Investigating the "Steric Gate" of Human Immunodeficiency Virus Type 1 (HIV-1) Reverse Transcriptase by Targeted Insertion of Unnatural Amino Acids
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- 作者：George J. Klarmann ; Brian M. Eisenhauer ; Yi Zhang ; Matthias Gotte ; Janice D. Pata ; Deb K. Chatterjee ; Sidney M. Hecht ; Stuart F. J. Le Grice
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：February 27, 2007
- 年：2007
- 卷：46
- 期：8
- 页码：2118 - 2126
- 全文大小：471K
- 年卷期：v.46,no.8(February 27, 2007)
- ISSN：1520-4995
文摘
To investigate how structural changes in the amino acid side chain affect nucleotide substrateselection 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 coupledtranscription/translation system. Mutant p66 subunits were reconstituted with wild-type (WT) p51 RTand purified by affinity chromatography. Each modified enzyme retained DNA polymerase activityfollowing this procedure. Aminomethyl-Phe115 RT incorporated dCTP more efficiently than the WT andwas resistant to the chain terminator (-)-
-2',3'-dideoxy-3'-thiacytidine triphosphate (3TCTP) whenexamined in a steady-state fidelity assay. However, 2-naphthyl-Tyr115 RT inefficiently incorporated dCTPat low concentrations and was kinetically slower with all dCTP analogues tested. Models of RT containingthese side chains suggest that the aminomethyl-Phe115 substitution provides new hydrogen bonds throughthe minor groove to the incoming dNTP and the template residue of the terminal base pair. These hydrogenbonds likely contribute to the increased efficiency of dCTP incorporation. In contrast, models of HIV-1RT containing 2-naphthyl-Tyr115 reveal significant steric clashes with Pro157 of the p66 palm subdomain,necessitating rearrangement of the active site.
-2',3'-dideoxy-3'-thiacytidine triphosphate (3TCTP) whenexamined in a steady-state fidelity assay. However, 2-naphthyl-Tyr115 RT inefficiently incorporated dCTPat low concentrations and was kinetically slower with all dCTP analogues tested. Models of RT containingthese side chains suggest that the aminomethyl-Phe115 substitution provides new hydrogen bonds throughthe minor groove to the incoming dNTP and the template residue of the terminal base pair. These hydrogenbonds likely contribute to the increased efficiency of dCTP incorporation. In contrast, models of HIV-1RT containing 2-naphthyl-Tyr115 reveal significant steric clashes with Pro157 of the p66 palm subdomain,necessitating rearrangement of the active site.