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Xinhua Ji, Ph.D.

Portait Photo of Xinhua Ji
Macromolecular Crystallography Laboratory
Head, Biomolecular Structure Section
Senior Investigator
Center for Cancer Research
National Cancer Institute
1050 Boyles Street
Building 538, Room 103
Frederick, MD 21702-1201
Phone:  
301-846-5035
Fax:  
301-846-6073
E-Mail:  
jix@mail.nih.gov

Biography

Dr. Ji earned his Ph.D. degree at the University of Oklahoma (1985-1990) and performed postdoctoral research at the University of Maryland (1991-1995), where he became a Research Assistant Professor before joining the National Cancer Institute, National Institutes of Health (NIH). As a group leader, Dr. Ji established his laboratory in the ABL-Basic Research Program in 1995, moved to the Center for Cancer Research as a Section Chief in 1999, and in 2001 gained tenure as an NIH Senior Investigator.

Research

Biomolecular Structure and Mechanism, Structure-Based Drug Design

Our research is focused on the structural biology of RNA biogenesis, with an emphasis on RNA-processing proteins and RNA polymerase-associated transcription factors, and structure-based development of therapeutic agents. Our goal for structural analysis is to map the reaction trajectory or functional cycle of selected biological macromolecules (RNase III, Era, RapA, and Nus), and that for drug development is to design, synthesize, and characterize novel anticancer and antimicrobial agents (PABA/NO and HPPK).

RNase III proteins, including Dicer, produce small interfering RNAs that mediate RNA interference. For mechanistic studies, bacterial enzyme has been a valuable model system for the family. We have shown how the dimerization of the endonuclease domain of RNase III creates a catalytic valley where two catalytic sites are located, how the catalytic valley accommodates a dsRNA in a manner such that each of the two RNA strands is aligned with one of the two catalytic sites, how the hydrolysis of each strand involves both subunits, and how RNase III uses the two catalytic sites to create the 2-nucleotide 3' overhangs in its products (View). We have also shown how magnesium is essential for the formation of a catalytically competent protein-RNA complex, how the use of two magnesium ions can drive the hydrolysis of each phosphodiester bond, and how conformational changes in both the substrate and the protein are critical elements for assembling the catalytic complex. Moreover, we have modeled a protein-substrate complex and a protein-reaction intermediate (transition state) complex in a meaningful way. Together, the models and structures suggest a stepwise mechanism for the enzyme to execute the phosphoryl transfer reaction (View).

Era, essential for bacterial cell viability, is composed of a GTPase domain and an RNA-binding KH domain (View). Present in nearly every bacterial species and essential for both cell growth and division, Era is unique among all other known protein functions of bacteria. We have characterized the functional cycle of Era (View), providing structural basis for its essential roles in the maturation of 16S rRNA and assembly of the 30S ribosomal subunit (View). We have shown that Era recognizes 10 nucleotides (nt 1530-1539, GAUCACCUCC) near the 3' end of 16S rRNA, and that this recognition stimulates the GTP-hydrolyzing activity of the protein. The GAUCA sequence and the upstream helix 45 (h45, nt 1506-1529) are highly conserved in all three kingdoms of life. We have shown that Era also binds h45. Among the 10 nucleotides, however, G1530 does not stimulate Era's GTPase activity. Rather, A1531 and A1534 are most important for stimulation and h45 further contributes to the stimulation. Although G1530 does not contribute to the GTPase activity, its interaction with Era is essential for the protein to function, leading to the discovery of a cold-sensitive phenotype of the protein.

RapA, as abundant as sigma 70 in the cell, is an RNA polymerase (RNAP)-associated Swi2/Snf2 protein with ATPase activity (View). It stimulates RNAP recycling during transcription. We have determined the structure of RapA, which is the first full-length structure for the entire Swi2/Snf2 family. RapA contains seven domains, two of which exhibit novel protein folds. Our model of RapA in complex with ATP and dsDNA suggests that RapA may bind to and translocate on dsDNA (View). Kinetic template-switching assay shows that RapA facilitates the release of sequestered RNAP from a posttranscrption/posttermination complex for transcription reinitiation. In vitro competition experiment indicates that RapA binds to core RNAP but is readily displaceable by sigma 70. RapA is likely another general transcription factor, the structure of which provides a framework for future studies of this bacterial Swi2/Snf2 protein and its important roles in RNAP recycling during transcription.

Nus (N-utilizing substances) A, B, E, and G (NusA, NusB, NusE, and NusG) play essential roles in the regulation of gene expression. Processive transcription antitermination requires the assembly of a complete antitermination complex, which is initiated by the formation of the ternary NusB-NusE-BoxA RNA complex. We have elucidated the crystal structure of this complex, demonstrating that BoxA is composed of eight nucleotides that are recognized by the NusB-NusE heterodimer (View). Functional data support the structural observations and establish the relative significance of key protein-protein and protein-RNA interactions. Further crystallographic investigation of a NusB-NusE-dsRNA complex reveals a heretofore unobserved dsRNA-binding site (View), which is contiguous with BoxA-binding site (View). We propose that the observed dsRNA represents the BoxB RNA, as both single-stranded BoxA and double-stranded BoxB components are present in the classical lambda antitermination site. Combining these data with known interactions amongst antitermination factors suggests a specific model for the assembly of the complete antitermination complex (View).

PABA/NO represents a new family of anticancer prodrugs that kill cancer cells from within by releasing cytotoxic levels of nitric oxide. It is activated by glutathione S-transferase (GST), a superfamily of detoxification enzymes, represented by GST-alpha and GST-pi. GST-alpha is the predominant isoform of GST in human liver, playing important roles for our well being, whereas GST-pi is overexpressed in many forms of cancer. Our structure-based design yielded PABA/NO, which exhibited anticancer activity both in vitro and in vivo with a potency similar to that of cisplatin. The design was based on GST structures at both ground state and transition state. The ground-state structures outlined the shape and property of the substrate-binding site in different isozymes, and the structural information at the transition-state provided guidance for structural modifications of prodrug molecules. Two key alterations of a GST-alpha-selective compound led to the GST-pi-selective PABA/NO (View).

HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase) catalyzes magnesium-dependent pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). The reaction follows a bi-bi mechanism with ATP as the first substrate and AMP and HP pyrophosphate (HPPP) as the two products. HPPK is a key enzyme in the folate biosynthetic pathway and is essential for microorganisms but absent from mammals. We have constructed a reaction coordinate on the basis of the thermodynamic and transient kinetic data and mapped out the reaction trajectory with five crystal structures of the enzyme at various liganded states (View). On the basis of structural and mechanistic information, we have been attempting the development of HPPK inhibitors as novel antimicrobial agents.

Our collaborators include Sherif Abou Elela, University of Sherbrooke (Quebec, Canada); Andrew Byrd, NCI; Donald Court, NCI; Dimiter Dimitrov, NCI; Ding Jin, NCI; Larry Keefer, NCI; Shivendra Singh, University of Pittsburgh; Joseph Tropea, NCI; David Waugh, NCI; Allan Weissman, NCI; and Honggao Yan, Michigan State University.

This page was last updated on 6/3/2014.