Aids Related Nmr Research

Environmental Health Sciences

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Abstract

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) studies of model nuclease and polymerase systems, and 3) studies related to agents producing adventitious infections in immunocompromised patients. [unreadable] [unreadable] Project 1. 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. During the past year, we have determined the solution structure of the BRCT domain for a protein involved in NHEJ, human DNA polymerase. BRCT domains are typically involved in protein-protein interactions between factors required for the cellular response to DNA damage. The pol BRCT domain is atypical in that, unlike other reported BRCT structures, the pol Mu BRCT is neither part of a tandem grouping, nor does it appear to form stable homodimers. Although the sequence of the pol BRCT domain has some unique characteristics particularly the presence of > 10% proline residues - it forms the characteristic alpha-beta-alpha sandwich, in which three alpha helices lie above and below a central four-stranded beta-sheet. The structure of helix alpha1 is characterized by two solvent-exposed hydrophobic residues, F46 and L50, suggesting that this element may play a role in mediating interactions of pol with other proteins. Consistent with this argument, mutation of these residues, as well as the proximal, conserved residue R43, specifically blocked the ability of pol Mu to efficiently work together with NHEJ factors Ku and XRCC4-ligase IV to join non-complementary ends together in vitro. The structural, dynamic, and biochemical evidence reported here identifies a functional surface in the pol BRCT domain critical for promoting assembly and activity of the NHEJ machinery. Further, the similarity between the interaction regions of the BRCT domains of pol Mu and TdT support the conclusion that they participate in NHEJ as alternate polymerases.[unreadable] [unreadable] [unreadable] Project 2. The C-terminal domain of HIV Reverse Transcriptase exhibits ribonuclease H activity, and plays an essential role in the viability and pathogenicity of the virus. The role of this domain is to remove the genomic template RNA strand from the reverse transcribed RNADNA hybrid. This activity is not targeted by any current AIDS pharmaceutical. Confounding the ability to develop inhibitors is the enigma that the isolated RNase H domain is not active. A chimeric construct of the HIV and E. coli RNase H domains has been reported developed that is active in the presence of Mn2+ ions. With the goal of determining the structure of the chimeric protein, we have expressed unlabeled and U-15N labeled protein. Since most of the protein expressed in E. coli took the form of inclusion bodies, a refolding procedure needed to be developed. The refolded protein was judged to be folded based on circular dichroism (CD) spectroscopy. Further, the resulting CD spectrum is similar to the wild type HIV RT RNase H domain. However NMR spectra of the 15N labeled protein show only half the number of expected resonances, indicating that significant portions of the protein are subject to significant exchange broadening. Additionally, sample stability appears to be somewhat problematic. Crystallization screens were initiated to determine if crystals suitable for X-ray crystallography could be obtained. Continued effort will be centered on over-expressing the protein in a soluble form, so that it does not need to be refolded. Additionally, more crystallization trials will be attempted, and NMR spectra will be screened under different solution conditions in the hope of obtaining more suitable NMR spectra.[unreadable] [unreadable] Project 3. In contrast with the extensive literature on proteinase inhibition, only a limited amount of information has been available in the area of nuclease inhibition. We recently have been studying the structure of NucA, a cyanobacterial, non-specific nuclease that in many respects serves as a model for the RNase H domain of HIV reverse transcriptase. In particular, both the reverese transcriptase RNase H domain and NucA are sequence-independent, degrade double stranded substrates, and require Mg2+ for activity. Hence, a determination of the molecular basis for the inhibition of NucA by NuiA could provide useful insights for the development of inhibitors of HIV RNase H activity. We have determined the structure of the complex formed between NucA and its specific inhibitor, NuiA. The NucA-NuiA complex is characterized by an unusual divalent metal ion bridge that connects the nuclease with its inhibitor. The C-terminal Thr135NuiA hydroxyl oxygen is directly coordinated with the catalytic Mg2+ of the nuclease active site, and Glu24NuiA also extends into the active site, mimicking the charge of a scissile phosphate. [unreadable] [unreadable] Project 4. [unreadable] Individuals whose immune system has been compromised by HIV suffer from reduced resistance to a host of bacterial pathogens. As noted above, our interest in the bacterial nuclease NucA is based in part on the role that analogous nucleases play as a countermeasure to the host neutrophil defense system. The nuclease inhibitor NuiA may be considered as a model for the inhibition of such extracellular nucleases. A second line of research in this area has been the study of a plasmid-encoded, Type II dihydrofolate reductase (DHFR). Bacterial resistance to antibiotic therapy is an increasing problem particularly for AIDS patients. The Type II DHFR is resistant to anti-folate drugs that target bacterial DHFR, and hence represents an emerging bacterial resistance mechanism. During the past year, we refined our structure of the catalytic complex formed from the Type II DHFR, NADP and dihydrofolate. The structure indicates that the type II DHFR is able to recognize the substrate and cofactor by a parallel mode of binding that involves interaction of Ile68 carbonyl and amide groups with the carboxamide group of NADP and with the N3-O4 amide group of the pteridine ring system. Crystallography and inter-ligand Overhauser effect studies show that the two ring systems adopt a relative endo geometry, characterized by tilted aromatic rings which approach each other most closely at nicotinamide C4-pteridine C6, corresponding to the reactive positions on the corresponding substrates. This binding mode differs from that observed in Type I DHFR, but is similar to that recently observed in pteridine reductase. This study provides a basis for understanding trimethoprim resistance conferred by the enzyme and for the development of Type II DHFR-targeted inhibitors.