In my laboratory, nuclear magnetic resonance (NMR) spectroscopy is used to elucidate structures and mechanisms of biological macromolecules.
Structural Studies of Human APOBEC3G and HIV Vif
Human APOBEC3G is capable of altering the HIV genome by deaminating single stranded DNA cytosines to uracils. This activity can genetically inactivate HIV. As a counter-defense mechanism, HIV promotes protein degradation of APOBEC3G. Degradation requires that the HIV virion infectivity factor (Vif) protein interacts with APOBEC3G. Current studies have revealed substantial genetic and biochemical details of this host-pathogen conflict, but an atomic level understanding is still lacking. Therefore, the major objectives of this research are to obtain a structural understanding of the DNA cytosine deaminase activity of APOBEC3G and of the APOBEC3G -Vif interaction. These primary objectives will be met by combining genetic, biochemical, biophysical and structural (NMR) approaches. These studies will produce the first structure of a polynucleotide cytosine deaminase, and the resulting structural insights will take us several steps closer to achieve our long-term goal of developing therapeutic methods for enhancing APOBEC3G function.
RNA in Viral DNA packaging
During replication, viruses and bacteriophage package their lengthy genomic DNA into a protein shell. This geometrically challenging process is performed rapidly via an ATP-dependent motor. The Bacillus subtilis bacteriophage 29 has become the model system for studying this DNA packaging mechanism. Essential to the 29 DNA packaging motor is the prohead RNA (pRNA), which forms a multimer that attaches to the bottom of the 29 precursor protein shell (prohead). The ATPase gp16 protein binds to the attached pRNA multimer to form a functional motor, which can pass the 19-kilobase genomic DNA of 29 into the prohead. Although the essential components of the motor have been identified, the mechanisms by which genomic DNA passes through the motor have not yet been elucidated. The long-term goal of this research is to reveal the molecular architecture of the 29 motor. Our objective, which is the next step towards achieving this goal, is to reveal the structure of pRNA.
Mechanisms of Atypical Drug Kinetics and Interactions
The goals of this research are to understand the biochemical mechanisms resulting in atypical kinetic profiles and develop computer models capable of predicting the occurrence of atypical kinetics. The objectives of this project are to explore the active site interactions of substrates (drugs) with a common drug metabolizing enzyme, cytochrome P450 2C9, establish the relevant key amino acid residues with which the substrates interact and assess changes in substrate binding in the presence of concurrently binding effector molecules. These interactions will then be modeled using computer software programs to predict substrate-amino acid interactions for drugs metabolized by cytochrome P450 2C9.
Structural studies of human DEK protein
The goal of this research is to provide structural information of human DEK protein. The highly abundant 43 kDa nuclear protein DEK has been associated with acute myeloid leukemia (AML), ataxia-telangiectasia (A-T), as well as brain, liver and skin cancers. Recent studies have shown that DEK can alter the topology of chromosome, and this could be the key function that links DEK to various cancers. Our objectives are to determine the structure of this DNA-supercoiling domain of DEK, and identify amino acids that are important for DNA-supercoiling.
Harjes S, Solomon C. W, Li M, Chen K-M, Harjes E, Harris S. R and Matsuo H (2013) “Impact of H216 on the DNA binding and catalytic activities of the HIV restriction factor APOBEC3G”. Jun; 87(12):7008-14. doi: 10.1128/JVI.03173-12. Epub 2013 Apr 17. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3676121/
Chiba J, Kouno T, Aoki S, Sato H, Zhang J, Matsuo H and Inouye M. (2012) “Electrochemical direct detection of DNA deamination catalyzed by APOBEC3G” Chem. Commun., 48, 12115–12117, DOI:10.1039/c2cc36779c. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3541039/
Fukunishi Y, Hongo S, XLintuluoto M and Matsuo H. (2012) “Free energy profile of APOBEC3G protein calculated by a molecular dynamics simulation” Biology 1(2), 245-259; PMC4009775 http://www.ncbi.nlm.nih.gov/pubmed/?term=PMC4009775
Harjes E, Kitamura A, Zhao W, Morais M, Jardine P.J, Grimes S and Matsuo H. (2012) “Structure of the RNA claw of the DNA packaging motor of bacteriophage f29” Nucleic Acids Research, Oct 1; 40(19):9953-63. doi: 10.1093/nar/gks724. Epub 2012 Aug 8. http://www.ncbi.nlm.nih.gov/pubmed/22879380
Kuan-Ming Chen, Elena Harjes, Phillip J. Gross , Amr Fahmy, Yongjian Lu, Keisuke Shindo, *Reuben S. Harris and *Hiroshi Matsuo, "Structure of the DNA deaminase domain of the HIV-1 restriction factor APOBEC3G." Nature, in press
Matthew Devany , Ferdinand Kappes, Kuan-Ming Chen, David M. Markovitz and *Hiroshi Matsuo , "Solution NMR structure of the N-terminal domain of the human protein DEK." Protein Science, (2008), 17, pp205-215. http://www.proteinscience.org/cgi/content/full/17/2/205
Aya Kitamura, Paul J Jardine, Dwight Anderson, Shelley Grimes and *Hiroshi Matsuo, "Analysis of intermolecular base pair formation in prohead RNA of the phage ø29 DNA packaging motor using NMR spectroscopy." Nucleic Acids Research, Nucleic Acids Research Advance Access published December 15, 2007. http://nar.oxfordjournals.org/cgi/content/full/36/3/839
Kuan-Ming Chen, Natalia A. Martemyanova, Keisuke Shindo, Yongjian Lu, *Hiroshi Matsuo and *Reuben S. Harris, "Extensive mutagenesis experiments corroborate a structural model for the DNA deaminase domain of APOBEC3G." FEBS letters, (2007), Volume 581, Issue 24, Pages 4761-4766. http://www.febsletters.org/article/S0014-5793(07)00963-5/abstract
Brandie J. Kovaleski, Robert Kennedy, Ahmad Khorchid, Lawrence Kleiman, Hiroshi Matsuo, and *Karin Musier-Forsyth, "Critical role of helix4 of HIV-1 Capsid C-terminal domain in interactions with human Lysl-tRNA sythetase." J. B. C. (2007), Nov 2; 282(44):32274-9. http://www.jbc.org/cgi/content/full/282/44/32274