Scientific Report 2006

High-Throughput Approaches to Protein Structure and Function

S.A. Lesley, M. Deller, D. Carlton, H. Johnson, Y. Elias, T. Clayton

Genomic information from the large number of sequenced species has provided as many questions as it has answered. Evaluating protein structure and function is of primary importance for understanding the basic biology of the cell and is a challenge in the context of the genome. To address this challenge, we have established high-throughput approaches for evaluating structural and functional diversity of proteins. We have developed the capacity to clone, express, purify, and crystallize large numbers of proteins in parallel as part of our structural genomics effort with the Joint Center for Structural Genomics, and we hope to apply these same tools to characterize the molecular basis of the specificity of enzyme substrates.

The goals of the Joint Center for Structural Genomics are to develop a high-throughput and cost-effective structure pipeline and to use the pipeline to determine novel protein folds and explore protein structure-function relationships. We have used this approach in the extensive study of the thermophilic bacterium Thermotoga maritima and for targets from mouse and other bacterial genomes. Our technologies have enabled us to perform comprehensive structural studies of these proteomes. To date, these efforts have resulted in more than 300 novel protein structures from the center.

Functional studies of selected targets have been performed. For example, in collaboration with A. Kohen, University of Iowa, Iowa City, we explored the mechanism of thymidylate synthase from T maritima. This protein has a novel fold and a unique flavin-dependent biochemical mechanism. The gene for thymidylate synthase is an essential one, and the protein is an important potential antibacterial target because of its structural dissimilarity with the human protein.

We have also developed the method of deuterium exchange by mass spectrometry in collaboration with V. Woods, University of California, San Diego, to characterize protein regions with highly flexible regions that interfere with crystallization. Subsequent elimination of these regions dramatically improves crystallization and has resulted in structures for several problematic structures.

Expression of membrane proteins continues to be one of the most difficult challenges in studying this important protein class. Our structural genomics efforts in collaboration with S. Eshaghi, Karolinska Institutet, Stockholm, Sweden, have led to the structure of the integral membrane protein CorA, a magnesium transporter from T maritima. In collaboration with P. Schultz, Department of Chemistry, we are exploring the use of unnatural amino acids to enhance the purification and crystallization of integral membrane proteins.

Publications

Arndt, J.W., Schwarzenbacher, R., Page, R., et al. Crystal structure of an α/β serine hydrolase (YDR428C) from Saccharomyces cerevisiae at 1.85 Å resolution. Proteins 58:755, 2005.

Chamberlain, P.P., Sandberg, M.L., Sauer, K., Cooke, M.P., Lesley, S.A., Spraggon, G. Structural insights into enzyme regulation for inositol 1,4,5-trisphosphate 3-kinase B. Biochemistry 44:14486, 2005.

Columbus, L., Lipfert, J., Klock, H., Millett, I., Doniach, S., Lesley, S.A. Expression, purification, and characterization of Thermotoga maritima membrane proteins for structure determination. Protein Sci. 15:961, 2006.

DiDonato, M., Krishna, S.S., Schwarzenbacher, R., et al. Crystal structure of a single-stranded DNA-binding protein (TM0604) from Thermotoga maritima at 2.60 Å resolution. Proteins 63:256, 2006.

Eshaghi, S., Niegowski, D., Kohl, A., Martinez Molina, D., Lesley, S.A., Nordlund, P. Crystal structure of a divalent metal ion transporter CorA at 2.9 Å resolution [published correction appears in Science 313:1389, 2006]. Science 313:354, 2006.

Han, G.W., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of an apo mRNA decapping enzyme (DcpS) from mouse at 1.83 Å resolution. Proteins 60:797, 2005.

Han, G.W., Schwarzenbacher, R., Page, R., et al. Crystal structure of an alanine-glyoxylate aminotransferase from Anabaena sp. at 1.70 Å resolution reveals a noncovalently linked PLP cofactor. Proteins 58:971, 2005.

Han, S., Zhou, V., Pan, S., Liu, Y., Hornsby, M., McMullan, D., Klock, H., Lesley, S.A., Gray, N., Caldwell, J., Gu, X.J. Identification of coumarin derivatives as a novel class of allosteric MEK1 inhibitors. Bioorg. Med. Chem. Lett. 15:5467, 2005.

