Daniel McMullan

Structural Genomics of the Thermotoga maritima Proteome Implemented in a High-throughput Structure Determination Pipeline

Structural genomics is emerging as a principal approach to define protein structure–function relationships. To apply this approach on a genomic scale, novel methods and technologies must be developed to determine large numbers of structures. We describe the design and implementation of a high-throughput structural genomics pipeline and its application to the proteome of the thermophilic bacterium Thermotoga maritima. By using this pipeline, we successfully cloned and attempted expression of 1,376 of the predicted 1,877 genes (73%) and have identified crystallization conditions for 432 proteins, comprising 23% of the T. maritima proteome. Representative structures from TM0423 glycerol dehydrogenase and TM0449 thymidylate synthase-complementing protein are presented as examples of final outputs from the pipeline.

Functional Analysis of Substrate and Cofactor Complex Structures of a Thymidylate Synthase-Complementing Protein

Like thymidylate synthase (TS) in eukaryotes, the thymidylate synthase-complementing proteins (TSCPs) are mandatory for cell survival of many prokaryotes in the absence of external sources of thymidylate. Details of the mechanism of this novel family of enzymes are unknown. Here, we report the structural and functional analysis of a TSCP from Thermotoga maritima and its complexes with substrate, analogs, and cofactor. The structures presented here provide a basis for rationalizing the TSCP catalysis and reveal the possibility of the design of an inhibitor. We have identified a new helix-loop-strand FAD binding motif characteristic of the enzymes in the TSCP family. The presence of a hydrophobic core with residues conserved among the TSCP family suggests a common overall fold.

Crystal Structure of the Human Neuropilin-1 b1 Domain

Neuropilin-1 (Npn-1) is a type I cell surface receptor involved in a broad range of developmental processes, including axon guidance, angiogenesis, and heterophilic cell adhesion. We have determined the crystal structure of the human Npn-1 b1 domain to 1.9 A. The overall structure resembles coagulation factor V and VIII (F5/8) C1 and C2 domains, exhibiting a distorted jellyroll fold. Details of the structure provide insight to b1 domain regions responsible for ligand binding and facilitate rationalization of existing biochemical binding data. A polar cleft formed by adjacent loops at one end of the molecule in conjunction with flanking electronegative surfaces may represent the binding site for the positively charged tails of semaphorins and VEGF(165). The nature of the cell adhesion binding site of the b1 domain can be visualized in context of the structure.

Crystal structures of two novel dye-decolorizing peroxidases reveal a beta-barrel fold with a conserved heme-binding motif.

BtDyP from Bacteroides thetaiotaomicron (strain VPI‐5482) and TyrA from Shewanella oneidensis are dye‐decolorizing peroxidases (DyPs), members of a new family of heme‐dependent peroxidases recently identified in fungi and bacteria. Here, we report the crystal structures of BtDyP and TyrA at 1.6 and 2.7 Å, respectively. BtDyP assembles into a hexamer, while TyrA assembles into a dimer; the dimerization interface is conserved between the two proteins. Each monomer exhibits a two‐domain, α+β ferredoxin‐like fold. A site for heme binding was identified computationally, and modeling of a heme into the proposed active site allowed for identification of residues likely to be functionally important. Structural and sequence comparisons with other DyPs demonstrate a conservation of putative heme‐binding residues, including an absolutely conserved histidine. Isothermal titration calorimetry experiments confirm heme binding, but with a stoichiometry of 0.3:1 (heme:protein). Proteins 2007.

Crystal structure of thy1, a thymidylate synthase complementing protein from Thermotoga maritima at 2.25 Å resolution

Peter Kuhn, Scott A. Lesley, Irimpan I. Mathews, Jaume M. Canaves, Linda S. Brinen, Xiaoping Dai, Ashley M. Deacon, Marc A. Elsliger, Said Eshaghi, Ross Floyd, Adam Godzik, Carina Grittini, Slawomir K. Grzechnik, Chittibabu Guda, Keith O. Hodgson, Lukasz Jaroszewski, Cathy Karlak, Heath E. Klock, Eric Koesema, John M. Kovarik, Andreas T. Kreusch, Daniel McMullan, Timothy M. McPhillips, Mark A. Miller, Mitchell Miller, Andrew Morse, Kin Moy, Jie Ouyang, Alyssa Robb, Kevin Rodrigues, Thomas L. Selby, Glen Spraggon, Raymond C. Stevens, Susan S. Taylor, Henry van den Bedem, Jeff Velasquez, Juli Vincent, Xianhong Wang, Bill West, Guenter Wolf, John Wooley, and Ian A. Wilson* The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California The Genomics Institute of Novartis Foundation, San Diego, California The San Diego Supercomputer Center, La Jolla, California The University of California, San Diego, La Jolla, California The Scripps Research Institute, La Jolla, California

Structural Basis of Murein Peptide Specificity of a γ-D-glutamyl-L-diamino Acid Endopeptidase

The crystal structures of two homologous endopeptidases from cyanobacteria Anabaena variabilis and Nostoc punctiforme were determined at 1.05 and 1.60 A resolution, respectively, and contain a bacterial SH3-like domain (SH3b) and a ubiquitous cell-wall-associated NlpC/P60 (or CHAP) cysteine peptidase domain. The NlpC/P60 domain is a primitive, papain-like peptidase in the CA clan of cysteine peptidases with a Cys126/His176/His188 catalytic triad and a conserved catalytic core. We deduced from structure and sequence analysis, and then experimentally, that these two proteins act as gamma-D-glutamyl-L-diamino acid endopeptidases (EC 3.4.22.-). The active site is located near the interface between the SH3b and NlpC/P60 domains, where the SH3b domain may help define substrate specificity, instead of functioning as a targeting domain, so that only muropeptides with an N-terminal L-alanine can bind to the active site.

