Chancellor's Professor of Biochemistry
and Chemistry, Ingram Professor of
Cancer Research
Ph.D., Concordia University (Montreal), 1983
walter.chazin@Vanderbilt.Edu
Chazin Lab

Research in our laboratory seeks to characterize the structure and motions of proteins and nucleic acids, and the way in which they interact with other proteins, nucleic acids and drugs. We are in essence using the power of the chemistry approach to address key problems in biology and medicine. NMR spectroscopy is the primary experimental tool, though in studying these complex biomolecules, we make use of other biophysical and structural techniques, including X-ray crystallography, calorimetry, fluorescence spectroscopy and X-ray scattering.

The Structural Basis for Protein Function

The sequence of a protein specifies its structure, which in turn determines how it functions. While much has been learned about the structure of proteins in isolation, one of the great challenges today is to understand how proteins act together to perform the major processes in a cell such as DNA replication. A process like DNA replication is complex, involving a sequence of many chemical steps. Our lab is trying to understand how these multiple steps (i.e. the activity of a number of proteins) are coordinated? What we have learned is that groups of proteins work side by side and communicate with each other, functioning much like a machine. Our lab currently studies two types of multi-protein machinery, one group involved in DNA replication, damage response and repair, and a second involved in protein ubiquitination.

NMR spectroscopy has proven to be remarkably powerful as an approach to investigate the dynamic nature of multi-protein machinery. It is being used to study a large (116 kDa) protein, Replication Protein A (RPA), the major single-strand DNA (ssDNA) binding protein that is essential for most transactions in a cell that involve DNA. The protein itself is very complex and is comprised of three separate polypeptide chains with eight different domains. Thus, RPA is a small protein machine and serves as an excellent model system for developing techniques. But more importantly, RPA is a central player in many multi-protein machines involved in processing DNA. RPA performs its functions by constantly adjusting its binding of ssDNA and other proteins through structural changes within its domains as well as by altering the organization of its eight domains.

Our work has delineated the way in which RPA helps to orchestrate the intricate dance of proteins that is required to replicate DNA, respond when DNA is damaged, and repair the damage. This has been achieved by using a mix of biochemical and NMR experiments to identify and structurally characterize the interactions of RPA with specific proteins required for each of these processes. Our studies have focused on structurally characterizing the contacts between specific RPA domains and the corresponding regions of the partner proteins. More recently, NMR and X-ray scattering studies have been undertaken on intact RPA and we have made considerable progress in understanding the global architecture of RPA and how this is changed upon binding DNA. By piecing together these aspects of RPA structure and interactions, we are building a basic understanding of how the RPA molecule functions in mediating DNA processes. In so doing, we are laying the foundation to determine how mutations in the constituent proteins cause defects that lead to cancer and other diseases.

The second type of multi-protein machinery that we study is complex E3 ubiqutin ligases. Ubiquitin is itself a small protein that is used in the cell as a signal through its covalent attachment to target proteins. Its most common use is to poly-ubiquitinate the target, which is a signal that the target protein should be destroyed. Defects in this signaling process are associated with cancer because the target proteins become overabundant when they are not removed from the cell on a regular basis. The process of attaching ubquitin to a substrate protein involves a series of E1, E2 and E3 enzymes. The E3 ubiquitin ligase catalyzes the final attachment step by recruiting both activated ubiquitin molecules and the target protein so that they are in close proximity. The E3 ubiquitin ligase therefore has multiple activities that are performed through the coordinated action of multiple proteins. Our laboratory was the first to experimentally determine the structure of one class of E3 ligase, those termed U-boxes. We are currently studying three different U-box proteins to better understand how target proteins are recognized and what factors control the type of ubiquitin attachment that occurs. In addition, investigations have been ongoing of the complex, multi-protein SCFTBL1 E3 ligase, malfunction of which is implicated as a factor in certain breast cancers.

Ca2+ Signal Transduction by EF-hand Proteins

Change in the level of calcium inside a cell is a common means for regulating biochemical signaling cascades and biomechanical actions- ranging from controlling the opening and closing of ion channels to the contraction of muscles. The EF-hand family of calcium binding proteins plays a central role in virtually every aspect of calcium signaling, so studies of how EF-hand proteins respond to the binding of calcium are the key to understanding how this ion influences so many aspects of health and disease.

