Retroviral gag polyproteins and their proteolytic nucleic acid binding protein (NABP) products contain, without exception, conserved """"""""zinc finger"""""""" amino acid sequences: -Cys-X2-Cys-X3-Gly- His-X4-Cys- (X = variable amino acid). These sequences are responsible for differentiation of viral RNA from cellular RNA during viral growth. Although zinc fingers from DNA-binding proteins have received much recent attention, important questions exist regarding the structural and physicochemical properties of retroviral NABPs and retroviral zinc fingers, including: (1) Do retroviral fingers bind zinc stoichiometrically as proposed (Berg, J. M., Science 232, 485, (1986))? (2) What are the structural features of retroviral zinc fingers? (3) What is the relationship of the zinc finger to the rest of the NABP molecule? and (2) What amino acid residues in retroviral NABPs are involved in sequence specific binding to RNA? Answers to these questions will provide insights into the major issues of retroviral gene recognition and facilitate the design of therapeutic inhibitor agents for the control of cancer and other retroviral disease. A two-pronged approach is proposed involving studies of (i) synthetic peptides with amino acid sequences of retroviral NABP zinc fingers, and (ii) retroviral NABPs isolated from virus. Having established from preliminary studies that retroviral fingers form highly stable, stoichiometric zinc adducts, our specific initial objectives are to (1) determine the complete three-dimensional structure of a retroviral zinc finger complex, (2) determine the structure of a retroviral NABP, and (3) determine the fundamental structural features and dynamic properties of nucleic acid complexes with finger peptides and NABPs. Quantitative information regarding metal and nucleic acid binding, the nature of coordinated ligands, and 3D structural features will be determined using advanced two- dimensional NMR spectroscopic methods (ROESY, NOESY, HOHAHA, 1H- 113Cd HMQC) in combination with molecular dynamics and distance geometry calculations. Where necessary, new NMR pulse sequences and techniques will be developed. Other spectroscopic methods (UV- VIS, CD, Laser Fluorescence) will also be employed. In addition to providing fundamental structural and physicochemical information on retroviral NABPs, this work will lay the groundwork for studies of more complex nucleic acid binding proteins that are larger and contain multiple zinc finger domains.
