activity. NTA-MoB from M. thermoresistibile, a rare but infectious close relative of M. tuberculosis which can thrive at elevated temperatures, has been expressed, purified and crystallized. The 1.6 Å resolution crystal structure of component B of NTA-Mo presented here is one of the first crystal structures determined from the organism M. thermoresistibile. The NTA-MoB crystal structure reveals a homodimer with the characteristic split-barrel motif typical of flavin reductases. Surprisingly, NTA-MoB from M. thermoresistibile contains a C-terminal tail that is highly conserved among mycobacterial orthologs and resides in the active site of the other protomer. Based on the structure, the C-terminal tail may modulate NTA-MoB activity in mycobacteria by blocking the binding of flavins and NADH.

NEWT, a new taxonomy portal.

NEWT is a new taxonomy portal to the SWISS-PROT protein sequence knowledgebase. It contains taxonomy data, which is updated daily, for the complete set of species represented in SWISS-PROT, as well as those stored at the NCBI. Users can navigate through the taxonomy tree and access corresponding SWISS-PROT protein entries. In addition, a manually curated selection of external links allows access to specific information on selected species. NEWT is available at http://www.ebi.ac.uk/newt/.

Interactive InterPro-based comparisons of proteins in whole genomes.

MOTIVATION: The SWISS-PROT group at the EBI has developed the Proteome Analysis Database utilizing existing resources and providing comprehensive and integrated comparative analysis of the predicted protein coding sequences of the complete genomes of bacteria, archaea and eukaryotes. The Proteome Analysis Database is accompanied by a program that has been designed to carry out interactive InterPro proteome comparisons for any one proteome against any other one or more of the proteomes in the database.

A thermostable endonuclease III homolog from the archaeon Pyrobaculum aerophilum.

Pyrimidine adducts in cellular DNA arise from modification of the pyrimidine 5,6-double bond by oxidation, reduction or hydration. The biological outcome includes increased mutation rate and potential lethality. A major DNA N:-glycosylase responsible for the excision of modified pyrimidine bases is the base excision repair (BER) glycosylase endonuclease III, for which functional homologs have been identified and characterized in Escherichia coli, yeast and humans. So far, little is known about how hyperthermophilic Archaea cope with such pyrimidine damage. Here we report characterization of an endonuclease III homolog, PaNth, from the hyperthermophilic archaeon Pyrobaculum aerophilum, whose optimal growth temperature is 100 degrees C. The predicted product of 223 amino acids shares significant sequence homology with several [4Fe-4S]-containing DNA N:-glycosylases including E.coli endonuclease III (EcNth). The histidine-tagged recombinant protein was expressed in E.coli and purified. Under optimal conditions of 80-160 mM NaCl and 70 degrees C, PaNth displays DNA glycosylase/ss-lyase activity with the modified pyrimidine base 5,6-dihydrothymine (DHT). This activity is enhanced when DHT is paired with G. Our data, showing the structural and functional similarity between PaNth and EcNth, suggests that BER of modified pyrimidines may be a conserved repair mechanism in Archaea. Conserved amino acid residues are identified for five subfamilies of endonuclease III/UV endonuclease homologs clustered by phylogenetic analysis.

Proteome Analysis Database: online application of InterPro and CluSTr for the functional classification of proteins in whole genomes.

The SWISS-PROT group at EBI has developed the Proteome Analysis Database utilising existing resources and providing comparative analysis of the predicted protein coding sequences of the complete genomes of bacteria, archaea and eukaryotes (http://www.ebi.ac. uk/proteome/). The two main projects used, InterPro and CluSTr, give a new perspective on families, domains and sites and cover 31-67% (InterPro statistics) of the proteins from each of the complete genomes. CluSTr covers the three complete eukaryotic genomes and the incomplete human genome data. The Proteome Analysis Database is accompanied by a program that has been designed to carry out InterPro proteome comparisons for any one proteome against any other one or more of the proteomes in the database.

Drosophila dumpy is a gigantic extracellular protein required to maintain tension at epidermal-cuticle attachment sites.

