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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010326-14
Application #
8763074
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
14
Fiscal Year
2013
Total Cost
$1,463,239
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Li, Shengjian; Liang, Yu-He; Mariano, Jennifer et al. (2015) Insights into Ubiquitination from the Unique Clamp-like Binding of the RING E3 AO7 to the E2 UbcH5B. J Biol Chem 290:30225-39
Kakar, Smita; Fang, Xianyang; Lubkowska, Lucyna et al. (2015) Allosteric Activation of Bacterial Swi2/Snf2 (Switch/Sucrose Non-fermentable) Protein RapA by RNA Polymerase: BIOCHEMICAL AND STRUCTURAL STUDIES. J Biol Chem 290:23656-69
Blaszczyk, Jaroslaw; Lu, Zhenwei; Li, Yue et al. (2014) Crystallographic and molecular dynamics simulation analysis of Escherichia coli dihydroneopterin aldolase. Cell Biosci 4:52
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 β-tubulin. J Biol Chem 289:1852-65
Maciag, Anna E; Holland, Ryan J; Kim, Youseung et al. (2014) Nitric oxide (NO) releasing poly ADP-ribose polymerase 1 (PARP-1) inhibitors targeted to glutathione S-transferase P1-overexpressing cancer cells. J Med Chem 57:2292-302
Court, Donald L; Gan, Jianhua; Liang, Yu-He et al. (2013) RNase III: Genetics and function; structure and mechanism. Annu Rev Genet 47:405-31
Das, Ranabir; Liang, Yu-He; Mariano, Jennifer et al. (2013) Allosteric regulation of E2:E3 interactions promote a processive ubiquitination machine. EMBO J 32:2504-16
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; Shaw, Gary; Li, Yue et al. (2012) Bisubstrate analog inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: new lead exhibits a distinct binding mode. Bioorg Med Chem 20:4303-9
Zhao, Jianfei; Stagno, Jason R; Varticovski, Lyuba et al. (2012) P6981, an arylstibonic acid, is a novel low nanomolar inhibitor of cAMP response element-binding protein binding to DNA. Mol Pharmacol 82:814-23

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