John BushwellerJohn H. Bushweller

Structural Biology, NMR Spectroscopy, Protein Structure and Dynamics, Structure-Based Drug Design

Principal Research Interests

Our lab is fundamentally interested in understanding, from a structural and biophysical perspective, the functioning of proteins involved in regulating transcription, particularly those involved in the dysregulation associated with the development of cancer. Structural and functional characterization of the native forms of these proteins and their relevant complexes via NMR spectroscopy and a variety of other techniques provides a baseline of understanding. Subsequent characterization of the oncoprotein forms then provides a detailed understanding of the molecular mechanism of oncogenesis associated with altered forms of these proteins. Such knowledge leads to novel avenues for the design of therapeutic agents to treat the cancers associated with these par-ticular oncoproteins.

One current focus is structural studies of a novel transcriptional enhancer referred to as the core-binding factor (CBF). This heterodimeric protein is essential for hematopoietic development. Gene translocations associated with the genes coding for the two subunits of CBF produce novel fusion proteins which have been implicated as playing a role in more than 30% of acute leukemias. We are carrying structural (NMR spectroscopy and X-ray crystallography) and functional studies of the oncoprotein forms of the two subunits of CBF that are associated with leukemia to gain an understanding of their roles in the development of leukemia.

A second focus is the development of highly targeted small molecule inhibitors of the oncoprotein forms of CBF. Using structural information on the proteins, virtual screening, NMR and fluorescence-based assays, and medicinal chemistry, we have developed the first small molecule inhibitors of these proteins. This is a collaborative effort with the lab of Dr. Milton Brown here at UVa and outside investigators at Harvard, Dartmouth, and NIH which is supported by a Specialized Center of Research (SCOR) grant from the Leukemia and Lymphoma Society.

A third focus for the lab is the application of solution NMR methods to the structure determination of membrane proteins. The vast majority of drug targets are membrane- embedded proteins. This class of proteins has presented significant challenges for structure determination by any method. We completed the structure determination of the largest membrane protein to be solved by NMR spectroscopy thus far. This structure established a paradigm for tackling this class of proteins by solution NMR. We are currently examining additional technical improvements in this area as well as targeting several new systems for structure determination.

Representative Publications

High-Resolution NMR Structure and Functional Characterization of the PWWP Domain from Hepatoma Derived Growth Factor (HDGF). Stephen M. Lukaisk, Tomasz Cierpicki, Allen Everett,  and John H. Bushweller. Protein Science, in press.

Comparative Molecular Field Analysis of the Interaction Between 2-Aminothiazoles and Core Binding Factor β. Michael J. Gorczynski,  Miki Newman, Jolanta Grembecka, Takeshi Corpora, Yunpeng Zhou, John H. Bushweller, Milton L. Brown. J. Med. Chem., in press.

Probing the supramodular architecture of a multidomain protein by NMR: the solution structure of syntenin. Tomasz Cierpicki, John H. Bushweller, and Zygmunt Derewenda. (2005), Structure, 13, 1-9.

Membrane Structures of the Hemi-Fusion-Inducing Fusion Peptide Mutant G1S and the Fusion-Blocking Mutant G1V of Influenza Hemagglutinin Suggest A Mechanism for Pore Opening in Membrane Fusion. Yinling Li, Xing Han, Alex L. Lai, John H. Bushweller, David S. Cafiso and Lukas K. Tamm. J. Virol. 79, 12065-12076 (2005).

Allosteric Activation of Protein Phosphatase 2C by Mn2+-D-Chiro-Inositol-Galactosamine, a Putative Mediator-Mimetic of Insulin Action. Brautigan D. L., Brown, M., Grindrod, S., Chinigo, G., Kruszewski, A., Lukasik, S., Bushweller, J. H., Horal, M., Keller, S., Tamura, S., Heimark, D. B., Price, J., Larner, A. N., and Larner, J. Biochemistry, 44, 11067-11073 (2005).

