HDX and NMR Core

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Abstract Proteins are dynamic molecules that undergo constant flexing and motion. Changes in protein conformation and dynamics are important to molecular recognition and these changes occur during many key biological processes including activation and inhibition of enzymes, ligand binding to receptors, posttranslational modifications, and allosteric communication across protein-protein interfaces. All of these processes are regulated through the structure and dynamics of the proteins within the functional complex. Thus, techniques to study protein structure and dynamics are critical to the understanding of biological processes and to aid in developing strategies to target proteins for therapeutic intervention in disease. The HDX-NMR Core will address central goals of the HIVE Center that include characterization of HIV protein interactions among themselves, with host molecules, or with small molecule probes. A key focus of HDX efforts will be the structure and dynamics of the HIV-1 reverse transcriptase (RT) initiation complex, or RTIC, a large protein-nucleic acid complex comprising RT (p66/p51 heterodimer), the primer-binding site region of the 5’ untranslated viral RNA (vRNA) and a human tRNA (Project 3: Arnold, Musier-Forsyth, Griffin, Lyumkis, and Sarafianos). We will also investigate the interactions of HIV-1 RT with members of the APOBEC3 (A3) family of restriction factors (most notably A3G), which have been reported to suppress the polymerase activity of RT and will also be studied in Project 3. In addition to the RTIC from lentivirus HIV-1, we will also investigate the RTIC of spumavirus prototype foamy virus (PFV). We are taking a multifaceted approach towards understanding these important virus-host complex interactions, using cryo-EM, X-ray crystallography, HDX, SAXS, and pre-steady state kinetic analyses. Among the main questions addressable by HDX are: How does the vRNA/tRNA interact with RT compared to DNA/DNA and DNA/RNA substrates (cryo-EM, X-ray, HDX, kinetics)? What is the role of RNA structure in this complex and how does it interact with RT (cryo-EM, X-ray, HDX)? What are the dynamic properties of the initiation complex (cryo-EM, SAXS, HDX, kinetics)? How do interactions in the HIV-1 and PFV RTICs differ? How does HIV-1 RT interact with A3 proteins to contribute to HIV-1 restriction? HDX has been instrumental for studying quinoline-based allosteric integrase (IN) inhibitor (ALLINIs) induced aberrant multimerization of full-length wild type (WT) IN and its application will now be extended to examine how recently discovered (Core 4) thiophene- based inhibitors alter the IN structure (Project 1). HDX-MS will also be used to probe inter-protein interactions among HIV-1 Gag and Gag-Pol proteins (Project 2), the effect of resistance mutations or inter-clade differences in inter-protein interactions in HIV-1 proteins (Project 5), and interactions between HIV-1 proteins and SuFEx or other small molecule probes (Project 6). NMR will be used to support Project 6 and Core 4.