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. 2012 Feb 3;287(6):4066-75.
doi: 10.1074/jbc.M111.314781. Epub 2011 Nov 21.

Mutagenesis of Human Immunodeficiency Virus Reverse Transcriptase p51 Subunit Defines Residues Contributing to Vinylogous Urea Inhibition of Ribonuclease H Activity

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Mutagenesis of Human Immunodeficiency Virus Reverse Transcriptase p51 Subunit Defines Residues Contributing to Vinylogous Urea Inhibition of Ribonuclease H Activity

Suhman Chung et al. J Biol Chem. .
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Abstract

The vinylogous urea, NSC727447, was proposed to allosterically inhibit ribonuclease H (RNase H) activity of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) by interacting with the thumb subdomain of its non-catalytic p51 subunit. Proximity of the p51 thumb to the p66 RNase H domain implied that inhibitor binding altered active site geometry, whereas protein footprinting suggested a contribution from α-helix I residues Cys-280 and Lys-281. To more thoroughly characterize the vinylogous urea binding site, horizontal alanine scanning mutagenesis between p51 residues Lys-275 and Thr-286 (comprising α-helix I and portions of the neighboring αH/αI and αI/αJ connecting loops) was combined with a limited vertical scan of Cys-280. A contribution from Cys-280 was strengthened by our observation that all substitutions at this position rendered selectively mutated, reconstituted p66/p51 heterodimers ∼45-fold less sensitive to inhibition. An ∼19-fold reduced IC(50) for p51 mutant T286A coupled with a 2-8-fold increased IC(50) when intervening residues were substituted supports our original proposal of p51 α-helix I as the vinylogous urea binding site. In contrast to these allosteric inhibitors, mutant enzymes retained equivalent sensitivity to the natural product α-hydroxytropolone inhibitor manicol, which x-ray crystallography has demonstrated functions by chelating divalent metal at the p66 RNase H active site. Finally, reduced DNA strand-transfer activity together with increased vinylogous urea sensitivity of p66/p51 heterodimers containing short p51 C-terminal deletions suggests an additional role for the p51 C terminus in nucleic acid binding that is compromised by inhibitor binding.

Figures

FIGURE 1.
FIGURE 1.
a, shown is the structure of the p66/p51 HIV-1 RT heterodimer. p66 subunits are color-coded as follows: blue, fingers; red, palm; green, thumb; yellow, connection (Conn.); gold, RNase H. Only the p51 thumb subdomain has been color-coded in green. b, expanded illustration of the p66 RNase H domain and p51 thumb is shown. Thumb residues substituted with alanine are denoted in white, active site carboxylates of the RNase H domain are in red, and the conserved His-539 is in magenta. Data were based the RT/manicol cocrystal of Chung et al. (24). c, procedures were as b but illustrate residues of p51 thumb (Cys-280—Thr-290, white) and the p66 RNase H domain (Pro-537—Glu-546, gold) that contribute to the buried surface at the dimer interface. The conserved His-539 of the RNase H domain is indicated in magenta.
FIGURE 2.
FIGURE 2.
a, PPT cleavage activity of selectively mutated reconstituted p51 thumb mutants is shown. The hydrolysis product indicating cleavage at the PPT/U3 junction is indicated. b, shown is an evaluation of DNA polymerase and RNase H activity of p51 thumb mutants via their ability to support DNA strand transfer between 40-nt RNA templates sharing 20 nt of homology. DNA polymerase and RNase H hydrolysis products are indicated in green and red, respectively. STI40 and STP60 indicate the 40-nt strand transfer intermediate and the 60-nt strand transfer product, respectively. Lane 1, migration positions of the 20-nt Cy3-labeled DNA primer (P20) and 40-nt Cy5-lebeled donor RNA template (T40), respectively. Lane 2, wild type RT. Rn denotes the size of the RNase H hydrolysis product.
FIGURE 3.
FIGURE 3.
DNA strand transfer activity of selectively deleted p66/p51 HIV-1 RT heterodimers. The extent of the p51 C-terminal deletion is indicated above the figure. Nucleic acid substrate, DNA synthesis, and RNase H hydrolysis product notations are described in the legend of Fig. 2.

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