Synthesis and Evaluation of Substituted 4-Aryloxy- and 4-Arylsulfanyl-phenyl-2-aminothiazoles as Inhibitors of Human Breast Cancer Cell Proliferation. Michael J. Gorczynski, Susan L. Mooberry, John H. Bushweller,and Milton L. Brown. (2004) Bio. Med. Chem. Lett., 12, 1029-1036.

CBFβ Allosterically Regulates the Runx1 Runt domain by Shifting a  Dynamic Conformational Equilibrium in the Runt Domain and DNA. Jiangli Yan, Yizhou Liu, Stephen M. Lukasik, Nancy A. Speck, and John H. Bushweller. (2004) Nat. Struc. Mol. Biol., 11, 901-906.

Charged Gels as Orienting Media for Measurement of Residual Dipolar Couplings in Soluble and Integral Membrane Proteins. Tomasz Cierpicki and John H. Bushweller. (2004) J. Am. Chem. Soc., 126, 16259-66.

The DCX-domain tandems of doublecortin and doublecortin-like kinase. Myung Hee Kim, Tomasz Cierpicki, Urszula Derewenda, Daniel Krowarsch, Yuanyi Feng, Yancho Devedjiev, Zbigniew Dauter, Christopher A. Walsh, Jacek Otlewski, John H. Bushweller, Zygmunt S. Derewenda. (2003). Nat. Struc. Biol., 10, 324-333.

Mutagenesis of the Runt Domain Defines Two Energetic Hotspots for Heterodimerization with the Core Binding Factor β Subunit. Lina Zhang, Zhe Li, Jiangli Yan, Padmanava Pradhan, Takeshi Corpora, Jerónimo Bravo, Alan J. Warren, John Bushweller, and Nancy A. Speck. (2003), J. Biol. Chem., 278, 33097-33104.

Energetic Contribution of Residues in the Runx1 Runt Domain to DNA Binding. Zhe Li, Jiangli Yan, Christina J. Matheny, Takeshi Corpora, Jerónimo Bravo, Alan J. Warren, John H. Bushweller, and Nancy A. Speck. (2003), J. Biol. Chem., 278, 33088-33096.

Structure, Dynamics and Function of the Outer Membrane Protein A and Influenza Hemagglutinin Fusion Domain in Detergent Micelles by Solution NMR. Lukas K. Tamm, Frits Abildgaard, Ashish Arora, Heike Blad, and John H. Bushweller. (2003), FEBS Letters, 555, 139-143.

Altered Affinity of CBFβ–SMMHC for Runx1 Explains Its Role in Leukemogenesis. Stephen M. Lukasik, Lina Zhang, Takeshi Corpora, Sarah Tomanicek, Yuanhong Li, Mondira Kundu, Kari Hartman, P. Paul Liu, Thomas M. Laue, Rodney L. Biltonen, Nancy A. Speck, and John H. Bushweller. (2002) Nat. Struc. Biol. 9, 674-679.

Structure of the Outer Membrane Protein A Transmembrane Domain in Detergent Micelles by NMR Spectroscopy. Ashish Arora, Frits Abildgaard, John H. Bushweller, and Lukas K. Tamm. (2001) Nat. Struc. Biol., 8, 334-338.

Membrane Structure and Fusion-Triggering Conformational Change of the Fusion Domain from Influenza Hemagglutinin. Xing Han, John H. Bushweller, David S. Cafiso, and Lukas K. Tamm. (2001)Nat. Struc. Biol., 8, 715-720.

MQ-HCN-based Pulse Sequences for the Measurement of 13C1’-1H1’, 13C1’-15N, 1H1’-15N, 13C1’-13C2’, 1H1’-13C2’, 13C6/8-1H6/8, 13C6/8-15N, 1H6/8’-15N, 13C6-13C5, 1H6-13C5 Dipolar Couplings in 13C, 15N-labeled DNA (and RNA). Jiangli Yan , Takeshi Corpora, Padmanava Pradhan, and John H. Bushweller. (2001) J. Biomol. NMR, 22, 9-20.