Background: Our 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 entry and integration. Results during the past year: DNA replication - We have determined the crystal structures at very high resolution of two forms of the T4 bacteriophage GP59 helicase assembly protein. Retroviral proteins - We have completed the refinement of a high resolution crystal structure of the ectodomain of the GP41 envelope protein from Simian Immunodeficiency Virus. Significance and future directions: GP59 structure--Bacteriophage T4 gene 59 helicase assembly protein accelerates the loading of the hexameric gene 41 helicase, especially when the gene 32 single stranded DNA binding protein is present. The 59 protein has been previously shown to interact with both single and double-stranded DNA, with gp32 protein, and with gp41 helicase. Recently it has been discovered that 59 protein preferentially binds to fork DNA, binding to the double stranded and to both of the single-stranded regions of the fork. The high resolution crystal structure of the full length 59 protein has been completed to 1.45 Angstrom resolution in the adsence of DNA substrate. The 59 structure is of a monomeric protein, with no evidence of higher order assembly such as hexamerization. The structure reveals a novel fold of a two domain protein. The N-terminal domain appears to be responsible for double-stranded DNA binding and has structural homology with the high-mobility group DNA binding domains. The C-terminal domain has two surface regions with hydrophobic and basic residues that may be responsible for binding the single stranded regions of the fork DNA molecules. These results suggest that the 59 helicase assembly protein is responsible for displacing 32 protein and assembling the 41 helicase specifically at the replication fork and on forked recombination intermediates. The protein model of GP59 suggests how it can simultaneously bind to both single and double-stranded DNA and provide directionality for assembly of the helicase. The model will provide a starting point for future mutagenesis studies of its function. Retroviral GP41 ectodomain structure- Envelope glycoproteins, gp120 and gp41 in human (HIV) and simian (SIV) immunodeficiency viruses mediate viral entry via membrane fusion. Peptides of the gp41 ectodomain have proven to be potent inhibitors of viral fusion, presumably by interfering with a change in the conformation or the oligomerization state of gp41. Understanding the active conformations and oligomeric state will be key to successful vaccine development. The crystal structure of the SIV gp41 ectodomain was determined at 1.47 angstrom resolution. The rod-like trimeric structure of SIV gp41 comprises three parallel N-terminal -helices assembled as a central coiled-coil with three antiparallel C-terminal -helices packed on the outside connected by highly flexible loops. The structure of GP41 ectodomain is the most complete and accurate structure now available. It confirms models of GP41 derived from crystal structures of smaller, discontinuous peptide fragments. Work is underway to extend these studies to include larger complexes and other conformations. Lay Summary: Knowledge of a macromolecule's three-dimensional structure is vital to understanding its biological function. 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.
| 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 |