BACKGROUND: Growth and morphogenesis during development depend both on patterning genes, which assign positional information, and on genes that regulate mechanical forces. The dumpy gene of the fruit fly Drosophila melanogaster is an example of the latter class, with mutant phenotypes affecting size and shape of the limbs, thoracic cuticle, trachea and mouthparts. RESULTS: The genetically complex dumpy locus was found to span over 100 kb and encode a gigantic 2.5 MDa extracellular matrix protein. Dumpy represents an extreme form of modular protein evolution, containing 308 epidermal growth factor (EGF) modules, interspersed with a new module class, DPY, and terminating in a crosslinking zona pellucida domain and membrane anchor sequence. We determined the three-dimensional structure of the DPY module by nuclear magnetic resonance (NMR) spectroscopy and found that it forms a disulphide-stabilised beta sheet motif, capable of linking end-to-end with EGF modules to form a fibre. Consistent with its cuticle phenotypes, dumpy is expressed at several sites of cuticle-epidermal cell attachment, including the trachea and the muscle tendon cells, which mediate anchorage of the muscles to the cuticle. CONCLUSIONS: The dumpy gene encodes a gigantic extracellular molecule that we predict to be a membrane-anchored fibre of almost a micrometer in length. Insertion and crosslinking of this fibre within the cuticle may provide a strong anchor for the underlying tissue, allowing it to maintain mechanical tension at sites under stress. This would explain its contribution to tissue morphogenesis through its regulation of mechanical properties.

Cloning and characterization of a new member of the Nudix hydrolases from human and mouse.

Proteins containing the Nudix box "GX(5)EX(7)REUXEEXGU" (where U is usually Leu, Val, or Ile) are Nudix hydrolases, which catalyze the hydrolysis of a variety of nucleoside diphosphate derivatives. Here we report cloning and characterization of a human cDNA encoding a novel nudix hydrolase NUDT5 for the hydrolysis of ADP-sugars. The deduced amino acid sequence of NUDT5 contains 219 amino acids, including a conserved Nudix box sequence. The recombinant NUDT5 was expressed in Escherichia coli and purified to near homogeneity. At the optimal pH of 7, the purified recombinant NUDT5 catalyzed hydrolysis of two major substrates ADP-ribose and ADP-mannose with K(m) values of 32 and 83 microM, respectively; the V(max) for ADP-mannose was about 1.5 times that with ADP-ribose. The murine NUDT5 homolog was also cloned and characterized. mNudT5 has 81% amino acid identity to NUDT5 with catalytic activities similar to NUDT5 under the optimal pH of 9. Both NUDT5 and mNudT5 transcripts were ubiquitously expressed in tissues analyzed with preferential abundance in liver. The genomic structures of both NUDT5 and mNudT5 were determined and located on human chromosome 10 and mouse chromosome 2, respectively. The role of NUDT5 in maintaining levels of free ADP-ribose in cells is discussed.

Solution structure of a type 2 module from fibronectin: implications for the structure and function of the gelatin-binding domain.

BACKGROUND: Fibronectin is an extracellular matrix glycoprotein involved in cell adhesion and migration events in a range of important physiological processes. Aberrant adhesion of cells to the matrix may contribute to the breakdown of normal tissue function associated with various diseases. The adhesive properties of fibronectin may be mediated by its interaction with collagen, the most abundant extracellular matrix protein. The collagen-binding activity of fibronectin has been localized to a 42 kDa proteolytic fragment on the basis of this fragment's affinity for denatured collagen (gelatin). This gelatin-binding domain contains the only type 2 (F2) modules found in the protein. The F2 modules of the matrix metalloproteinases MMP2 and MMP9 are responsible for the affinity of these proteins for gelatin. Knowledge of the structure of fibronectin will provide insights into its interactions with other proteins, and will contribute to our understanding of the structure and function of the extracellular matrix, in both normal and disease-altered tissues. RESULTS: We have determined the solution structure of the first F2 (1F2) module from human fibronectin by two-dimensional NMR spectroscopy. The tertiary structure of the 1F2 module is similar to that of a shorter F2 module, PDC-109b, from the bovine seminal plasma protein PDC-109. The 1F2 module has two double-stranded antiparallel beta sheets oriented approximately perpendicular to each other, and enclosing a cluster of highly conserved aromatic residues, five of which form a solvent-exposed hydrophobic surface. The N-terminal extension in 1F2 brings the N and C termini of the module into close proximity. CONCLUSIONS: The close proximity of the N and C termini in 1F2 allows for interactions between non-contiguous modules in the gelatin-binding domain. Thus, instead of forming an extended, linear chain of modules, the domain may have a more compact, globular structure. A pocket in the module's solvent-exposed hydrophobic surface may bind nonpolar residues in the putative fibronectin-binding site of the extracellular matrix component type I collagen.

