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Walter Chazin

Title and Contact Information

Chancellor's Professor of Biochemistry and Chemistry, Ingram Professor of Cancer Research

Office: 5140 MRB III
Phone: (615) 936-2210


Ph.D., Concordia University (Montreal), 1983


Physical Chemistry
Chemical Biology
Center for Structural Biology
Biophysical Chemistry

In the News

Reporter- New view of DNA processing 'hub'

Reporter- Antibacterial protein's molecular workings revealed

Research News @ Vanderbilt- Repair protein's DNA recognition motif



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

Chen J, Le S, Basu A, Chazin WJ, Yan J. Mechanochemical regulations of RPA's binding to ssDNA. Scientific Reports. 2015, 5:9296.

Ning B, Feldkamp MD, Cortez D, Chazin WJ, Friedman KL, Fanning E. Simian Virus Large T Antigen Interacts with the N-Terminal Domain of the 70 kD Subunit of Replication Protein A in the Same Mode as Multiple DNA Damage Response Factors. PLoS One. 2015, 10(2):e0116093.

Waterson AG, Kennedy JP, Patrone JD, Pelz NF, Feldkamp MD, Frank AO, Vangamudi B, Souza-Fagundes EM, Rossanese OW, Chazin WJ, Fesik SW. Diphenylpyrazoles as replication protein a inhibitors. ACS Medicinal Chemistry Letters. 2014, 6(2):140-5.

Topolska-Woś AM, Shell SM1, Kilańczyk E, Szczepanowski RH, Chazin WJ, Filipek A. Dimerization and phosphatase activity of calcyclin-binding protein/Siah-1 interacting protein: the influence of oxidative stress. FASEB Journal. 2015, pii: fj.14-264770. [Epub ahead of print]

Guilliam TA, Jozwiakowski SK, Ehlinger A, Barnes RP, Rudd SG, Bailey LJ, Skehel JM, Eckert KA, Chazin WJ, Doherty AJ. Human PrimPol is a highly error-prone polymerase regulated by single-stranded DNA binding proteins. Nucleic Acids Research. 2015 . 43(2):1056-68.

Sugitani N, Chazin WJ. Characteristics and concepts of dynamic hub proteins in DNA processing machinery from studies of RPA. Progress Biophysics and Molecular Biology. 2014, [Epub ahead of print]

Vaithiyalingam S., Arnett D.R., Aggarwal A., Eichman B.F., Fanning E., Chazin W.J. Insights into eukaryotic primer synthesis from structures of the p48 subunit of human DNA primase. Journal of Molecular Biology. 2014, 426 (3): 558-69.

Frank A.O., Vangamudi B., Feldkamp M.D., Souza-Fagundes E.M., Luzwick J.W., Cortez D., Olejniczak E.T., Waterson A.G., Rossanese O.W., Chazin W.J., Fesik S.W. Discovery of a Potent Stapled Helix Peptide That Binds to the 70N Domain of Replication Protein A. Journal of Medicinal Chemistry. 2014, 0 (0): [Epub ahead of print].

Hwang H.S., Nitu F.R., Yang Y., Walweel K., Pereira L., Johnson C.N., Faggioni M., Chazin W.J., Laver D.R., George A. Jr., Cornea R.L., Bers D.M., Knollmann B.C. Divergent Regulation of RyR2 Calcium Release Channels by Arrhythmogenic Human Calmodulin Missense Mutants. Circulation Research. 2014, 0 (0): [Epub ahead of print].

Soss S.E., Klevit R.E., Chazin W.J. Activation of UbcH5c similar to Ub Is the Result of a Shift in Interdomain Motions of the Conjugate Bound to U-Box E3 Ligase E4B. Biochemistry. 2013, 52 (17): 2991-2999.

Damo S.M., Kehl-Fie T.E., Sugitani N., Holt M.E., Rathi S., Murphy W.J., Zhang Y.F., Betz C., Hench L., Fritz G., Skaar E.P., Chazin W.J. Molecular basis for manganese sequestration by calprotectin and roles in the innate immune response to invading bacterial pathogens. Proceedings of the National Academy of Sciences of the United States of America. 2013, 110 (10): 3841-3846.

Brosey C.A., Yan C.L., Tsutakawa S.E., Heller W.T., Rambo R.P., Tainer J.A., Ivanov I., Chazin W.J. A new structural framework for integrating replication protein A into DNA processing machinery. Nucleic Acids Research. 2013, 41 (4): 2313-2327.

Damo S., Chazin W.J., Skaar E.P., Kehl-Fie T.E. Inhibition of bacterial superoxide defense A new front in the struggle between host and pathogen. Virulence. 2012, 3 (3): 325-328.

Pruneda J.N., Littlefield P.J., Soss S.E., Nordquist K.A., Chazin W.J., Brzovic P.S., Klevit R.E. Structure of an E3:E2 similar to Ub Complex Reveals an Allosteric Mechanism Shared among RING/U-box Ligases. Molecular Cell. 2012, 47 (6): 933-942.

Shell S.M., Chazin W.J. XPF-ERCC1: On the Bubble. Structure. 2012, 20 (4): 566-568.

Liu J.Z., Jellbauer S., Poe A.J., Ton V., Pesciaroli M., Kehl-Fie T.E., Restrepo N.A., Hosking M.P., Edwards R.A., Battistoni A., Pasquali P., Lane T.E., Chazin W.J., Vogl T., Roth J., Skaar E.P., Raffatellu M. Zinc Sequestration by the Neutrophil Protein Calprotectin Enhances Salmonella Growth in the Inflamed Gut. Cell Host & Microbe. 2012, 11 (3): 227-239.

Souza-Fagundes E.M., Frank A.O., Feldkamp M.D., Dorset D.C. Chazin W.J., Rossanese O.W., Olejniczak E.T., Fesik S.W. A high-throughput fluorescence polarization anisotropy assay for the 70N domain of replication protein A. Analytical Biochemistry. 2012, 421 (2): 742-749.

Guler G.D., Liu H.J., Vaithiyalingam S., Arnett D.R., Kremmer E., Chazin W.J., Fanning E. Human DNA Helicase B (HDHB) Binds to Replication Protein A and Facilitates Cellular Recovery from Replication Stress. Journal of Biological Chemistry. 2012, 287 (9): 6469-6481.

Williams C.K., Vaithiyalingam S., Hammel M., Pipas J., Chazin W.J. Binding to retinoblastoma pocket domain does not alter the inter-domain flexibility of the J domain of SV40 large T antigen. Archives of Biochemistry and Biophysics. 2012, 518 (2): 111-118.