Investigating the “Steric Gate” of Human Immunodeficiency Virus Type 1 (HIV-1) Reverse Transcriptase by Targeted Insertion of Unnatural Amino Acids
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-...
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Published in | Biochemistry (Easton) Vol. 46; no. 8; pp. 2118 - 2126 |
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Main Authors | , , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Chemical Society
27.02.2007
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Subjects | |
Online Access | Get full text |
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Summary: | 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. |
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Bibliography: | This research was supported in part by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research (to G.J.K. and S.F.J.LeG.) and by the Canadian Institutes of Health Research (to M.G.). This project has been funded in whole or part with federal funds from the National Cancer Institute, National Institutes of Health, under contract number N01-CO-12400 (to D.K.C.). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. ark:/67375/TPS-XQX16FGH-3 istex:3D17DD3A82F14FF5FCA088374634AB66D05DEDC1 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0006-2960 1520-4995 |
DOI: | 10.1021/bi061772w |