Over the past few years we have been determining the structural basis for how changes in calcium levels in cells control inactivation of the human sodium cardiac channel Nav1.5. These studies revealed a complex mechanism involving an EF-hand domain in Nav1.5 that directly binds calcium, and an equally critical role for the ubiquitous EF-hand protein calmodulin. These two calcium sensing mechanisms act in concert to re-position a flap at the edge of the pore that controls movement of sodium ions from outside to inside the cell. Mutations in the corresponding regions of Nav1.5 have been shown to lead to cardiac arrhythmia syndromes and are being investigated in an effort to determine if new therapeutic strategies for these diseases can be developed based on our structural insights.

A second area of emphasis involves the unique S100 class of EF-hand proteins, the first structures of which were determined in our laboratory. These proteins are distinguished by their ability to exert activity both inside and outside cells. We currently focus on calprotectin (CP), a dimer of S100A8 and S100A9 that plays a role in mediating inflammation and serves as an integral part of the innate immune response. CP exhibits a remarkable ability to suppress infections by S. aureus and other bacteria by starving them of essential metals needed for survival. Our ultimate goal is to develop new approaches for antimicrobial agents that are based on the mechanism of action of CP. A second CP project involves determining the structural basis of CP activity in inflammatory processes, which results from its ability to activate the cell surface receptor RAGE (receptor for advanced glycation end products). The structure of RAGE is not known, so characterization of RAGE alone is underway in parallel to analyzing the structural basis for RAGE activation by CP. These studies will provide critical insights for understanding chronic inflammation and atherosclerosis in diabetics and have the potential to reveal new avenues for treating these and other chronic inflammatory disorders.


 

Selected Publications

Chagot B., Potet F., Balser J.R., and Chazin W.J. Solution NMR Structure of the C-terminal EF-hand Domain of Human Cardiac Sodium Channel Na(V)1.5. Journal of Biological Chemistry. 2009, 284 (10): 6436-6445.

Potet F., Chagot B., Anghelescu M., Viswanathan P.C., Stepanovic S.Z., Kupershmidt S., Chazin W.J. and Balser J.R. Functional Interactions between Distinct Sodium Channel Cytoplasmic Domains through the Action of Calmodulin. Journal of Biological Chemistry. 2009, 284 (13): 8837-8845.

Chazin W.J. Evolution of the NIGMS Protein Structure Initiative. Structure. 2008, 16 (1): 12-14.

Ghavami S., Rashedi I., Dattilo B.M., Eshraghi M., Chazin W.J., Hashemi M., Wesselborg S., Kerkhoff C. and Los M. S100A8/A9 at low concentration promotes tumor cell growth via RAGE ligation and MAP kinase-dependent pathway. Journal of Leukocobiology. 2008, 83 (6): 1484-1492.

Lee Y.T., Dimitrova Y.N., Schneider G., Ridenour W.B., Bhattacharya S., Soss S.E., Caprioli R.M., Filipek A., and Chazin W.J. Structure of the S100A6 complex with a fragment from the C-terminal domain of Siah-1 interacting protein: A novel mode for S100 protein target recognition. Biochemistry. 2008, 47 (41): 10921-10932.

B.D. Corbin, E.H. Seeley, A. Raab, J. Feldman, M.R. Miller, V.J. Torres, K.L. Anderson, B.M. Datillo, P.M. Dunman, R. Gerads, R.M. Caprioli, W. Nacken, W.J. Chazin, and E.P. Skaar. Metal chelation and inhibition of bacterial growth in tissue abscesses . Science. 2008, 319: 962-965.

W.J. Chazin. The impact of X-ray crystallography and NMR on intracellular calcium signal transduction by EF-hand proteins: Crossing the threshold from structure to biology and medicine. Science STKE. 2007, 388: pe27
(3 pages).

Dattilo B.M., Fritz G., Leclerc E., Vander Kooi C.W., Heizmann C.W., and Chazin W.J. The extracellular region of the receptor for advanced glycation end products is composed of two independent structural units. Biochemistry. 2007, 46 (23): 6957-6970.

Weiner B.E., Huang H., Dattilo B.M., Nilges M.J., Fanning E., Chazin W.J. An iron-sulfur cluster in the C-terminal domain of the p58 subunit of human DNA primase. Journal of Biological Chemistry. 2007, 282 (46): 33444-33451.