Jaroszewski, L., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of Hsp33 chaperone (TM1394) from Thermotoga maritima at 2.20 Å resolution.
Proteins 61:669, 2005.

Jin, K.K., Krishna, S.S., Schwarzenbacher, R., et al. Crystal structure of TM1367 from Thermotoga maritima at 1.90 Å resolution reveals an atypical member of the cyclophilin (peptidylprolyl isomerase) fold. Proteins 63:1112, 2006

Klock, H.E., Schwarzenbacher, R., Xu, Q., et al. Crystal structure of a conserved hypothetical protein (gi: 13879369) from mouse at 1.90 Å resolution reveals a new fold. Proteins 61:1132, 2005.

Klock, H.E., White, A., Koesema, E., Lesley, S.A. Methods and results for semi-automated cloning using integrated robotics. J. Struct. Funct. Genomics 6:89, 2005.

Kreusch, A., Han, S., Brinker, A., Zhou, V., Choi, H., He, Y., Lesley, S.A., Caldwell, J., Gu, X. Crystal structures of a new class of HSP90 inhibitors, dihydroxyphenylpyrazoles. Bioorg. Med. Chem. Lett. 15:1475, 2005.

Kreusch, A., Han, S., Brinker, A., Zhou, V., Choi, H.S., He, Y., Lesley, S.A., Caldwell, J., Gu, X.J. Crystal structures of human HSP90α complexed with dihydroxyphenylpyrazoles. Bioorg. Med. Chem. Lett. 15:1475, 2005.

Lesley, S.A., Wilson, I.A. Protein production and crystallization at the Joint Center for Structural Genomics. J. Struct. Funct. Genomics 6:71, 2005.

Levin, I., Miller, M.D., Schwarzenbacher, R., et al. Crystal structure of an indigoidine synthase A (IndA)-like protein (TM1464) from Thermotoga maritima at 1.90 Å resolution reveals a new fold. Proteins 59:864, 2005.

Mason, A., Agrawal, N., Washington, M.T., Lesley, S.A., Kohen, A. A lag-phase in the reduction of flavin dependent thymidylate synthase (FDTS) revealed a mechanistic missing link. Chem. Commun. (Camb.) 1781, 2006, Issue 16.

Mathews, I., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA) from Thermotoga maritima at 2.0 Å resolution reveals a new fold. Proteins 59:869,2005.

Mathews, I.I., Krishna, S.S., Schwarzenbacher, R., et al. Crystal structure of phosphoribosylformylglycinamidine synthase II (smPurL) from Thermotoga maritima at 2.15 Å resolution. Proteins 63:1106, 2006.

McMullan, D., Canaves, J.M., Quijano, K., Abdubek, P., Nigoghossian, E., Haugen, J., Klock, H.E., Vincent, J., Hale, J., Paulsen, J., Lesley, S.A. High-throughput protein production for x-ray crystallography and use of size-exclusion chromatography to validate computational biological unit predictions. J. Struct. Funct. Genomics 6:135, 2005.

Page, R., Deacon, A.M., Lesley, S.A., Stevens, R.C. Shotgun crystallization strategy for structural genomics, II: crystallization conditions that produce high resolution structure for T maritima proteins. J. Struct. Funct. Genomics 6:209, 2005.

Rife, C., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of the global regulatory protein CsrA from Pseudomonas putida at 2.05 Å resolution reveals a new fold. Proteins 61:449, 2005.

Rife, C., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of a putative modulator of DNA gyrase (pmbA) from Thermotoga maritima at 1.95 Å resolution reveals a new fold. Proteins 61:444, 2005.

Wang, Y., Klock, H., Yin, H., Wolff, K., Bieza, K., Niswonger, K., Matzen, J., Gunderson, D., Hale, J., Lesley, S., Kuhen, K., Caldwell, J., Brinker, A. Homogeneous high-throughput screening assays for HIV-1 integrase 3β-processing and strand transfer activities. J. Biomol. Screen. 10:456, 2005.

Xu, Q., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of a formiminotetrahydrofolate cyclodeaminase (TM1560) from Thermotoga maritima at 2.80 Å resolution reveals a new fold. Proteins 58:976, 2005.

Xu, Q., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of virulence factor CJ0248 from Campylobacter jejuni at 2.25 Å resolution reveals a new fold. Proteins 62:292, 2006.