Implementation of semi-automated cloning and and prokaryotic expression screening: the impact of SPINE

The implementation of high-throughput (HTP) cloning and expression screening in Escherichia coli by 14 laboratories in the Structural Proteomics In Europe (SPINE) consortium is described. Cloning efficiencies of greater than 80% have been achieved for the three non-ligation-based cloning techniques used, namely Gateway, ligation-indendent cloning of PCR products (LIC-PCR) and In-Fusion, with LIC-PCR emerging as the most cost-effective. On average, two constructs have been made for each of the approximately 1700 protein targets selected by SPINE for protein production. Overall, HTP expression screening in E. coli has yielded 32% soluble constructs, with at least one for 70% of the targets. In addition to the implementation of HTP cloning and expression screening, the development of two novel technologies is described, namely library-based screening for soluble constructs and parallel small-scale high-density fermentation

Physical estimation of triplet phases from two new proteins

International audienceThree-beam interference experiments have been performed with crystals of two glycosidases: guinea-fowl hexagonal lysozyme, MW 14.3 kDa, and C. thermocellum endoglucanase CelA, MW 40 kDa. In both cases triplet phases could be estimated. Experimental parameters and details of the procedure are presented along with some examples of the results. The average differences between the estimated phases and those calculated from the crystallographic refinements were 17.9 and 15.9 degrees, respectively. A brief discussion of alternative methods for physical phase acquisition is given, including possible strategies for the measurement and application of experimental phases in macromolecular crystallography

Neutron diffraction analysis of barium nitroprusside trihydrate at room temperature

International audienceThe crystal structure of barium nitroprusside trihydrate, Ba[Fe(CN)5NO].3H2O, at room temperature has been refined using neutron diffraction measurements (F(000) = 41.8, Dx = 2.13 g cm−3, μ = 0.6 cm−1 (evaluated), space group Pbcm (57), orthorhombic, Z = 4, a = 7.620(7), b = 19.394(17), and c = 8.631(8) Å, V = 1276(4) Å3). A final R factor of 0.060 was obtained using 1349 observed structure factors. The nitroprusside ion in Ba[Fe(CN)5NO].3H2O presents a distorted octahedral configuration similar to that found in other nitroprusside salts determined by X-ray and neutron diffraction methods. Its polar axis lies in a symmetry plane, which also includes the Ba2+ and the oxygen of one of the water molecules. A positional disorder of the other two water molecules is observed. Two structural phase transitions were found at 130(3) and 112(4) K; they were analyzed from the evolution of selected reflections as a function of temperature in the range between 295 and 77 K

Proteomic Identification of M.tuberculosis Protein Kinase Substrates: PknB Recruits GarA, a FHA Domain-containing Protein, Through Activation Loop-mediated Interactions

International audienceGenes for functional Ser/Thr protein kinases (STPKs) are ubiquitous in prokaryotic genomes, but little is known about their physiological substrates and their actual involvement in bacterial signal transduction pathways. We report here the identification of GarA (Rv1827), a Forkhead-associated (FHA) domain-containing protein, as a putative physiological substrate of PknB, an essential Ser/Thr protein kinase from Mycobacterium tuberculosis. Using a global proteomic approach, GarA was found to be the best detectable substrate of the PknB catalytic domain in non-denatured whole-cell protein extracts from M. tuberculosis and the saprophyte Mycobacterium smegmatis. Enzymological and binding studies of the recombinant proteins demonstrate that docking interactions between the activation loop of PknB and the C-terminal FHA domain of GarA are required to enable efficient phosphorylation at a single N-terminal threonine residue, Thr22, of the substrate. The predicted amino acid sequence of the garA gene, including both the N-terminal phosphorylation motif and the FHA domain, is strongly conserved in mycobacteria and other related actinomycetes, suggesting a functional role of GarA in putative STPK-mediated signal transduction pathways. The ensuing model of PknB-GarA interactions suggests a substrate recruitment mechanism that might apply to other mycobacterial kinases bearing multiple phosphorylation sites in their activation loops

3-keto-5-aminohexanoate Cleavage Enzyme: A Common Fold For An Uncommon Claisen-type Condensation

