Our basic research has been focused on RNA-processing proteins [RNase III (model system for a family of dsRNA-specific endonucleases exemplified by bacterial RNase III and eukaryotic Rnt1p, Drosha, and Dicer), KsgA (universally conserved methyltransferase that functions as a ribosomal biogenesis factor), and Era (conserved GTPase that couples cell growth with cell division)] and RNA polymerase (RNAP)-associated transcription factors [SspA (stringent starvation protein A), RapA (ATP-dependent dsDNA translocase that recycles RNAP during transcription), and N-utilizing substances A, B, E, and G (NusA, NusB, NusE, and NusG)]. Previously, we made pioneering contribution to the mechanism of RNase III action, significant progress in KsgA-RNA interactions, a breakthrough advance in the structure and functional cycle of Era. We also determined the crystal structure of SspA, RapA, and NusG, and provided structural insight into the phage lambda N protein-mediated transcription antitermination by determining crystal structures of the ternary NusB-NusE-BoxA RNA and NusB-NusE-dsRNA complexes. This year, our most significant discovery is the crystal structure of a plectonemic RNA supercoil. Genome packaging is an essential housekeeping process in virtually all organisms for proper storage and maintenance of genetic information. Although the extent and mechanisms of packaging vary, the process involves the formation of nucleic-acid superstructures. Crystal structures of DNA coiled coils indicate that their geometries can vary according to sequence and/or the presence of stabilizers such as proteins or small molecules. However, such superstructures have not been revealed for RNA. We have determined the crystal structure of an RNA supercoil, which displays one level higher molecular organization than previously reported structures of DNA coiled coils. In the presence of the NusB protein from Aquifex aeolicus, two interlocking RNA coiled coils of double-stranded RNA, a coil of coiled coils, form a plectonemic supercoil. Molecular dynamics simulations suggest that protein-RNA interaction is required for the stability of the supercoiled RNA. The supercoiled RNA in the crystal lattice has a nucleic acid density of 42 bp/100 cubic nm. Intriguingly, the average genome packing density of dsRNA viruses is 40 bp/100 cubic nm. This study provides structural insight into higher-order packaging mechanisms of nucleic acids. Furthermore, the A. aeolicus NusB protein, given its sequence-independent interactions with supercoiled RNA, could potentially be utilized to promote the formation and/or crystallization of other nucleic-acid superstructures, or in the construction of novel nanostructures. Our effort in structure-based drug development has been focused on Glutathione S-transferase (GST)-activated, nitric oxide-releasing, anticancer prodrugs and bisubstrate analog inhibitors of 6-hydroxymethyl-7,8-dihydroptein pyrophosphokinase (HPPK) useful as antibacterial agents. Previously, our structure-based design of prodrugs yielded PABA/NO, which exhibits anticancer activity both in vitro and in vivo with potency similar to that of cisplatin. We also designed, synthesized, and characterized a group of HPPK inhibitors as lead compounds for novel antibiotics, and optimized the synthetic route of HPPK inhibitors, leading to the invention of a novel intermediate and a new method for the synthesis of a known intermediate with a yield of 95%. This year, we have synthesized and characterized another lead inhibitor of HPPK, which exhibits a distinct binding mode to the enzyme and represents a new direction for further development.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Investigator-Initiated Intramural Research Projects (ZIA)
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National Cancer Institute Division of Basic Sciences
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Antony, Marie L; Lee, Joomin; Hahm, Eun-Ryeong et al. (2014) Growth arrest by the antitumor steroidal lactone withaferin A in human breast cancer cells is associated with down-regulation and covalent binding at cysteine 303 of ?-tubulin. J Biol Chem 289:1852-65
Metzger, Meredith B; Liang, Yu-He; Das, Ranabir et al. (2013) A structurally unique E2-binding domain activates ubiquitination by the ERAD E2, Ubc7p, through multiple mechanisms. Mol Cell 50:516-27
Shi, Genbin; Ji, Xinhua (2011) New ways to derivatize at position 6 of 7,7-dimethyl-7,8-dihydropterin. Tetrahedron Lett 52:6174-6176
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
Das, Ranabir; Mariano, Jennifer; Tsai, Yien Che et al. (2009) Allosteric activation of E2-RING finger-mediated ubiquitylation by a structurally defined specific E2-binding region of gp78. Mol Cell 34:674-85
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
Prabakaran, Ponraj; Vu, Bang K; Gan, Jianhua et al. (2008) Structure of an isolated unglycosylated antibody C(H)2 domain. Acta Crystallogr D Biol Crystallogr 64:1062-7