This paper presents the first solution structure of the RNase H domain of HIV-1 reverse transcriptase (RT) determined by NMR methods. The solution conditions in this study were at physiological pH in the presence of Mg(2+). An investigation of the dependence of the (1)H-(15)N HSQC spectrum of the RNase H domain on [Mg(2+)] indicates that Mg(2+) produces significant, global effects on the amide chemical shifts, implying that divalent metal ion binding is important for stabilizing the structure of the isolated domain in solution. Analysis of amide shift data as a function of MgCl(2) concentration using either a single- or two-site binding model indicated that the latter provided a significantly improved fit, with the K(D) for site A = 2.7-3.2 mM and K(D) for site B approximately 35 mM, calculated on the assumption that site A is already occupied. Resonances of the [U-(13)C,(15)N]RNase H domain, measured at pH 6.8, in 80 mM MgCl(2), were assigned and NOESY data collected in order to determine the structure. Assignment of the NOESY spectra using the ARIA program resulted in a high-resolution structure for residues 6-114 which was similar to the crystal structure of the isolated domain,. The data were insufficient to define a compact structure for the C-terminal residues after 114. Residues I134-L138 located at the C-terminus are highly disordered and give rise to relatively sharp and intense amide resonances, while the amide resonances for the segment from E124 to A132 appear to be largely absent and are presumably subject to significant exchange broadening between different conformational states. Comparisons with crystal structure data for the full reverse transcriptase molecule indicate that the corresponding region is absent in nearly all of the crystal structures determined for the P2(1)2(1)2(1) space group, while these residues adopt an alpha-helix in structures determined for other symmetry groups. This structural heterogeneity indicates that significant conformational variability exists for this segment of the full reverse transcriptase enzyme as well, and the structure of the C-terminal peptide can be selected or deselected, depending on crystallization conditions. This analysis, along with the structural characterization contained herein, challenges the previous paradigm that the dynamic behavior of the isolated RNase H domain differs substantially from the behavior in the intact enzyme. The poor Mg(2+) binding and conformational flexibility of residues located near the active site indicate that substrate binding is a precondition for metal ion binding and for selecting the active site conformation of the RNase H domain.
Crystallographic characterization of DNA polymerase beta (pol beta) has suggested that multiple-domain and subdomain motions occur during substrate binding and catalysis. NMR studies of [methyl-(13)C]methionine-labeled pol beta were conducted to characterize the structural and dynamic response to ligand binding. The enzyme contains seven methionine residues, one of which is at the amino terminus and is partially removed by the expression system. Three of the methyl resonances were readily assigned using site-directed mutants. Assignment of the resonances of Met155, Met158, and Met191 was more difficult due to the spatial proximity of these residues, so that assignments were based on NOESY-HSQC data and on the response to paramagnetic Co(2+) addition, as well as shift perturbations observed for the site-directed mutants. The response of the methyl resonances to substrate binding was evaluated by the serial addition of a template oligonucleotide, a downstream 5'-phosphorylated oligonucleotide, and a primer oligonucleotide to create a two-nucleotide-gapped DNA substrate. Addition of the single-stranded template DNA resulted in selective broadening of the methyl resonance of Met18 in the 8 kDa lyase domain, and this resonance then shifted and sharpened upon addition of a 5'-phosphate-terminated downstream complementary oligonucleotide. Conversion of the two-nucleotide-gapped DNA substrate to a single-nucleotide-gapped substrate by incorporation of ddCMP produced a small perturbation of the Met236 resonance, which makes contact with the primer strand in the crystal structure. The addition of a second equivalent of ddCTP to form the pol beta-DNA-ddCTP ternary complex resulted in significant shifts for the resonances corresponding to Met155, Met191, Met236, and Met282. The Met155 methyl resonance is severely broadened, while the Met191 and Met282 resonances exhibit significant but less extreme broadening. Since only Met236 makes contact with the substrate, the effects on Met155, Met236, and Met282 result from indirect conformational and dynamic perturbations. Previous crystallographic characterization of this abortive complex indicated that a polymerase subdomain or segment (alpha-helix N) repositions itself to form one face of the binding pocket for the nascent base pair. Met282 serves as a probe for motion in this segment. Addition of Mg(2+)-dATP to pol beta in the absence of DNA produced qualitatively similar but much smaller effects on Met191 and Met155, but did not strongly perturb Met282, leading to the conclusion that Mg(2+)-dATP alone is insufficient to produce the large conformational changes that are observed in the abortive complex involving the gapped DNA with a blocked primer and ddNTP. Thus, the NMR data indicate that the nucleotide-DNA interaction appears to be essential for conformational activation.
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