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

George J. Klarmann, Brian M. Eisenhauer, Yi Zhang, Matthias Gotte, Janice D. Pata, Deb K. Chatterjee, Sidney Hecht, Stuart F J Le Grice

Research output: Contribution to journalArticle

6 Citations (Scopus)

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 (-)-β-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.

Original languageEnglish (US)
Pages (from-to)2118-2126
Number of pages9
JournalBiochemistry
Volume46
Issue number8
DOIs
StatePublished - Feb 27 2007
Externally publishedYes

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RNA-Directed DNA Polymerase
HIV-1
Amino Acids
Hydrogen
Hydrogen bonds
Affinity chromatography
DNA-Directed DNA Polymerase
Transcription
Affinity Chromatography
Base Pairing
Escherichia coli
Tyrosine
Human immunodeficiency virus 1 reverse transcriptase
Assays
Catalytic Domain
Substitution reactions
Nucleotides
2'-deoxycytidine 5'-triphosphate
Substrates
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Klarmann, G. J., Eisenhauer, B. M., Zhang, Y., Gotte, M., Pata, J. D., Chatterjee, D. K., ... Le Grice, S. F. J. (2007). Investigating the "steric gate" of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase by targeted insertion of unnatural amino acids. Biochemistry, 46(8), 2118-2126. https://doi.org/10.1021/bi061772w

Investigating the "steric gate" of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase by targeted insertion of unnatural amino acids. / Klarmann, George J.; Eisenhauer, Brian M.; Zhang, Yi; Gotte, Matthias; Pata, Janice D.; Chatterjee, Deb K.; Hecht, Sidney; Le Grice, Stuart F J.

In: Biochemistry, Vol. 46, No. 8, 27.02.2007, p. 2118-2126.

Research output: Contribution to journalArticle

Klarmann, George J. ; Eisenhauer, Brian M. ; Zhang, Yi ; Gotte, Matthias ; Pata, Janice D. ; Chatterjee, Deb K. ; Hecht, Sidney ; Le Grice, Stuart F J. / Investigating the "steric gate" of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase by targeted insertion of unnatural amino acids. In: Biochemistry. 2007 ; Vol. 46, No. 8. pp. 2118-2126.
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