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Macromolecular Crystallography Our research is directed toward understanding macromolecular interactions and their roles in biological function. Through an integrated approach including x-ray crystallography, site-directed mutagenesis, and enzymology, we are working to understand the structural basis of nucleic acid recognition and the mechanism of polymerization by Moloney murine leukemia virus reverse transcriptase (MMLV RT). Reverse transcriptase is a relatively simple polymerase by comparison with its mammalian counterparts and is therefore an ideal enzyme for studying the complicated process of polymerization. The retroviral enzyme reverse transcriptase (RT) has two enzymatic activities, RNA- and DNA- directed DNA polymerase and ribonuclease H (RNase H), that produce a double-stranded DNA copy of the single-stranded RNA genome of the retrovirus. This retroviral DNA is then integrated into the host genome, and the host's cellular machinery is utilized to produce more retrovirus. The epidemic outbreak of AIDS caused by human immunodeficiency virus (HIV) has focused a great deal of research efforts on HIV RT. Drugs that are presently being used to treat AIDS patients include four inhibitors of HIV-1 RT, which continues to be a target for development of new inhibitors. We have focused our efforts on the Moloney murine leukemia virus (MMLV) RT, a related retroviral RT, with the goal of understanding the mechanism of the polymerization and interactions with nucleic acid. Our approach is to compare structures of functionally related enzymes, HIV-1 RT and MMLV RT, in order to understand how the enzyme works. Basic and detailed knowledge of catalysis and substrate interactions in RT will further efforts in development of effective inhibitors. We are currently working on complexes of nucleic acid with a catalytic fragment from MMLV RT in addition to the full-length enzyme. The catalytic fragment was initially defined by limited proteolysis of the full-length MMLV RT and found to retain polymerase activity. We have cloned, expressed, and purified large quantities of the catalytic fragment for use in our crystallographic studies and have shown that this bacterially-expressed catalytic fragment also retains polymerase activity. The catalytic fragment includes the N-terminal fingers and palm domains from MMLV RT. Our goal in these experiments is to obtain high resolution structures of relevant nucleic acid complexes that will provide sufficiently detailed information to answer questions regarding mechanism of polymerization, processivity, and nucleic acid binding. Nucleic acid substrates for RT include a template-primer duplex with overhanging single-stranded template. For crystallographic studies, we have used catalytic fragment produced by limited proteolysis as well as the bacterially-expressed catalytic fragment. Complexes have been made with duplex DNA substrates including single-stranded template overhangs in addition to blunt-ended DNA duplexes. Three different crystal forms have been obtained. We have determined the x-ray crystallographic structures of two of the three complex crystals at 2.3Å resolution. Form I crystals have been solved by molecular replacement using the model of the uncomplexed catalytic fragment previously reported. Form II crystals were solved by single isomorphous replacement and anomalous scattering phasing in addition to molecular replacement. Data for Form III crystals have been obtained to 4Å resolution and a molecular replacement solution obtained. Work continues on the comparison and analyses of these three crystal forms. A preliminary analysis of the complex structures has indicated a basis for the observed nucleic acid binding that involves at least two conserved amino acid residues. We have made point mutations for the conserved residues and purified substituted catalytic fragments and full-length MMLV RTs. Two crystal forms have been obtained of nucleic acid complexes with a substituted catalytic fragment. One crystal form is the same as the previously characterized Form II crystals of the wild-type catalytic fragment, but the second is a new crystal form that exhibits an entirely different packing of the protein molecules. We are currently determining these structures at 2.0Å resolution. In addition, we plan to characterize the enzymatic properties of the substituted MMLV RTs and catalytic fragments and compare them to the wild-type enzymes. We hope to correlate the structural information with the enzymatic properties of these enzymes We are also pursuing the crystal structure of a nucleic acid complex with the full-length MMLV RT. Several different duplex oligonucleotides have been screened in our efforts to improve the size and diffraction quality of crystals obtained initially from PEG-containing precipitant solutions. Recently, significantly larger crystals were obtained from a screen based on the length of the duplex DNA substrate. The presence of MMLV RT and oligonucleotide in these crystals has been verified by native PAGE, and initial x-ray diffraction experiments indicate that the crystals diffract. We are also pursuing crystallization experiments with the substituted full-length RTs with the goal of improving the complex crystals. Although retroviral RTs perform the same functional activities, there are obvious differences in the architectures of the enzymes. HIV-1 RT and Rous sarcoma virus RT, representing the other two retroviral families, are heterodimeric enzymes with two subunits of identical amino acid sequence in which one subunit has been proteolytically processed removing a single functional domain. It has been proposed based on gel retardation assays that MMLV RT, which is monomeric as isolated, dimerizes in the presence of substrate. We hope to address remaining questions regarding the interactions of nucleic acid with RT in the complex of the full-length RT such as the role of the RNaseH domain in modulating polymerase activity and dimerization of MMLV RT in the presence of substrate.
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