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Quinolinic acid phosphoribosyltransferase (QAPRTase; NadC) catalyzes an indispensable step in NAD biosynthesis, one that is essential for cell survival in prokaryotes, which makes it an attractive target for antibacterial drug therapy. We recently reported the crystal structures of Helicobacter pylori QAPRTase with bound quinolinic acid, nicotinamide mononucleotide, and phthalic acid. The enzyme exists as a hexamer organized as a trimer of dimers, which is essential for full enzymatic activity. The loop between helix alpha7 and strand beta8 contributes significantly to the hydrophobic dimer-dimer interactions. Phe181Pro mutation within the alpha7-beta8 loop disrupts the hexamerization of QAPRTase, and the resultant dimer shows dramatically reduced protein stability and no activity. Our findings thus suggest that compounds able to disrupt its proper oligomerization could potentially function as selective inhibitors of Helicobacter pylori QAPRTase and represent a novel set of antibacterial agents.

Homologs of the Escherichia coli surE gene are present in many eubacteria and archaea. Despite the evolutionary conservation, little information is available on the structure and function of their gene products. We have determined the crystal structure of the SurE protein from Thermotoga maritima. The structure reveals the dimeric arrangement of the subunits and an active site around a bound metal ion. We also demonstrate that the SurE protein exhibits a divalent metal ion-dependent phosphatase activity that is inhibited by vanadate or tungstate. In the vanadate- and tungstate-complexed structures, the inhibitors bind adjacent to the divalent metal ion. Our structural and functional analyses identify the SurE proteins as a novel family of metal ion-dependent phosphatases.

In the yeast two-hybrid screening, we have isolated a cDNA clone from a human heart library using Nck Src homology 3 (SH3) domains as bait. The full-length cDNA, which encoded 722 amino acids, was identified as a VIP54-related gene containing an SH3 domain, proline-rich motifs, a serine/threonine-rich region, and a long C-terminal hydrophobic region. We refer to this protein as SPIN90 (SH3 Protein Interacting with Nck, 90 kDa). The amino acid sequence of the SH3 domain has the highest homology with those of Fyn, Yes, and c-Src. SPIN90 was broadly expressed in human tissues; in particular, it was highly expressed in heart, brain, and skeletal muscle, and its expression was developmentally regulated during cardiac myocyte differentiation. SPIN90 is able to bind to the first and third SH3 domains of Nck, in vitro, and is colocalized with Nck at sarcomere Z-discs within cardiac myocytes. Moreover, treatment with antisera raised against SPIN90 disrupted sarcomere structure, suggesting that this protein may play an important role in the maintenance of sarcomere structure and/or in the assembly of myofibrils into sarcomeres.

The surE protein from Thermotoga maritima is a 247-residue protein of unknown function. Its homologues are well conserved among both the eubacteria and the archaea. It has been overexpressed in soluble form in Escherichia coli. The protein has been crystallized at 296 K using 2-propanol as a precipitant. X-ray diffraction data have been collected to 1.9 A resolution using synchrotron radiation. The crystals belong to the trigonal space group P3(1)21 (or P3(2)21), with unit-cell parameters a = b = 115.96, c = 78.60 A, alpha = beta = 90, gamma = 120 degrees. The asymmetric unit contains two monomers of the surE protein, with a corresponding V(M) of 2.72 A(3) Da(-1) and a solvent content of 54.7%.

The key response-regulator gene of light regulation, rcp1, from Synechocystis sp. has been overexpressed, purified and subsequently crystallized using ammonium sulfate as a precipitant in forms suitable for X-ray crystallographic studies. A native data set was collected to a resolution of 2.5 A at cryogenic temperature. The crystals belong to the hexagonal space group P6(3), with unit-cell parameters a = b = 89.04 (5), c = 60.29 (3) A. The Matthews parameter suggests that Rcp1 crystallizes with two molecules per asymmetric unit.

A highly thermostable DNA ligase from Thermus filiformis has been crystallized at room temperature using methoxypolyethylene glycol 5000 as a precipitant. The crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 90.63, b = 117.80, c = 98. 65 A, beta = 115.56 degrees. Two molecules of DNA ligase are present in the asymmetric unit, giving a crystal volume per protein mass (V(m)) of 3.1 A(3) Da(-1) and a solvent content of 61%. A native data set extending to 3.0 A resolution has been collected at 100 K using synchrotron X-rays.

Ribosome recycling factor (RRF) catalyzes the disassembly of a termination complex during the final stage of protein synthesis. RRF from Escherichia coli has been crystallized with PEG 400 as precipitant at 287 K. The crystal belongs to the trigonal space group P3(1)21 (or P3(2)21), with unit-cell parameters a = b = 48.08, c = 141.67 A. Native data were collected from a frozen crystal to a resolution of 3.0 A on a Cu Kalpha rotating-anode X-ray source.