Background: This working group studies the three-dimensional structure and function of biological macromolecules (proteins, enzymes, and nucleic acids) using the method of single-crystal x-ray diffraction. Objective: Major emphasis is given to macromolecular complexes and complex interactions between macromolecules. Systems studied include a model system for DNA replication. Structural studies of retroviral proteins and enzymes are also underway to understand the mechanism of retroviral integration as well as the structure of human autoantigens. Results during the past year: DNA replication: Our high-resolution structures of the helicase assembly protein GP59 from phage T4 were analyzed to propose a model for the binding of forked DNA substrates. Six site-specific mutant forms were constructed for solution studies to test our hypotheses. Other work was directed towards finding forms of phage RNA primases, helicases, and multiprotein complexes suitable for crystallization studies. Promising crystalline forms of full-length primase and its truncated forms have been discovered. Retroviral proteins: We have completed the structure solution and refinement of three crystal forms of an active two-domain form of Rous sarcoma virus integrase. Multiprotein complexes: were have obtained diffraction quality crystals of the human mitochondrial CLP P protease complex and have determined the structure by molecular replacement methods. Autoantigens: we have constructed several recombinatly engineered forms of the human La protein and have begun testing them for crystallization. Conclusions and Significance: A long term goal of our research is to understand better the process of DNA replication by studying the structures of the molecular machinery involved. We believe that our recently determined structure of GP59 will be important for understanding how other proteins and enzymes assemble at a replication fork. The structure of the two-domain form suggests models by which the protein interacts with both viral and host cell DNA and provides a new view of the critically important active center. Lay Summary: Knowledge of a macromolecules three-dimensional structure is vital to understanding its biological function. The study of crystals of these molecules is the best way to obtain detailed pictures. DNA replication is indispensable to all living cells. Errors in DNA replication and repair can lead to cancer and genetic diseases. Structures of retroviral target enzymes are essential for future structure-based drug development and other potential therapeutic strategies for diseases such as AIDS. - Structural Biology, x-ray crystallography, protein structure, DNA replication, AIDS
| Mueser, T C; Jones, C E; Nossal, N G et al. (2000) Bacteriophage T4 gene 59 helicase assembly protein binds replication fork DNA. The 1.45 A resolution crystal structure reveals a novel alpha-helical two-domain fold. J Mol Biol 296:597-612 |
