This project utilizes state-of-the-art NMR spectroscopy to study problems that are of continuing interest to the area of environmental health. The primary emphasis during the recent review period involves several different research projects: 1) characterization of the structural and dynamic behavior of the bacterial nucleotide excision repair protein UvrB in order to understand the molecular basis for damage recognition; 2) fundamental studies of the relationships between the 13C shifts of amino acids and their conformation in proteins; 3) structural work on dust mite allergens; and 4) structural analysis of type II Dihydrofolate reductase (DHFR), a plasmid-encoded enzyme that confers resistance to bacteria-targeted antifolate antibiotics. ? ? Project 1. Nucleotide excision repair (NER) is an essential DNA repair mechanism which is highly conserved among all biological systems. Of all the repair mechanisms, it exhibits the greatest versatility, characterized by the extremely heterogeneous types of damage shown to be recognized and corrected. In prokaryotes, NER is accomplished by three enzymes: Uvr A, B and C. Structural information has been limited by the apparent disorder of the C-terminal domain 4 in crystal structures of intact UvrB; in solution, the isolated domain 4 is found to form a helix-loop-helix dimer. In order to gain insight into the solution behavior of UvrB, we have performed NMR studies on methyl-13C-methionine-labeled UvrB (MW = 75 kD) from B. caldotenax. The 13 methyl resonances were assigned on the basis of site-directed mutagenesis and domain deletion. Analysis of the resonance assigned to M632, located at the interface of the domain 4 dimer, demonstrated that a UvrB-UvrB-domain 4 heterodimer could form, but that a UvrB-UvrB homodimer appeared to be disfavored in solution. In order to characterize the nature of the interaction between UvrB and DNA, subsequent studies have investigated the interaction of UvrB with DNA hairpins. Based on crystal structure data showing the involvement of the UvrB hairpin (residues S91-D117), we have introduced methionine probe residues at positions 109 and 115 using site-directed mutagenesis. Initial results demonstrate that the spectral changes observed in the presence of hairpin DNA are consistent with expectations based on the crystal structure. In order to extend these conclusions, studies of other modified DNAs are currently in progress.? ? Project 2. Chemical shift data from the BiomagResDataBank and conformational data derived from the protein data bank have been correlated in order to explore the conformational dependence of sidechain 13C resonance shifts. Consistent with predictions based on steric compression, upfield shifts for Cγ resonances of Thr, Val, Ile, Leu, Met, Arg, Lys, Glu, and Gln residues correlate with both the number of heavy atom (non-proton) γ-substituents and with gauche conformational orientations of γ-substituents. The 13C shift/conformation correlations are most apparent for Cγ carbons, but also can be observed at positions further from the backbone. Intra-residue steric conflict leads to a correlation between upfield-shifted sidechain 13C resonances and statistically lower probabilities in surveys of protein sidechain conformation. Illustrative applications to the DNA pol lambda lyase domain, and to dihydrofolate reductase are discussed. In the latter case, 13C shift analysis indicates that the conformation of the remote residue V119 on the βF-βG loop is correlated with the redox state of the bound pyridine nucleotide cofactor, providing one basis for discrimination between substrate and product. Anlaysis of 13C shift data for protein sidechains should provide a useful basis for the analysis of conformational changes even in large, deuterated proteins. Additionally, the large dependence of the leucine methyl shift difference, δCδ1-δCδ2, on both χ1 and χ2 is sufficient to allow this parameter to be used as a restraint in structure calculations if stereospecific assignment data are available.? ? Project 3. Inhalation allergy to house dust mite allergens is among the most prevalent allergic diseases. Our studies of the structure of dust mite allergens have focused in two specific examples: Der p 5 and Der p 7. We found that Der p 5 can be overexpressed with a GST-tag and purified after TEV cleavage. NMR spectra of the protein were of marginal quality, however, the protein crystallized readily and gave data to 3.2 A. Unfortunately, even