Investigating the "steric gate" of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase by targeted insertion of unnatural amino acids

Biochemistry. 2007 Feb 27;46(8):2118-26. doi: 10.1021/bi061772w. Epub 2007 Feb 3.

Abstract

To investigate how structural changes in the amino acid side chain affect nucleotide substrate selection in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), a variety of non-natural tyrosine analogues were substituted for Tyr115 of p66 RT. RT variants containing meta-Tyr, nor-Tyr, aminomethyl-Phe, and 1- and 2-naphthyl-Tyr were produced in an Escherichia coli coupled transcription/translation system. Mutant p66 subunits were reconstituted with wild-type (WT) p51 RT and purified by affinity chromatography. Each modified enzyme retained DNA polymerase activity following this procedure. Aminomethyl-Phe115 RT incorporated dCTP more efficiently than the WT and was resistant to the chain terminator (-)-beta-2',3'-dideoxy-3'-thiacytidine triphosphate (3TCTP) when examined in a steady-state fidelity assay. However, 2-naphthyl-Tyr115 RT inefficiently incorporated dCTP at low concentrations and was kinetically slower with all dCTP analogues tested. Models of RT containing these side chains suggest that the aminomethyl-Phe115 substitution provides new hydrogen bonds through the minor groove to the incoming dNTP and the template residue of the terminal base pair. These hydrogen bonds likely contribute to the increased efficiency of dCTP incorporation. In contrast, models of HIV-1 RT containing 2-naphthyl-Tyr115 reveal significant steric clashes with Pro157 of the p66 palm subdomain, necessitating rearrangement of the active site.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Binding Sites
  • Cell-Free System
  • Cytidine Triphosphate / analogs & derivatives
  • Cytidine Triphosphate / metabolism
  • Cytidine Triphosphate / pharmacology
  • DNA / biosynthesis
  • DNA / drug effects
  • Deoxycytosine Nucleotides / metabolism
  • Dideoxynucleotides
  • Escherichia coli
  • HIV Reverse Transcriptase / chemistry*
  • HIV Reverse Transcriptase / metabolism*
  • HIV-1 / enzymology*
  • Hydrogen Bonding
  • Kinetics
  • Lamivudine / analogs & derivatives
  • Lamivudine / metabolism
  • Lamivudine / pharmacology
  • Models, Molecular
  • Protein Biosynthesis
  • Protein Engineering*
  • Reverse Transcriptase Inhibitors / metabolism
  • Reverse Transcriptase Inhibitors / pharmacology
  • Transcription, Genetic
  • Tyrosine / analogs & derivatives

Substances

  • Deoxycytosine Nucleotides
  • Dideoxynucleotides
  • Reverse Transcriptase Inhibitors
  • lamivudine triphosphate
  • 2'-deoxycytidine 5'-triphosphate
  • Lamivudine
  • Tyrosine
  • Cytidine Triphosphate
  • DNA
  • HIV Reverse Transcriptase