This project utilizes NMR spectroscopy to study the molecular components of HIV and model systems. The primary research areas are: 1) analysis of the structure, dynamics and ligand binding behavior of the Ribonuclease H domain of HIV reverse transcriptase, 2) spectroscopic studies of specifically labeled HIV reverse transcriptase; 3) studies of model nuclease and polymerase systems, and 4) protein engineering studies of the cyanobacterial nuclease inhibitor NuiA, in order to determine whether this inhibitor can target nucleases of infectious bacteria. ? ? ? Project 1. The C-terminal domain of HIV Reverse Transcriptase contains the RNase H active site, and the isolated RNase H domain can be expressed as a stable, folded protein. However, the isolated domain is inactive and its C-terminal helix exhibits significant conformational exchange broadening. We have studied various RNase H mutations and solution conditions in order to understand how these influence structure and activity. Mutations have been introduced based on related nucleases such as that derived from B halodurans, as well as structural analyses. Unexpectedly, nearly all of the mutations introduced resulted in significantly greater lability of the protein, as indicated by the presence of proteolyzed fragments. In general, the first proteolytic cleavage results in the loss of the C-terminal 20 residues. These effects are currently being analyzed. We have also obtained HSQC spectra for RT containing methyl-13Cmethionine residues in the P66 subunit. Most of these resonances have been assigned using site-directed mutants. These studies will ultimately provide information into the subtle long range effects of drug resistance mutations, drug binding effects, as well as substrate interactions. ? ? Project 2. Immunocompromised individuals suffering from AIDS are particularly susceptible to nosocomial infections. It has recently been demonstrated that the host defense mechanism includes the release of neutrophil extracellular traps or NETs, that immobilize and sometimes kill the invading bacteria. These extracellular traps utilize a DNA scaffold, and thus can be digested by the extracellular nucleases secreted by bacteria. NuiA is an inhibitor of a non-specific nuclease that is a member of the beta-beta-alpha metal superfamily secreted by Anabaena. It is also a weak inhibitor of a homologous nuclease excreted by the infectious agent, Serratia marcescens. Since serratia is a source of nosocomical infections, we are have, in collaboration with the Pingoud group, initiated studies designed to modify NuiA to increase its potency against the Serratia nuclease. Initial studies have demonstrated that substitution of the Arg69 residue of NuiA with any of a number of residues increase the potency of the inhibitor. Further studies are in progress in order to examine the effects of additional mutations suggested by modeling and calculational studies, and to determine the structure of the NuiA-Serratia nuclease complex. Subsequent to these studies, we hope to extend the application of this approach to inhibit the extracellular nucleases of other pathogens, e.g., S. pneumoniae. In addition to the objective described above, the NucA-NuiA complex also serves as a structural model for inhibition of a Mg-dependent nuclease such as the HIV RNase H domain of RT. ? ? Project 3. Non-homologous end joining (NHEJ) is a pathway that can be used to repair double-strand breaks in DNA. It is referred to as """"""""non-homologous"""""""" because the break ends are directly ligated without the need for a homologous template, in contrast to homologous recombination, which requires a homologous sequence to guide repair. NHEJ is evolutionarily conserved throughout all kingdoms of life and is the predominant double-strand break repair pathway in many organisms, including higher eukaryotes such as human and mouse. Additionally, NHEJ may play a role in the integration of the HIV-1 genome into the host genome. Three of the four family X polymerases, DNA polymerase mu, DNA polymerase lambda, and terminal deoxynucleotidyl transferase (TdT), have been associated with repair of double-strand DNA breaks by nonhomologous endjoining (NHEJ). Their involvement in this DNA repair process requires an N-terminal BRCT domain that mediates interaction with other protein factors required for recognition and binding of broken DNA ends. During the past year, we determined the NMR solution structure of the BRCT domain of DNA polymerase lambda, completing the structural portrait for this family of enzymes. Analysis of the overall fold of the polymerase lambda BRCT domain reveals substantial structural similarity to the BRCT domains of polymerase mu and TdT, yet exhbits some key sequence and structural differences that may account for important differences in the biological activities of these enzymes and their roles in nonhomologous end-joining. Mutagenesis studies indicate that the conserved Arg57 residue of Pol mu plays a more critical role for binding to the XRCC4-Ligase IV complex than its structural homolog in Pol mu, Arg43. In contrast, the hydrophobic Leu60 residue of Pol lambda contributes less significantly to binding than the structurally homologous Phe46 residue of Pol mu. A third leucine residue involved in the binding and activity of Pol mu, is nonconservatively replaced by a glutamine in Pol lambda (Gln64) and, based on binding and activity data, is apparently unimportant for Pol lambda interactions with the NHEJ complex. Overall, both the structure of the Pol lambda BRCT domain and its mode of interaction with the other components of the NHEJ complex significantly differ from the two previously studied homologs, Pol mu and TdT.? ? ? Project 4. DNA polymerase IV has been considered as a model for the behavior of HIV reverse transcriptase. During the past year, we studied how interactions with various ligands alters the enzyme conformation utilizing methionine probes distributed throughout the enzyme. These results demonstrate characteristic perturbations that accompany DNA binding and conformational activation upon formation of an abortive ternary complex. In order to extend these results, we recently have worked toward the complete resonance assignment of the U-2H,13C,15N labeled enzyme. In general, we have found that many of the techniques developed for the assignment of selectively protonated methyl groups in proteins do not work well for Pol beta, probably due to its limited solubility. However, the use of residue-correlated labeling has proven more useful for this project. We also have investigated several of the cancer-associated mutations that have been identified in Pol beta. In particular, the E295K Pol beta mutant shows typical spectral perturbations in the presence of gapped DNA substrates, but fails to become conformationally activated upon addition of complementary nucleotides. Thus, the inactivity of this mutant can be understood in terms of a conformational effect. Further studies are in progress to elucidate how the presence of this inactive form might interfere with normal DNA repair.
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