Eileen Ambing

Crystal structure of the ALK (anaplastic lymphoma kinase) catalytic domain.

ALK (anaplastic lymphoma kinase) is an RTK (receptor tyrosine kinase) of the IRK (insulin receptor kinase) superfamily, which share an YXXXYY autophosphorylation motif within their A-loops (activation loops). A common activation and regulatory mechanism is believed to exist for members of this superfamily typified by IRK and IGF1RK (insulin-like growth factor receptor kinase-1). Chromosomal translocations involving ALK were first identified in anaplastic large-cell lymphoma, a subtype of non-Hodgkins lymphoma, where aberrant fusion of the ALK kinase domain with the NPM (nucleophosmin) dimerization domain results in autophosphosphorylation and ligand-independent activation. Activating mutations within the full-length ALK kinase domain, most commonly R1275Q and F1174L, which play a major role in neuroblastoma, were recently identified. To provide a structural framework for understanding these mutations and to guide structure-assisted drug discovery efforts, the X-ray crystal structure of the unphosphorylated ALK catalytic domain was determined in the apo, ADP- and staurosporine-bound forms. The structures reveal a partially inactive protein kinase conformation distinct from, and lacking, many of the negative regulatory features observed in inactive IGF1RK/IRK structures in their unphosphorylated forms. The A-loop adopts an inhibitory pose where a short proximal A-loop helix (alphaAL) packs against the alphaC helix and a novel N-terminal beta-turn motif, whereas the distal portion obstructs part of the predicted peptide-binding region. The structure helps explain the reported unique peptide substrate specificity and the importance of phosphorylation of the first A-loop Tyr1278 for kinase activity and NPM-ALK transforming potential. A single amino acid difference in the ALK substrate peptide binding P-1 site (where the P-site is the phosphoacceptor site) was identified that, in conjunction with A-loop sequence variation including the RAS (Arg-Ala-Ser)-motif, rationalizes the difference in the A-loop tyrosine autophosphorylation preference between ALK and IGF1RK/IRK. Enzymatic analysis of recombinant R1275Q and F1174L ALK mutant catalytic domains confirms the enhanced activity and transforming potential of these mutants. The transforming ability of the full-length ALK mutants in soft agar colony growth assays corroborates these findings. The availability of a three-dimensional structure for ALK will facilitate future structure-function and rational drug design efforts targeting this receptor tyrosine kinase.

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.

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.

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

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

Crystal structure of a PIN (PilT N‐terminus) domain (AF0591) from Archaeoglobus fulgidus at 1.90 Å resolution

Inna Levin, Robert Schwarzenbacher, Rebecca Page, Polat Abdubek, Eileen Ambing, Tanya Biorac, Linda S. Brinen, Jamison Campbell, Jaume M. Canaves, Hsiu-Ju Chiu, Xiaoping Dai, Ashley M. Deacon, Mike DiDonato, Marc-André Elsliger, 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, Daniel McMullan, Timothy M. McPhillips, Mitchell D. Miller, Andrew Morse, Kin Moy, Jie Ouyang, Kevin Quijano, Ron Reyes, Fred Rezezadeh, Alyssa Robb, Eric Sims, Glen Spraggon, Raymond C. Stevens, Henry van den Bedem, Jeff Velasquez, Juli Vincent, Frank von Delft, 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

Crystal structure of acireductone dioxygenase (ARD) from Mus musculus at 2.06 Å resolution

Qingping Xu, Robert Schwarzenbacher, S. Sri Krishna, Daniel McMullan, Sanjay Agarwalla, Kevin Quijano, Polat Abdubek, Eileen Ambing, Herbert Axelrod, Tanya Biorac, Jaume M. Canaves, Hsiu-Ju Chiu, Marc-André Elsliger, Carina Grittini, Slawomir K. Grzechnik, Michael DiDonato, Joanna Hale, Eric Hampton, Gye Won Han, Justin Haugen, MichaelHornsby, Lukasz Jaroszewski, Heath E. Klock, Mark W. Knuth, Eric Koesema, Andreas Kreusch, Peter Kuhn, Mitchell D. Miller, Kin Moy, Edward Nigoghossian, Jessica Paulsen, Ron Reyes, Chris Rife, Glen Spraggon, Raymond C. Stevens, Henry van den Bedem, Jeff Velasquez, Aprilfawn White, Guenter Wolf, Keith O. Hodgson, John Wooley, Ashley M. Deacon, Adam Godzik, Scott A. Lesley, 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

Crystal structure of an Udp-n-acetylmuramate-alanine ligase MurC (TM0231) from Thermotoga maritima at 2.3 Å resolution

Crystal Structure of an Udp-n-acetylmuramate-alanine Ligase MurC (TM0231) from Thermotoga maritima at 2.3 Å Resolution Glen Spraggon, Robert Schwarzenbacher, Andreas Kreusch, Christian C. Lee, 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, Peter Kuhn, Inna Levin, Daniel McMullan, Timothy M. McPhillips, Mitchell D. Miller, Andrew Morse, Kin Moy, Jie Ouyang, Rebecca Page, Kevin Quijano, Alyssa Robb, Raymond C. Stevens, Henry van den Bedem, Jeff Velasquez, Juli Vincent, Frank von Delft, Xianhong Wang, Bill West, Guenter Wolf, Qingping Xu, Keith O. Hodgson, John Wooley, Scott A. Lesley, and Ian A. Wilson* The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, California 92121 The San Diego Supercomputer Center, La Jolla, California 92093 Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California The Scripps Research Institute, La Jolla, California 92037 The University of California, San Diego, La Jolla, California 92093

Crystal structure of a putative PII-like signaling protein (TM0021) from Thermotoga maritima at 2.5 A resolution

Robert Schwarzenbacher, Frank von Delft, Polat Abdubek, Eileen Ambing, Tanja Biorac, Linda S. Brinen, Jaume M. Canaves, Jamison Cambell, Hsui-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, Mitchell D. Miller, 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

Structure of a tryptophanyl-tRNA synthetase containing an iron–sulfur cluster

The crystal structure of tryptophanyl-tRNA synthetase from T. maritima unexpectedly revealed an iron–sulfur cluster bound to the tRNA anticodon-binding region.