Xinhua  Ji, Ph.D.
Xinhua Ji, Ph.D.
Senior Investigator
Head, Biomolecular Structure Section

Center for Cancer Research
National Cancer Institute

Building 538, Room 103
Frederick, MD 21702-1201
301-846-5035

Dr. Ji pioneered the structural analysis of double-stranded RNA (dsRNA) in complex with RNase III enzymes. Over the past decade, he has provided the structural view of dsRNA processing by bacterial RNase III. Recently, his structure of a eukaryotic RNase III has revealed a picking motif in structured RNA and a double-ruler mechanism for substrate selection.

As Head, Dr. Ji directs the Section’s basic and translational research program that is focused on the structural biology of RNA biogenesis, especially post-transcriptional RNA processing, and structure-based development of anticancer and antimicrobial agents.

Areas of Expertise
1) X-ray crystallography, 2) structural biology, 3) chemical biology, 4) structure-based drug design, 5) RNA biogenesis, 6) RNase III enzymes

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. The goal for structural analysis is to map the reaction trajectory or functional cycle of selected biological macromolecules, and that for drug development is to design, synthesize, and characterize novel anticancer and antimicrobial agents. See Research Gallery and Drug Discovery Patents for the scope and depth of our science. Examples of  biomolecular systems under investigation are described below.

RNase III (ribonuclease III) enzymes, exemplified by bacterial RNase III and eukaryotic Rnt1p, Drosha, and Dicer, play important  roles in RNA processing and maturation, post-transcriptional gene silencing, and defense against viral infection. For mechanistic  studies, the bacterial enzyme is a valuable model system for the  entire family. We have shown how the dimerization of the  endonuclease domain of RNase III creates a catalytic valley where  two cleavage 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 cleavage sites, how the  hydrolysis of each strand involves both subunits, and how RNase  III uses the two cleavage 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 provided a stepwise mechanism for the enzyme to execute  the phosphoryl transfer reaction (View). As informative as the bacterial enzyme for the mechanism of RNase III action, yeast  Rnt1p is a valuable model system for eukaryotic RNase III enzymes. Unlike bacterial enzyme that uses four catalytic side  chains, eukaryotic RNase III uses six. Also distinguished from the bacterial enzyme, every eukaryotic RNase III has an N-terminal extension. What is more, Rnt1p exhibits a strict guanine nucleotide specificity, which is unique among RNase III enzymes. We have shown how the substrate-binding mode of Rnt1p is  distinct from that of bacterial RNase III (View), how all of the six  catalytic side chains are engaged in the cleavage site (View), how a new RNA-binding  motif of Rnt1p functions as a guanine-specific clamp (View), and how the double-stranded RNA-binding domain and N-terminal domain of Rnt1p  function as two rulers measuring the distance between the guanine  nucleotide to the cleavage sites (View). This unusual mechanism of substrate selectivity represents an example of the evolution  of substrate selectivity and provides a framework for understanding the mechanism of action of other eukaryotic RNase  III enzymes, including Drosha, and Dicer.

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 product molecules. HPPK is a key enzyme in the folate biosynthetic pathway. It is essential for microorganisms but absent from mammals. Unlike dihydropteroate  synthase (another key enzyme in the folate pathway), which is the target of sulfonamides (the first widely used synthetic antibiotics), HPPK is not the target for any existing antibiotics. Therefore, HPPK is an attractive target for developing novel antimicrobial agents. We have constructed a reaction coordinate on the basis of thermodynamic and transient kinetic data and mapped out the reaction trajectory by determining 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 (View).

Scientific Focus Areas:
Cancer Biology, Chemical Biology, Microbiology and Infectious Diseases, Molecular Biology and Biochemistry, Structural Biology
Selected Key Publications
  1. Liang Y, Lavoie M, Comeau M, Abou Elela S, Ji X.
    Mol. Cell. 54: 431-444, 2014. [ Journal Article ]
  2. Stagno JR, Ma B, Li J, Altieri AS, Byrd RA, Ji X.
    Nat Commun. 3: 901, 2012. [ Journal Article ]
  3. Tu C, Zhou X, Tarasov SG, Tropea JE, Austin BP, Waugh DS, Court DL, Ji X.
    Proc. Natl. Acad. Sci. USA. 108: 10156-10161, 2011. [ Journal Article ]
  4. Shaw G, Gan J, Zhou YN, Zhi H, Subburaman P, Zhang R, Joachimiak A, Jin DJ, Ji X.
    Structure. 16: 1417-1427, 2008. [ Journal Article ]
  5. Gan J, Tropea JE, Austin BP, Court DL, Waugh DS, Ji X.
    Cell. 124: 355-66, 2006. [ Journal Article ]

Dr. Ji earned his Ph.D. degree at the University of Oklahoma (1985-1990) and performed his postdoctoral research at the University of Maryland (1991-1994), where he became a Research Assistant Professor (1994-1995) before joining the National Cancer Institute (NCI), National Institutes of Health (NIH). At the NCI at Frederick, Dr. Ji established his laboratory in the ABL-Basic Research Program in 1995, moved to the Center for Cancer Research in 1999, gained tenure in 2001 as an NIH Senior Investigator, and in 2008 became a member of the Senior Biomedical Research Service (SBRS). The SBRS, established under the Public Health Service Act, was created for scientists who are considered by their peers to be outstanding in their work.

