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Chazin Lab Chazin Lab Vanderbilt University
   
 

Research Overview

Research in my laboratory uses a multi-disciplinary, collaborative and structurally-oriented approach to address key problems in biology and medicine. At the molecular level, we study the structure and dynamics of proteins and their complexes with other proteins, nucleic acids and small molecule ligands. My independent career began as a biomolecular NMR spectroscopist, but has evolved to a point where I could be categorized as a structural biochemist. At the technical level, we now make use of many structural and biophysical techniques, including calorimetry, fluorescence spectroscopy, X-ray crystallography, X-ray/neutron scattering, cryo electron microscopy and computation.  These studies are integrated with in vitro and cell-based biochemistry, in our own lab and by numerous collaborators at our institution, around the country and across the world.  The following sections outline our three research programs.

The Structural Basis for Function of DNA Processing Machinery

One of the great scientific challenges today is to understand how proteins act together to perform the major processes in a cell such as DNA replication, all of which involve a sequence of multiple biochemical steps.  Much of our work has revolved around human Replication Protein A (RPA), the major eukaryotic single-strand DNA (ssDNA) binding protein, which is essential for most DNA transactions in all cells. RPA is structurally very complex with three subunits containing eight different domains. It 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 domains.

Our work has helped delineate 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. Important insights have been obtained by identifying, and structurally characterizing, the interactions of RPA with specific proteins required for each of these processes. Recently, we have focused on the rearrangements in the global architecture of RPA, the mechanisms of binding and unbinding DNA, and how the binding of protein partners alter the landscape. As we have progressed, there has been an increasing need to determine structures of RPA binding partners.  Current efforts center on human DNA primase, XPA and XPC. Together, these studies are laying the foundation to determine how mutations in the DNA processing proteins cause defects that lead to cancer and other diseases. Moreover, we are well invested into exploring translation of this knowledge into potential therapeutics by using fragment-based inhibitor discovery targeted to suppressing RPA-mediated recruitment of proteins to sites of DNA damage.

Structure and Function of U-box E3 Ubiquitin Ligases.

Covalent attachment of ubiquitin to a target protein serves as a cellular signal. For example, poly-ubiquitination of a target typically signals for degradation in the proteasome. Defects in this process are associated with cancer, for example a target can become overabundant if it is not degraded at a sufficient rate. The process of attaching ubquitin to a substrate protein involves a dynamic multi-protein machine comprised of E1, E2 and E3 enzymes. Our laboratory was the first to experimentally determine the structure of the U-box class of E3 ligases. Our studies of U-box proteins have focused on the mechanism of activation of the E2~ubiquitin conjugates, understanding how target proteins are recognized, and what factors control the type of ubiquitin chain attached. We are also investigating how the U-box E3 CHIP differentially regulates targets in the cell stress response.

Ca2+ Signal Transduction by EF-hand Proteins

Change in levels of calcium inside a cell is a common means for regulating biochemical signaling cascades and stimulating biomechanical actions. EF-hand calcium binding proteins play a central role in virtually every aspect of calcium signaling.  Consequently, studies of their response to the binding of calcium and activation of targets are keys to understanding how this ion influences so many aspects of health and disease.  I have worked in this field for nearly 30 years, starting as a postdoctoral fellow.  Currently we have two active research programs.

The first program investigates the structural basis for how changes in intracellular calcium levels control inactivation gating of the human cardiac sodium channel NaV1.5. We found a complex mechanism involving both an EF-hand domain in NaV1.5 that directly binds calcium, and the ubiquitous EF-hand protein calmodulin. These two calcium sensors act in concert to re-position a flap at the edge of the channel pore that controls movement of sodium from outside to inside the cell. Mutations in the corresponding regions of NaV1.5 lead to cardiac arrhythmia syndromes and we are determining if new therapeutic strategies for these diseases can be developed based on our structural insights. We have also been working in concert with deep sequencing of patients suffering from cardiac arrhythmia syndromes to discern the physical basis of the defects caused by mutations in calmodulin genes, which came as a complete surprise because this protein is almost completely conserved in organisms from plants to man.

We also study the unique S100 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 to invading microorganisms. We have shown that CP exhibits a remarkable ability to suppress infections by S. aureus and other bacteria by using a mechanism known as nutritional immunity, i.e. starving the pathogen 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 project involves determining the structural basis of CP activity in inflammatory processes, which results from its activation of the cell surface receptor RAGE. We have determined the structure of the RAGE ligand-binding domain and are 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. To this end, we have initiated a CP-RAGE fragment-based inhibitor discovery program.