Dynamic studies of a fibronectin type I module pair at three frequencies: Anisotropic modelling and direct determination of conformational exchange.

A detailed analysis of the (15)N relaxation of a pair of modules from fibronectin is presented. The overall dimensions of the protein structure can be approximated by a cylinder with an axial ratio D(∥)/D(⊥) of 1.9. T(1), T(2) and NOE data, collected at three (15)M frequencies (50.6, 60.8 and 76 MHz), can be fitted satisfactorily to a Lipari-Szabo model, taking anisotropy into account. A method for analysing the exchange contribution to relaxation is presented. This contribution depends upon the predicted B (inf0) (sup2) frequency dependence in the fast exchange limit of these exchange terms. Using this analysis, relatively slow conformational exchange contributions are detected around one of the disulphide bonds in the first module of the pair.

High-resolution structural studies of the factor XIIIa crosslinking site and the first type 1 module of fibronectin.

The N-terminal domain of fibronectin undergoes factor XIIIa-catalysed crosslinking to fibrin, bacteria and collagen. The reactive glutamine residue is in an extended, random coil 'tail' of about 18 residues that would be accessible for crosslinking.

Solution structure of a pair of fibronectin type 1 modules with fibrin binding activity.

The tertiary structure of the fourth and fifth type 1 module pair from the N terminus of human fibronectin, has been determined by two-dimensional homonuclear 1H nuclear magnetic resonance (NMR) spectroscopy. Comparison of each module fold with those of two other type 1 modules shows that the type 1 "consensus" structure is conserved in the pair. The modules connect end-to-end to form an elongated structure with a limited clockwise twist around the long axis, from N to C terminus. The short five residue linker sequence forms a tight loop and the relative orientation of the two modules is maintained by fixed and intimate hydrophobic contacts, dominated by a non-conserved tryptophan residue from the fourth type 1 module. The protein binds specifically to fibrin in an ELISA and surface accessible residues that may be involved in this and other protein interactions can be identified. The structure provides an insight into how chains of type 1 modules may link up in intact fibronectin.

Secondary structure of a pair of fibronectin type 1 modules by two-dimensional nuclear magnetic resonance.

The fourth and fifth type 1 module pair, corresponding to residues 151-244 from the amino terminus of human fibronectin, has been produced as a recombinant protein using a yeast expression system and studied by two-dimensional homonuclear 1H nuclear magnetic resonance (NMR) spectroscopy. The sequence-specific resonance assignment of the 1H NMR spectrum has been completed using a combination of 2D 1H nuclear Overhauser effect (NOE) spectroscopy, homonuclear Hartmann-Hahn, and correlated spectroscopy spectra recorded under a variety of pH and temperature conditions. Slow exchanging amide protons have been identified and estimates of many backbone 3JNH-C alpha H coupling constants were obtained by line shape fitting. The secondary structures of each module conform closely to the "consensus" fibronectin type 1 module structure determined previously for two other single type 1 modules. In the module pair described here, the two modules are linked by a short five-residue linker which appears to form a turn. The intermodule interface is defined by NOEs observed between a hydrophobic three-residue sequence from the fourth type 1 module and residues in the first double-stranded beta-sheet of the fifth type 1 module. The interaction is dominated by a tryptophan residue (unconserved in other type 1 sequences) within the fourth module, which causes large upfield ring current shifts for several proton resonances from the beta-sheet of the fifth module. The NMR data indicate that there is little or no relative reorientation of the two modules about the linker region but rather that the two modules combine with a fixed and intimate hydrophobic contact.