Beese Lab Research Interests
Structure Gallery
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Overview
The broad goal of our research is to understand biological processes in atomic detail. A multi-disciplinary strategy is employed using macromolecular X-ray crystallography to determine high resolution, three-dimensional images of proteins and appropriate complexes. The structural information is combined with biochemical, genetic, and computational analyses to address questions central to cancer biology. In addition, this approach may facilitate the development of new therapeutic agents for the treatment of human disease.
Signal transduction
Numerous essential signal transduction proteins, including members of the Ras GTPase superfamily, require posttranslational attachment of an isoprenoid lipid group for proper function. A major focus of the lab is on understanding the structural enzymology of the protein prenyltransferase family of lipid modifying enzymes (Fig1). We have determined high resolution, three-dimensional structures of human protein farnesyltransferase (FTase) and geranylgernyltransferase (GGTase-1) (Fig2a ; Fig2b), enzymes that are promising anticancer drug targets. Inhibitors of FTase cause tumor regression in animals and are currently being evaluated in phase II/III clinical trials for the treatment of human cancer. Subsequently, we have determined a complete series of structures representing major steps along the reaction coordinate of these enzymes (Fig3) (Animations 1. QuickTime 2. MPEG). From these observations can be deduced the structural determinants of substrate specificity and an unusual mechanism in which product release requires binding of substrate, analogous to classically processive enzymes. We are combining site-directed mutagenesis, enzyme kinetics, computation, and X-ray crystallography to test these hypotheses. Structural studies of other enzymes in the signaling pathway are ongoing.
Structure based drug design
Subtle structural differences among protein prenyltransferases are being used to develop highly specific inhibitors. Initially, we determined the mechanism of action of peptidomemetic inhibitors that showed tumor regression in animal studies (Fig4) and are currently investigating chemotherapeutics in clinical trials for treatment of human cancer (Fig5). These structures facilitate the design of new drugs targeting the prenyltransferase enzyme family. Additionally, inhibitors that specifically target prenyltransferases from parasites such as Plasmodium falciparum or Trypanosoma brucei show promise for the treatment of malaria and other diseases. Structural studies of prenyltransferases from human pathogens are in progress.
DNA replication
A major focus of the laboratory is on understanding the molecular mechanisms of DNA replication. We have determined high-resolution crystal structures of DNA polymerases with DNA primer-templates that capture different stages of the synthesis reaction (Fig8, Fig9). Of particular interest is the Bacillus DNA polymerase that retains its ability for processive, accurate DNA synthesis in the crystal (Fig10). We are using this polymerase as a model system to study molecular mechanisms of DNA fidelity (Polymerase Movie). DNA mispairs and carcinogenic DNA adducts captured at the active site suggest structural basis of mutations (Fig11). Structural studies of macromolecular assemblies central to DNA replication are ongoing.
DNA Mismatch Repair
Our laboratory is investigating protein-DNA assemblies involved in human mismatch repair (Fig6). Mismatch repair is essential for maintaining genomic stability of all organisms. Defects in genes involved in mismatch repair lead to elevated mutation rates, and in the case of humans confer a strong predisposition to tumorigenesis. Currently, experiments are focused on determining structures of protein-DNA assemblies involved in the initiation of the human mismatch repair reaction (Fig7). We are part of a consortium of scientists nationwide who are collaborating to study the Structural Cell Biology of DNA Repair machines.
Observing Enzymes in Action
A common theme of the laboratory is the study of enzyme mechanisms at near atomic resolution by determining three-dimensional structures that represent stages along the reaction pathway. Structural information is combined with biochemical, biophysical, and computational analyses to understand how enzymes function. An exciting new direction we are pursuing arises from our discovery of a DNA polymerase that retains catalytic activity in the crystal (see above). Currently, we are developing methodology to study the phosphoryl-transfer reaction using time-resolved crystallography (Fig12). Our goal is to observe DNA synthesis in real time. Ultimately, this many enable the dynamic process of accurate DNA replication to be viewed and constrasted with replication under mutagenic conditions.