Structure analysis of peptide deformylases from streptococcus pneumoniae,staphylococcus aureus, thermotoga maritima, and pseudomonas aeruginosa: snapshots of the oxygen sensitivity of peptide deformylase

Peptide deformylase (PDF) has received considerable attention during the last few years as a potential target for a new type of antibiotics. It is an essential enzyme in eubacteria for the removal of the formyl group from the N terminus of the nascent polypeptide chain. We have solved the X-ray structures of four members of this enzyme family, two from the Gram-positive pathogens Streptococcus pneumoniae and Staphylococcus aureus, and two from the Gram-negative bacteria Thermotoga maritima and Pseudomonas aeruginosa. Combined with the known structures from the Escherichia coli enzyme and the recently solved structure of the eukaryotic deformylase from Plasmodium falciparum, a complete picture of the peptide deformylase structure and function relationship is emerging. This understanding could help guide a more rational design of inhibitors. A structure-based comparison between PDFs reveals some conserved differences between type I and type II enzymes. Moreover, our structures provide insights into the known instability of PDF caused by oxidation of the metal-ligating cysteine residue.

Crystal structure of the global regulatory protein CsrA from Pseudomonas putida at 2.05 Å resolution reveals a new fold

Chris Rife, Robert Schwarzenbacher, Daniel McMullan, Polat Abdubek, Eileen Ambing, Herbert Axelrod, Tanya Biorac, Jaume M. Canaves, Hsiu-Ju Chiu, Ashley M. Deacon, Michael DiDonato, Marc-André Elsliger, Adam Godzik, Carina Grittini, Slawomir K. Grzechnik, Joanna Hale, Eric Hampton, Gye Won Han, Justin Haugen, Michael Hornsby, Lukasz Jaroszewski, Heath E. Klock, Eric Koesema, Andreas Kreusch, Peter Kuhn, Scott A. Lesley, Mitchell D. Miller, Kin Moy, Edward Nigoghossian, Jessica Paulsen, Kevin Quijano, Ron Reyes, Eric Sims, Glen Spraggon, Raymond C. Stevens, Henry van den Bedem, Jeff Velasquez, Juli Vincent, Aprilfawn White, Guenter Wolf, Qingping Xu, Keith O. Hodgson, John Wooley, and Ian A. Wilson* The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California The University of California, San Diego, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, California The Scripps Research Institute, La Jolla, California

A scaleable and integrated crystallization pipeline applied to mining the Thermotoga maritima proteome

The wealth of genomic data available for many organisms has set the stage for the next phase of structure—function analysis. High-throughput structural genomics is currently the method of choice for rapid analysis of protein structure—function relationships on a proteome-wide basis. The Joint Center for Structural Genomics (JCSG), established in 2000 under the NIH/NIGMS Protein Structure Initiative, has developed and implemented an integrated high-throughput structure pipeline and applied it in a 2-tiered approach to mining the proteome of the thermophilic bacterium Thermotoga maritima. In the first tier, the successful application of this integrated pipeline has resulted in the cloning and expression of 73% of the T. maritima proteome (1376 out of 1877 predicted genes), and has identified 465 proteins which produced crystal hits. These 465 proteins were compared with existing structural information and a subset of 269 targets were selected to process towards structure determination in a second tier effort. To date, the JCSG pipeline applied to the Thermotoga maritima proteome has resulted in 55 new structures and has identified 6 novel folds and continues to identify structures with novel features.

Crystal structure of a tandem cystathionine-β-synthase (CBS) domain protein (TM0935) from Thermotoga maritima at 1.87 Å resolution

Mitchell D. Miller, Robert Schwarzenbacher, Frank von Delft, Polat Abdubek, Eileen Ambing, Tanya Biorac, Linda S. Brinen, Jaume M. Canaves, Jamison Cambell, Hsiu-Ju Chiu, Xiaoping Dai, Ashley M. Deacon, Mike DiDonato, Marc-André Elsliger, Said Eshagi, Ross Floyd, Adam Godzik, Carina Grittini, Slawomir K. Grzechnik, Eric Hampton, Lukasz Jaroszewski, Cathy Karlak, Heath E. Klock, Eric Koesema, John S. Kovarik, Andreas Kreusch, Peter Kuhn, Scott A. Lesley, Inna Levin, Daniel McMullan, Timothy M. McPhillips, Andrew Morse, Kin Moy, Jie Ouyang, Rebecca Page, Kevin Quijano, Alyssa Robb, Glen Spraggon, Raymond C. Stevens, Henry van den Bedem, Jeff Velasquez, Juli Vincent, Xianhong Wang, Bill West, Guenter Wolf, Qingping Xu, Keith O. Hodgson, John Wooley, and Ian A. Wilson* Joint Center for Structural Genomics, Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park California Genomics Institute of the Novartis Research Foundation, San Diego, California San Diego Supercomputer Center, La Jolla, California University of California, San Diego, La Jolla, California Scripps Research Institute, La Jolla, California