Proceedings of the Thirteenth International Society of Sports Nutrition (ISSN) Conference and Expo

Meeting Abstracts: Proceedings of the Thirteenth International Society of Sports Nutrition (ISSN) Conference and Expo Clearwater Beach, FL, USA. 9-11 June 201

Structure-Function Studies of the Large Subunit of Ribonucleotide Reductase from Homo sapiens and Saccharomyces cerevisiae

Sufficient pools of deoxyribonucleotide triphophates (dNTPs) are essential for the high fidelity replication and repair of DNA, the hereditary material for a majority of living organisms. Ribonucleotide reductase (Rnr) catalyzes the rate-limiting step of de-novo DNA synthesis, the reduction of ribonucleosides to deoxyribonucleosides. Since the cell relies primarily upon ribonucleotide reductase for its dNTPs, both the cellular levels and activity of Rnr are heavily regulated, especially when DNA damage occurs or during replication blocks in the cell cycle. If dNTP pools become too high, too low, or imbalanced, genomic instability results, leading to either the formation of cancerous cells or cell death. High levels of dNTPs are required by actively propagating cells for the replication of new DNA molecules. Therefore, Rnr makes an excellent target for anti-cancer, anti-microbial, and anti-fungal chemotherapeutic agents. Deficiencies in the cellular mismatch repair (MMR) machinery have been linked to genetic instability and carcinogenesis. Two alleles of Rnr1 were recently discovered, Rnr1S269P and Rnr1S610F, which have a mismatch repair synthetic lethal (msl) phenotype in Saccharomyces cerevisiae cells with missing or defective MMR genes. To uncover the molecular mechanism of the msl phenotype in these two mutants, recombinant Rnr1p-S269P and Rnr1p-S610F were subjected to in vitro activity assays, X-ray crystallography, and in vitro nucleoside-binding assays (Chapter 3). The Rnr1S269P allele was shown to dysregulate specificity cross talk by X-ray crystallography experiments, leading to reduced levels of dATP in the cell. A 2-fold reduction in binding of ADP substrate was observed in the Rnr1S610F allele, however reduction of the kcat is believed to cause the observed msl phenotype in this mutant. The first X-ray crystal structures of the large subunit of ribonucleotide reductase from Homo sapiens (hRRM1) are also presented here (Chapter 4). The hRRM1●TTP and hRRM1●TTP●GDP structures describe the binding of effector and substrate to the specificity and catalytic sites. In addition, the two structures hRRM1●TTP●ATP and hRRM1●TTP●dATP are the first X-ray crystal structures of Rnr from any species with the allosteric activity site occupied with the natural ligands ATP and dATP. Size exclusion chromatography data and a low resolution X-ray crystal structure of hexameric S. cerevisiae Rnr provide a model for dATP-dependent oligomerization

KDOP synthase from <i>B. pseudomallei</i>.

<p>KDOP synthase (2-dehydro-3-deoxyphosphooctonate aldolase, BURPS1710b_3264, PDB: 3UND with bound D-arabinose-5-phosphate), a KDO2-lipid A biosynthesis enzyme with a TIM barrel structure, was one of five structures solved for orthologs of the putative essential <i>B. thailandensis</i> gene, Bth_I1893.</p

Isochorismatase from <i>B. thailandensis</i>.

<p>The isochorismatase family protein (BTH_II2229, PDB: 3TXY) from <i>B. thailandensis</i>, is shown in electrostatics surface representation with bound isochorismate taken from the <i>P. aeruginosa</i> ischorismatase, PhzD (PDB: 1NF8). 3TXY and 1NF8 have 30% sequence identity and an overall Cα RMSD of 1.7 Å. By aligning 1NF8 and 3TXY, the active site of 3TXY can be identified as a large pocket with a combination of hydrophobic (white) and positively charged (blue) amino acid residues.</p

FabH structures from <i>B. pseudomallei</i> and B. xenovorans.

<p>(A) FabH (3-oxoacyl-(acyl-carrier-protein) synthase III) from <i>B. pseudomallei 1710b</i> (BURPS1710b_0096, PDB: 3GWA, cyan) and <i>B. xenovorans LB400 B</i> (Bxe_A1072, PDB: 4DFE, magenta) have similar overall structures, with a Cα RMSD of 1.8 Å between individual chains of 3GWA and 4DFE. There is no close human homolog based on a BlastP search of the human proteome. (B) In 4DFE, a hydrophobic tunnel to the active site is adjacent to a positively-charged surface patch (marked in blue).</p

Thymidylate synthase (TS) from <i>B. thailandensis</i>, <i>E. coli</i> and <i>Homo sapiens</i>.

<p>TS from human (cyan, PDB: 1SYN) and <i>E. coli</i> (magenta, PDB: 1JU6) show similar active site structure as TS from <i>B. thailandensis</i> (green, PDB: 3V8H, C-terminal residues removed for clarity). A canonical active site tryptophan (W83 in <i>E. coli</i>) for bacterial sequences is replaced in <i>B. thailandensis</i> by asparagine, the residue observed in this position in human TS (side chains shown in stick representation, below and to the right of the bound ligand, citric acid).</p

Peptidyl-tRNA hydrolase from <i>B. thailandensis</i>.

<p>(A) The electrostatic surface of unliganded peptidyl-tRNA hydrolase (PTH, Bth_I0472, PDB: 3V2I) from <i>B. thailandensis</i> is superimposed with a cartoon representation of a structure from <i>P. aeruginosa</i> with bound adipic acid (PDB: 4DHW). The channel in unliganded 3V2I is closed due to adjacent flexible loops. (B) The electrostatics surface of 4DHW reveals an open, charged channel. 3V2I and 4DHW have 44% sequence identity and a similar overall fold (2.0 Å RMSD over all common Cα atoms). Discovery of a ligand that binds the alternately charged channel (positive/negative/positive) could block the reaction and prevent protein synthesis.</p

<i>Burkholderia</i> protein structures.

<p>An expanded version of this table is available in the Supporting Information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053851#pone.0053851.s002" target="_blank">Table S2</a>).</p><p>Structures described in detail in the manuscript are indicated in bold.</p>*<p>Best hit (if any) in a BlastP search against the human proteome, using an <i>E</i>-value cutoff of 1×10⁻¹⁰ (UniProtKB AC).</p>#<p>BURPS1710b_2511 was screened using a fragment-based approach, yielding 17 PDB structures and 16 unique ligand-bound complexes: 3F0D, 3F0E, 3F0F, 3F0G, 3IEQ, 3IEW, 3MBM, 3P0Z, 3P10, 3Q8H, 3QHD, 3IKE, 3IKF, 3JVH, 3K14, 3K2X, 3KE1.</p

Identification of essential genes using saturation transposon mutagenesis and Tn-seq.

<p>Identification of essential genes using saturation transposon mutagenesis and Tn-seq.</p