title: The amino acid Asn136 in HIV-1 reverse transcriptase (RT) maintains efficient association of both RT subunits and enables the rational design of novel RT inhibitors.
abstract: The highly conserved Asn136 is in close proximity to the nonnucleoside 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 Asn136 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 (denaturating) 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 Asn136 in HIV-1 RT with other amino acids resulted in notably increased amounts of free p51 and p66 monomers. Our findings identify a structural/functional role for Asn136 in stabilization of the RT p66/p51 dimer and provide hints for the rational design of novel NNRTIs or drugs targeting either Asn136 in the beta7-beta8 loop of p51 or its anchoring point on p66 (the peptide backbone of His96) so as to interfere with the RT dimerization 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.
authors.affiliation: Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium. jan.balzarini@rega.kuleuven.be
authors.forename: Jan
authors.initials: J
authors.lastname: Balzarini
authors.forename: Joeri
authors.initials: J
authors.lastname: Auwerx
authors.forename: Fátima
authors.initials: F
authors.lastname: Rodríguez-Barrios
authors.forename: Allel
authors.initials: A
authors.lastname: Chedad
authors.forename: Viktor
authors.initials: V
authors.lastname: Farkas
authors.forename: Francesca
authors.initials: F
authors.lastname: Ceccherini-Silberstein
authors.forename: Carlos
authors.initials: C
authors.lastname: García-Aparicio
authors.forename: Sonsoles
authors.initials: S
authors.lastname: Velázquez
authors.forename: Erik
authors.initials: E
authors.lastname: De Clercq
authors.forename: Carlo-Federico
authors.initials: CF
authors.lastname: Perno
authors.forename: María-José
authors.initials: MJ
authors.lastname: Camarasa
authors.forename: Federico
authors.initials: F
authors.lastname: Gago
mesh_terms: D001216:Asparagine / Q000737:chemistry / Q000235:genetics*; D002384:Catalysis; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D015195:Drug Design; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D009154:Mutation; D017433:Protein Structure, Secondary / Q000235:genetics; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D018894:Reverse Transcriptase Inhibitors / Q000138:chemical synthesis* / Q000494:pharmacology
chemical_list: D021122:Protein Subunits; D018894:Reverse Transcriptase Inhibitors; D001216:Asparagine; D054303:HIV Reverse Transcriptase; D004259:DNA-Directed DNA Polymerase