The primary goals of our Section are to address the structure and function of biomolecular systems with anticancer and antimicrobial significance and to explore the feasibility of drug design targeting such biomolecules. In our efforts to achieve these goals, we have established collaborations within NIH as well as with extramural experts in genetics, molecular biology, protein chemistry, enzymology, carcinogenesis, and medicinal chemistry. These collaborations have greatly extended our range of experiments. Glutathione S-transferase (GST): Structure-based Design of Electrophilic Diazeniumdiolates for Pharmacologic Delivery of Nitric Oxide GST represents a superfamily of detoxification enzymes. Many tumors become drug resistant by overexpressing pi-class GST (GSTP), one of the major GST isoenzymes in humans. We have been attempting to design agents that will overcome the drug resistance of cancer cells that overexpress GSTP. The comparison of the active site architectures and transition-state analogs of GST isozymes revealed a strategy to generate nitric oxide (NO) selectively in the active site of isoenzyme GSTP. Application of this strategy yielded a GSTP-selective NO donor that improves the potency of arsenite, a clinically useful anticancer agent, in cancer cells overexpressing GSTP. 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase (HPPK): Mechanism of Pyrophosphoryl Transfer and Structure-based Design of Novel Antimicrobial Agents Folate cofactors are essential for life. Mammals derive folates from diet; most microorganisms must synthesize folates de novo. HPPK is the first enzyme in the folate biosynthetic pathway. It is not the target for any existing antibiotics and is therefore an ideal target for developing novel antimicrobial agents to fight the worldwide crisis of antibiotic resistance. HPPK contains 158 amino acids and is thermostable, which also makes it an excellent model system for the mechanistic study of pyrophosphoryl transfer. Having elucidated high-resolution (up to 0.89 Angstrom) structures of well-chosen complexes, we have mapped out the trajectory of pyrophosphoryl transfer. This work also reveals unusual conformational changes of HPPK in its catalytic cycle. The structural information is now the basis of inhibitor design effort. We have synthesized a bisubstrate-mimicking inhibitor and a one-substrate analog and determined the crystal structures of HPPK in complex with these inhibitors. G Protein ERA and Ribonuclease III (RNase III): RNA Processing Control ERA and RNase III play key roles in the control of gene expression. ERA is an essential GTPase found in every bacterium sequenced to date. In these bacteria, ERA has a regulatory role in cell cycle control by coupling cell growth rate with cytokinesis. A highly conserved ERA homolog is also found in humans and is a candidate for a tumor suppressor. Our crystal structure of ERA reveals a novel protein architecture that consists of a Ras-like N-terminal domain and a K homology-module-containing C-terminal domain. Together with other observations, the structure indicates that ERA interacts with ribosomal RNA 16S. In the crystal lattice, ERA molecules form loosely associated dimers. Previously, however, no dimer had ever been detected in solution. Guided by our hypothesis of a monomer-dimer conversion mechanism, we demonstrated that dimerization is indeed essential for RNA binding in vivo. RNase III family members are among the few nucleases that show specificity toward double-stranded RNA (dsRNA). Evolutionarily, RNase III is conserved in bacteria, worms, flies, plants, fungi, and mammals. RNase III from bacteria is the simplest, containing an endonuclease domain and a dsRNA-binding domain. Our structure of the catalytic domain of Aquifex aeolicus RNase III reveals a new protein fold and suggests a mechanism for dsRNA cleavage. Every member of the RNase III family contains one or two copies of such endonuclease domain(s). Therefore, the information derived from our structure also sheds light on the structure and function of other RNase III enzymes, such as Dicer. Dicer plays an essential role in RNA interference, a broad class of RNA silencing phenomena found in fungi, plants, and animals.

Agency
National Institute of Health (NIH)
Institute
Division of Basic Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010326-03
Application #
6763532
Study Section
Mammalian Cell Lines Committee (MCL)
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2002
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Dabrazhynetskaya, Alena; Brendler, Therese; Ji, Xinhua et al. (2009) Switching protein-DNA recognition specificity by single-amino-acid substitutions in the P1 par family of plasmid partition elements. J Bacteriol 191:1126-31
Tu, Chao; Tropea, Joseph E; Austin, Brian P et al. (2009) Structural basis for binding of RNA and cofactor by a KsgA methyltransferase. Structure 17:374-85
Gan, Jianhua; Shaw, Gary; Tropea, Joseph E et al. (2008) A stepwise model for double-stranded RNA processing by ribonuclease III. Mol Microbiol 67:143-54
Shaw, Gary; Gan, Jianhua; Zhou, Yan Ning et al. (2008) Structure of RapA, a Swi2/Snf2 protein that recycles RNA polymerase during transcription. Structure 16:1417-27
Ji, Xinhua (2008) The mechanism of RNase III action: how dicer dices. Curr Top Microbiol Immunol 320:99-116
Ji, Xinhua; Pal, Ajai; Kalathur, Ravi et al. (2008) Structure-Based Design of Anticancer Prodrug PABA/NO. Drug Des Devel Ther 2:123-130
Tu, Chao; Tan, Yu Hong; Shaw, Gary et al. (2008) Impact of low-frequency hotspot mutation R282Q on the structure of p53 DNA-binding domain as revealed by crystallography at 1.54 angstroms resolution. Acta Crystallogr D Biol Crystallogr 64:471-7
Blaszczyk, Jaroslaw; Li, Yue; Gan, Jianhua et al. (2007) Structural basis for the aldolase and epimerase activities of Staphylococcus aureus dihydroneopterin aldolase. J Mol Biol 368:161-9
Gan, Jianhua; Wu, Yan; Prabakaran, Ponraj et al. (2007) Structural and biochemical analyses of shikimate dehydrogenase AroE from Aquifex aeolicus: implications for the catalytic mechanism. Biochemistry 46:9513-22
Saavedra, Joseph E; Srinivasan, Aloka; Buzard, Gregory S et al. (2006) PABA/NO as an anticancer lead: analogue synthesis, structure revision, solution chemistry, reactivity toward glutathione, and in vitro activity. J Med Chem 49:1157-64

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