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design. Mutagenesis studies have demonstrated the interdependence of DNA polymerase and RNase H
activities. Mutations that disrupt one of the two enzymatic activities of HIV-1 RT often also impair the
second activity [96–99]. Indeed, according to the crystal structure of HIV-1 RT, the polymerase active
site and the RNase H active site are separated by approximately 17–18 nucleotides [38] and the RNase
H domain has many contacts with the polymerase domain, especially with the connection subdomain of
p66 and the thumb and connection subdomains of p51 [31,38,100]. Interactions between the polymerase
domain and nucleic acid can modulate RNase H activity. Because the predominant contacts of HIV-1
RT with template-primer occur in the vicinity of the polymerase active site, precise placement of the
template strand relative to the RNase H active site may be regulated by the sequence and composition of
the template-primer. Mutagenesis experiments showed that mutations located at or near the “template
grip” in the polymerase domain of HIV-1 RT can have a greater effect on RNase H than on polymerase
activity [99,101,102]. It was also reported that binding of the NNRTI nevirapine alters the cleavage
specificity of RNase H [78]. Structural distortions in the position and conformation of template-primer
induced by NNRTI-binding may account for alteration of the cleavage specificity of RNase H [43].
Divalent metal ions such as Mg 2+ or Mn 2+ are essential for the RNase H activity [103–106]. The
structure of the isolated RNase H domain crystallized in the presence of MnCl 2 revealed two tightly
bound Mn 2+ ions in close proximity to four catalytically essential acidic residues, Asp443, Glu478,
Asp498, and Asp549, that form the active site [94]. Biochemical data have shown that mutations of
these conserved residues could either disrupt RNase H activity or lead to a highly unstable enzyme
[107–109]. Based on the crystal structures, a two-metal ion-dependent catalytic mechanism for RNase H
activity has been postulated [101], which is similar to that proposed for phosphoryl transfer reactions
catalyzed by polymerases and their associated nucleases [67,69–71]. In contrast, in the structure E. coli
RNase H reported by Katayanagi et al. [111] only one Mg 2+ ion was observed, and that led to the
proposal of a single metal-ion catalyzed hydrolysis [112]. Interestingly, in the structure of unliganded
HIV-1 RT reported by Rodgers et al. [41] and Hsiou et al. [43] only one Mg 2+ ion was found at the
RNase H active site. The mechanism of RNase H cleavage and the exact role of metal ion(s) in the
hydrolysis and formation of phosphodiester bonds are still under investigation (see review [89]).
Very few inhibitors specifically target HIV-1 RNase H activity. Illimaquinone, a natural marine product,
was shown to preferentially inhibit the HIV-1 RNase H activity [113,114]. However, this compound
appears to react with a sulfhydryl group in the polymerase domain and not with RNase H itself. It may
be possible to use the available information on structural and biochemi-
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