an NMR based homology model using the structure of Blo t 5 was unable to solve the phase problem. Currently the coding sequence has been moved into vectors containing an MBP tag that has been modified for enhanced crystallization. It is anticipated that co-crystals of MBP-Der p 5 should solve the phase problem. Similarly, various constructs of Der p 7 have been tested, and a construct that starts at residue 18 expresses the most soluble protein. NMR spectra were evaluated as poor. The protein can be concentrated to greater than 50 mg/ml and crystallizes readily. However, again, the crystals diffract from 3-11 A which is insufficient to solve the phase problem without a high resolution homology model. Again our current strategy is to try co-crystals using a modified MBP vector.? ? Project 4. Type II dihydrofolate reductase (DHFR) is a plasmid-encoded enzyme that confers resistance to bacterial DHFR-targeted antifolate drugs. It forms a symmetric homotetramer with a central pore which functions as the active site. Its unusual structure, which results in a promiscuous binding surface that accommodates either the Dihydrofolate (DHF) substrate or the NADPH cofactor, has constituted a significant limitation to efforts to understand its substrate specificity and reaction mechanism. We have determined the first structure of a ternary R67 DHFR-dihydrofolate-NADP+ catalytic complex, resolved to 1.26 A. This structure provides the first clear picture of how this enzyme, which lacks the active site carboxyl residue that is ubiquitous in Type I DHFRs, is able to function. In the catalytic complex, the polar backbone atoms of two symmetry-related I68 residues provide recognition motifs that interact with the carboxamide on the nicotinamide ring, and the N3-O4 amide function on the pteridine. This set of interactions orients the aromatic rings of substrate and cofactor in a relative endo geometry in which the reactive centers are held in close proximity. Additionally, a central, hydrogen-bonded network consisting of two pairs of Y69-Q67-Q67'-Y69' residues provides an unusually tight interface, which appears to serve as a molecular clamp holding the substrates in place in an orientation conducive to hydride transfer. In addition to providing the first clear insight regarding how this extremely unusual enzyme is able to function, the structure of the ternary complex provides general insights into how a mutationally-challenged enzyme, i.e., an enzyme whose evolution is restricted to four-residues-at-a-time active site mutations, overcomes this fundamental limitation.
| Wallace, Bret D; Berman, Zachary; Mueller, Geoffrey A et al. (2017) APE2 Zf-GRF facilitates 3'-5' resection of DNA damage following oxidative stress. Proc Natl Acad Sci U S A 114:304-309 |
| Gabel, Scott A; Smith, Cassandra E; Cuneo, Matthew J et al. (2014) Characterization of the redox transition of the XRCC1 N-terminal domain. Structure 22:1754-1763 |
| Gabel, Scott A; DeRose, Eugene F; London, Robert E (2013) XRCC1 interaction with the REV1 C-terminal domain suggests a role in post replication repair. DNA Repair (Amst) 12:1105-13 |
| Loeffler, Paul A; Cuneo, Matthew J; Mueller, Geoffrey A et al. (2011) Structural studies of the PARP-1 BRCT domain. BMC Struct Biol 11:37 |
| Butterfoss, Glenn L; DeRose, Eugene F; Gabel, Scott A et al. (2010) Conformational dependence of 13C shielding and coupling constants for methionine methyl groups. J Biomol NMR 48:31-47 |
| London, Robert E; Wingad, Brett D; Mueller, Geoffrey A (2008) Dependence of amino acid side chain 13C shifts on dihedral angle: application to conformational analysis. J Am Chem Soc 130:11097-105 |
| DeRose, Eugene F; Clarkson, Michael W; Gilmore, Steven A et al. (2007) Solution structure of polymerase mu's BRCT Domain reveals an element essential for its role in nonhomologous end joining. Biochemistry 46:12100-10 |
| DellaVecchia, Matthew J; Merritt, W Keither; Peng, Ye et al. (2007) NMR analysis of [methyl-13C]methionine UvrB from Bacillus caldotenax reveals UvrB-domain 4 heterodimer formation in solution. J Mol Biol 373:282-95 |
| Krahn, Joseph M; Jackson, Michael R; DeRose, Eugene F et al. (2007) Crystal structure of a type II dihydrofolate reductase catalytic ternary complex. Biochemistry 46:14878-88 |
| London, Robert E; Gabel, Scott A (2006) Photoactivated h/d exchange in tyrosine: involvement of a radical anion intermediate. J Am Chem Soc 128:2268-75 |
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