Ball H.L., Ehrhardt M.R., Mordes D.A., Glick G.G., and Chazin W.J., Cortez D. Function of a conserved checkpoint recruitment domain in ATRIP proteins. Molecular and Cellular Biology. 2007, 27 (9): 3367-3377.

Bunick C.G., Miller M.R., Fuller B.E., Fanning E., Chazin W.J. Biochemical and structural domain analysis of xeroderma pigmentosum complementation group C protein. Biochemistry. 2006, 45 (50): 14965-14979.

Jiang X.H., Klimovich V., Arunkumar A.I., Hysinger E.B., Wang Y.D., Ott R.D., Guler G.D., Weiner B., Chazin W.J., Fanning E. Structural mechanism of RPA loading on DNA during activation of a simple pre-replication complex. EMBO Journal. 2006, 25 (23): 5516-5526.

V.N. Shah, T.L. Wingo, K.L. Weiss, C.K. Williams, J.R. Balser and W.J. Chazin. Calcium-dependent regulation of the cardiac voltage-gated sodium channel hH1: Intrinsic and extrinsic sensors act through a common molecular switch. Proceedings of the National Academy of Sciences. 2006, 103: 3592-3597.

E. Johnson, W.J. Chazin and M. Rance. Effects of Calcium Binding on the Side-chain Methyl Dynamics of Calbindin D9k: A 2H NMR Relaxation Study. Journal of Molecular Biology. 2006, 357: 1237-1252.

J.H. Sheehan, C.G. Bunick, H. Hu, P.A. Fagan and W.J. Chazin. Structure of the N-terminal Calcium Sensor Domain of Centrin Reveals the Biochemical Basis for Domain-Specific Function. Journal of Biological Chemistry. 2006, 281: 2876-2881.

C.W. Vander Kooi, M.D. Ohi, J.A. Rosenberg, M.L. Oldham, M.E. Newcomer, K.L. Gould, and W.J. Chazin. The Prp19 U-box U-box Crystal Structure Suggests a Common Architecture for a Class of Oligomeric E3 Ubiquitin Ligases. Biochemistry. 2006, 45: 121-130.

Y. Nakatani, M. Yamazaki, W.J. Chazin and S. Yui. Regulation of S100A8/A9 (Calprotectin) Binding to Tumor Cells by Zinc Ion and its implication for apoptosis-inducing activity. Mediators of Inflammation. 2005, 5: 280-92.

S. Bhattacharya, Y.-T. Lee, M. Novotny, J. Kuznicki and W.J. Chazin. The modular structure of SIP facilitates its role in stabilizing multi-protein assemblies. Biochemistry. 2005, 44: 9462-9471.

C. Bunick, W.J. Chazin. Two Blades of the [Ex]Scissor. Structure. 2005, 13: 1740-1741.

M.D. Ohi, C.W. Vander Kooi, J.A. Rosenberg, L. Ren, J.P. Hirsch, W.J. Chazin, T. Walz, and K.L. Gould. Prp19p tetramerization is required for pre-mRNA splicing. Molecular and the Cellular Biology. 2005, 25: 451-460.

A.I. Arunkumar, V. Klimovich, X. Jiang, R.D. Ott, L. Mizoue, E. Fanning and W.J. Chazin. Insights into RPA32 C-terminal domain-mediated assembly of the simian virus 40 replisome. Nature Structural Molecular Biology. 2005, 12: 332-339.

C.G. Bunick, M.R. Nelson, S. Mangahas, M.J. Hunter, J.H. Sheehan, L.S. Mizoue, G.J. Bunick and W.J. Chazin. Designing sequence to control function in an EF-hand Protein. Journal of the American Chemical Society. 2004, 126: 5990-5998.

M.E. Stauffer and W.J. Chazin. Structural Mechanisms of DNA Replication, Recombination, and Repair. Journal of Biological Chemistry. 2004, 279: 30915-30918.

H. Hu, J.H. Sheehan and W.J. Chazin. The Mode of Action of Centrin: Binding of Ca2+ and a Peptide Fragment of Kar1p to the C-terminal Domain. Journal of Biological Chemistry. 2004, 279: 50895-50903.

T.L. Wingo, V.N. Shah, M.E. Anderson, T.P. Lybrand, W.J. Chazin, and J.R. Balser. An EF-Hand in the sodium channel couples intracellular calcium to cardiac excitability. Nature Structural Molecular Biology. 2004, 11: 219-225.

Specialties

• VICB
• Biophysical Chemistry
• Biochemistry

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