Replication of the Human Immunodeficiency virus is totally dependent on the reverse transcriptase activity of HIV-1 reverse transcriptase (HIV- RT). Because the reverse transcriptase activity of HIV-RT has no known cellular homolog it is an important target for the rational design of antiviral drugs. The basis of the rational design of specific enzyme inhibitors is a knowledge of the structure and catalytic mechanism of an enzyme. Although the three dimensional structure of HIV-RT has been elucidated little is known about the catalytic mechanism or the locations of residues that play critical roles in substrate binding before or during catalysis. The goal of the proposed research is to begin addressing these issues. We propose to identify amino acids in the nucleotide and primer/template binding domains of HIV-RT and to evaluate their importance using site-directed mutagenesis. Specifically, we propose to use a series of photoactive DNA probes in primer/template configurations, to label amino acids in the primer/template binding domain of HIV-RT. Isolation and amino acid sequencing of peptides crosslinked to these probes will allow the identification of amino acids that participate in primer/template binding or in catalysis. Two key features of the proposed labeling experiments include the use of both RNA and DNA templates, and the use of DNA primers containing a dideoxy photoactive nucleotide analog at the 3' end. A photoactive 3' dideoxy nucleotide will allow the labeling of amino acids near the 3' end of the primer while the enzyme is poised for catalysis but unable to incorporate dNTPs. Crosslinking to photoactive primer/templates having an A-form structure (RNA template) or a B-form structure (DNA template) will allow us to address the question of whether different protein conformations are used when HIV-RT is poised for catalysis in the DNA and RNA dependent modes. We also propose to use photoactive nucleotide analogs to label amino acids in the dNTP binding domain. Isolation and sequencing of peptides crosslinked to the nucleotide probes will identify amino acids likely to be involved in substrate binding and/or catalysis. To identify residues most likely to be involved in catalysis, photoaffinity labeling experiments will be performed while RT is in a catalytically competent ternary complex with a 3' dideoxy terminated primer/template and the next complementary dNTP. To determine the importance of the amino acids identified by the crosslinking experiments we propose to use site-directed mutagenesis to change amino acids at and near the sites of crosslinking. Our preliminary results suggest that Tyr-318 is in the dNTP binding domain. Therefore, we propose to change Tyr-318 to Phe, Thr and Gly to evaluate the importance of this amino acid. Mutant RTs will be characterized to determine the effects of each mutation on enzyme activity, the Kd for binding of primer/template and dNTPs and the Ki for AZT. The results of this study will be important toward the broad long-term objective, which is the rational design of potent and specific reverse transcriptase inhibitors.