The exponential increase in genome sequencing output has led to the accumulation of thousands of predicted genes lacking a proper functional annotation. Among this mass of hypothetical proteins, enzymes catalyzing new reactions or using novel ways to catalyze already known reactions might still wait to be identified. Here, we provide a structural and biochemical characterization of the 3-keto-5-aminohexanoate cleavage enzyme (Kce), an enzymatic activity long known as being involved in the anaerobic fermentation of lysine but whose catalytic mechanism has remained elusive so far. Although the enzyme shows the ubiquitous triose phosphate isomerase (TIM) barrel fold and a Zn 2+ cation reminiscent of metal-dependent class II aldolases, our results based on a combination of x-ray snapshots and molecular modeling point to an unprecedented mechanism that proceeds through deprotonation of the 3-keto-5-aminohexanoate substrate, nucleophilic addition onto an incoming acetyl- CoA, intramolecular transfer of the CoA moiety, and final retro-Claisen reaction leading to acetoacetate and 3-aminobutyryl- CoA. This model also accounts for earlier observations showing the origin of carbon atoms in the products, as well as the absence of detection of any covalent acyl-enzyme intermediate. Kce is the first representative of a large family of prokaryotic hypothetical proteins, currently annotated as the "domain of unknown function" DUF849. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.286312739927405Bateman, A., Coggill, P., Finn, R.D., (2010) Acta Crystallogr. F Struct. Biol. Cryst. Commun., 66, pp. 1148-1152Dayhoff, M.O., Schwartz, R., Orcutt, B.C., (1978) Atlas of Protein Sequence and Structure, pp. 345-352. , (Dayhoff, M. O., ed) National Biomedical Research Foundation, Washington, D.CEddy, S.R., (1996) Curr. Opin. Struct. Biol., 6, pp. 361-365Osterman, A., Overbeek, R., (2003) Curr. Opin. Chem. Biol., 7, pp. 238-251Zhang, C., Kim, S.H., (2003) Curr. Opin. Chem. Biol., 7, pp. 28-32Jaroszewski, L., Li, Z., Krishna, S.S., Bakolitsa, C., Wooley, J., Deacon, A.M., Wilson, I.A., Godzik, A., (2009) PLoS Biol., 7, pp. e1000205Kreimeyer, A., Perret, A., Lechaplais, C., Vallenet, D., Médigue, C., Salanoubat, M., Weissenbach, J., (2007) J. Biol. Chem., 282, pp. 7191-7197Barker, H.A., Kahn, J.M., Chew, S., (1980) J. Bacteriol., 143, pp. 1165-1170Barker, H.A., Kahn, J.M., Hedrick, L., (1982) J. Bacteriol., 152, pp. 201-207Yorifuji, T., Jeng, I.M., Barker, H.A., (1977) J. Biol. Chem., 252, pp. 20-31Kabsch, W., (2010) Acta Crystallogr. D Biol. Crystallogr., 66, pp. 125-132(1994) Acta Crystallogr. D Biol. Crystallogr., 50, pp. 760-763. , Collaborative Computational Project, Number 4Sheldrick, G.M., (2008) Acta Crystallogr. A, 64, pp. 112-122Terwilliger, T.C., Berendzen, J., (1999) Acta Crystallogr. D Biol. Crystallogr., 55, pp. 849-861Emsley, P., Lohkamp, B., Scott, W.G., Cowtan, K., (2010) Acta Crystallogr. D Biol. Crystallogr., 66, pp. 486-501Murshudov, G.N., Vagin, A.A., Dodson, E.J., (1997) Acta Crystallogr. D Biol. Crystallogr., 53, pp. 240-255Vagin, A., Teplyakov, A., (1997) J. Appl. Crystallogr., 30, pp. 1022-1025Blanc, E., Roversi, P., Vonrhein, C., Flensburg, C., Lea, S.M., Bricogne, G., (2004) Acta Crystallogr. D Biol. Crystallogr., 60, pp. 2210-2221Chen, V.B., Arendall III, W.B., Headd, J.J., Keedy, D.A., Immormino, R.M., Kapral, G.J., Murray, L.W., Richardson, D.C., (2010) Acta Crystallogr. D Biol. Crystallogr., 66, pp. 12-21Dolinsky, T.J., Nielsen, J.E., McCammon, J.A., Baker, N.A., (2004) Nucleic Acids Res., 32, pp. W665-W667Davis, F.A., Zhang, Y., Andemichael, Y., Fang, T., Fanelli, D.L., Zhang, H., (1999) J. Org. Chem., 64, pp. 1403-1406Trott, O., Olson, A.J., (2010) J. Comput. Chem., 31, pp. 455-461Hinsen, K., (2000) J. Comput. Chem., 21, pp. 79-85Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., Ferrin, T.E., (2004) J. Comput. Chem., 25, pp. 1605-1612Heath, R.J., Rock, C.O., (2002) Nat. Prod. Rep., 19, pp. 581-596Hermann, J.C., Ghanem, E., Li, Y., Raushel, F.M., Irwin, J.J., Shoichet, B.K., (2006) J. Am. Chem. Soc., 128, pp. 15882-15891Hermann, J.C., Marti-Arbona, R., Fedorov, A.A., Fedorov, E., Almo, S.C., Shoichet, B.K., Raushel, F.M., (2007) Nature, 448, pp. 775-779Guillén Schlippe, Y.V., Hedstrom, L., (2005) Arch. Biochem. Biophys., 433, pp. 266-278Burgi, H.B., Dunitz, J.D., Lehn, J.M., Wipff, G., (1974) Tetrahedron, 30, pp. 1563-1572Anstrom, D.M., Kallio, K., Remington, S.J., (2003) Protein Sci., 12, pp. 1822-1832Koon, N., Squire, C.J., Baker, E.N., (2004) Proc. Natl. Acad. Sci. U.S.A., 101, pp. 8295-8300Nagano, N., Orengo, C.A., Thornton, J.M., (2002) J. Mol. Biol., 321, pp. 741-76

Three-dimensional structure and antigen binding specificity of antibodies

International audienceA number of specific Fab and Fv fragments and their complexes with antigens (avian lysozymes), haptens, and anti-idiotopic Fabs have been studied by immunochemical and crystallographic techniques. Antigen and antibody interact through closely complementary contacting surfaces, without major conformational changes. An idiotopic determinant of a monoclonal antibody is shown to include parts of most of its complementarity determining regions. The specificity of antigen recognition resides in the close complementarity of the antigen determinant with the antibody combining site