The Amino Acid N136 in Hiv-1 Reverse Transcriptase (rt) Maintains Efficient Association of Both Rt Subunits and Enables the Rational Design of Novel Rt Inhibitors

The highly conserved N136 is in close vicinity of the non-nucleoside reverse transcriptase (RT) inhibitor (NNRTI)-specific lipophilic pocket of human immunodeficiency virus type 1 (HIV-1) RT. Site-directed mutagenesis has revealed that the catalytic activity of HIV-1 RT mutated at position N136 is heavily compromised. Only 0.07 to 2.1% of wild-type activity is retained depending on the nature of the amino acid change at position 136. The detrimental effect of the mutations at position 136 occurred when the mutated amino acid was present in the p51 subunit, but not in the p66 subunit of the p51/p66 RT heterodimer. All mutant enzymes could be inhibited by second-generation NNRTIs such as efavirenz. They were also markedly more sensitive to the inactivating (denaturing) effect of urea than wild-type RT, and the degree of increased urea sensitivity was highly correlated with the degree of (lower) catalytic activity of the mutant enzymes. Replacing wild-type N136 in HIV-1 RT by other amino acids resulted in notably increased amounts of free p51 and p66 monomers. Our findings identify a structural/functional role for N136 in stabilization of the RT p66/p51 dimer and provide hints for the rational design of novel NNRTIs or drugs targeting either N136 in the β7-β8 loop of p51 or its anchoring point on p66 (the peptide backbone of H96) so as to interfere with the RT dimerisation process and/or with the structural support that the p51 subunit provides to the p66 subunit and which is essential for the catalytic enzyme activity. This article has not been copyedited and formatted. The final version may differ from this version. Molecular Pharmacology Fast Forward. Published on April 15, 2005 as DOI: 10.1124/mol.105.012435 at A PE T Jornals on Jne 9, 2017 m oharm .aspeurnals.org D ow nladed from