Name Position
Lan Jin Ph.D. Research Fellow
Xing Jing Ph.D. Postdoctoral Fellow (Visiting)
Smita Kakar Ph.D. Postdoctoral Fellow (Visiting)
Vandana Kumari Ph.D. Postdoctoral Fellow (Visiting)
Gary Shaw Ph.D. Research Biologist
Genbin Shi Ph.D. Staff Scientist
He Song Ph.D. Postdoctoral Fellow (Visiting)
Chao Wang M.Eng. Special Volunteer

Summary

Dr. Ji pioneered the structural analysis of double-stranded RNA (dsRNA) in complex with RNase III enzymes. Over the past decade, he has provided the structural view of dsRNA processing by bacterial RNase III. Recently, his structure of a eukaryotic RNase III has revealed a picking motif in structured RNA and a double-ruler mechanism for substrate selection.

As Head, Dr. Ji directs the Section’s basic and translational research program that is focused on the structural biology of RNA biogenesis, especially post-transcriptional RNA processing, and structure-based development of anticancer and antimicrobial agents.

Areas of Expertise
1) X-ray crystallography, 2) structural biology, 3) chemical biology, 4) structure-based drug design, 5) RNA biogenesis, 6) RNase III enzymes

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. The goal for structural analysis is to map the reaction trajectory or functional cycle of selected biological macromolecules, and that for drug development is to design, synthesize, and characterize novel anticancer and antimicrobial agents. See Research Gallery and Drug Discovery Patents for the scope and depth of our science. Examples of  biomolecular systems under investigation are described below.

RNase III (ribonuclease III) enzymes, exemplified by bacterial RNase III and eukaryotic Rnt1p, Drosha, and Dicer, play important  roles in RNA processing and maturation, post-transcriptional gene silencing, and defense against viral infection. For mechanistic  studies, the bacterial enzyme is a valuable model system for the  entire family. We have shown how the dimerization of the  endonuclease domain of RNase III creates a catalytic valley where  two cleavage 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 cleavage sites, how the  hydrolysis of each strand involves both subunits, and how RNase  III uses the two cleavage 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 provided a stepwise mechanism for the enzyme to execute  the phosphoryl transfer reaction (View). As informative as the bacterial enzyme for the mechanism of RNase III action, yeast  Rnt1p is a valuable model system for eukaryotic RNase III enzymes. Unlike bacterial enzyme that uses four catalytic side  chains, eukaryotic RNase III uses six. Also distinguished from the bacterial enzyme, every eukaryotic RNase III has an N-terminal extension. What is more, Rnt1p exhibits a strict guanine nucleotide specificity, which is unique among RNase III enzymes. We have shown how the substrate-binding mode of Rnt1p is  distinct from that of bacterial RNase III (View), how all of the six  catalytic side chains are engaged in the cleavage site (View), how a new RNA-binding  motif of Rnt1p functions as a guanine-specific clamp (View), and how the double-stranded RNA-binding domain and N-terminal domain of Rnt1p  function as two rulers measuring the distance between the guanine  nucleotide to the cleavage sites (View). This unusual mechanism of substrate selectivity represents an example of the evolution  of substrate selectivity and provides a framework for understanding the mechanism of action of other eukaryotic RNase  III enzymes, including Drosha, and Dicer.

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 product molecules. HPPK is a key enzyme in the folate biosynthetic pathway. It is essential for microorganisms but absent from mammals. Unlike dihydropteroate  synthase (another key enzyme in the folate pathway), which is the target of sulfonamides (the first widely used synthetic antibiotics), HPPK is not the target for any existing antibiotics. Therefore, HPPK is an attractive target for developing novel antimicrobial agents. We have constructed a reaction coordinate on the basis of thermodynamic and transient kinetic data and mapped out the reaction trajectory by determining 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 (View).

Scientific Focus Areas:
Cancer Biology, Chemical Biology, Microbiology and Infectious Diseases, Molecular Biology and Biochemistry, Structural Biology

Publications

Selected Key Publications
  1. Liang Y, Lavoie M, Comeau M, Abou Elela S, Ji X.
    Mol. Cell. 54: 431-444, 2014. [ Journal Article ]
  2. Stagno JR, Ma B, Li J, Altieri AS, Byrd RA, Ji X.
    Nat Commun. 3: 901, 2012. [ Journal Article ]
  3. Tu C, Zhou X, Tarasov SG, Tropea JE, Austin BP, Waugh DS, Court DL, Ji X.
    Proc. Natl. Acad. Sci. USA. 108: 10156-10161, 2011. [ Journal Article ]
  4. Shaw G, Gan J, Zhou YN, Zhi H, Subburaman P, Zhang R, Joachimiak A, Jin DJ, Ji X.
    Structure. 16: 1417-1427, 2008. [ Journal Article ]
  5. Gan J, Tropea JE, Austin BP, Court DL, Waugh DS, Ji X.
    Cell. 124: 355-66, 2006. [ Journal Article ]

Biography

Dr. Ji earned his Ph.D. degree at the University of Oklahoma (1985-1990) and performed his postdoctoral research at the University of Maryland (1991-1994), where he became a Research Assistant Professor (1994-1995) before joining the National Cancer Institute (NCI), National Institutes of Health (NIH). At the NCI at Frederick, Dr. Ji established his laboratory in the ABL-Basic Research Program in 1995, moved to the Center for Cancer Research in 1999, gained tenure in 2001 as an NIH Senior Investigator, and in 2008 became a member of the Senior Biomedical Research Service (SBRS). The SBRS, established under the Public Health Service Act, was created for scientists who are considered by their peers to be outstanding in their work.

Team

Name Position
Lan Jin Ph.D. Research Fellow
Xing Jing Ph.D. Postdoctoral Fellow (Visiting)
Smita Kakar Ph.D. Postdoctoral Fellow (Visiting)
Vandana Kumari Ph.D. Postdoctoral Fellow (Visiting)
Gary Shaw Ph.D. Research Biologist
Genbin Shi Ph.D. Staff Scientist
He Song Ph.D. Postdoctoral Fellow (Visiting)
Chao Wang M.Eng. Special Volunteer