Mutational sensitivity patterns define critical residues in the palm subdomain of the reverse transcriptase of human immunodeficiency virus type 1.
We have analyzed 154 single amino acid replacement mutants within a 40 amino acid region (residues 164-203) of the reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1). This region consists of two antiparallel beta-strands (strands 9 and 10) flanked by two alpha helices (E and F). The structure of this region of the 'palm' subdomain is conserved in a variety of DNA and RNA polymerases, indicating a critical role in enzyme structure and function. Functional assays were performed by screening RT activity of mutants expressed in E. coli. A functionally important region corresponding closely to beta-strands 9 and 10 and the loop joining them was revealed by its mutational sensitivity. Structural analysis of mutants was performed by using Western blots to assay correct folding, which is required for processing to produce the mature p66 and p51 RT species. This analysis indicates that beta-strand 10 is a structurally important region. Combined analysis of these two assays revealed diagnostic patterns of mutational sensitivity which identify key positions in the RT sequence at which a specific amino acid side chain is critical, either for structure or function, as well as residues which are external to the RT structure. This work illustrates the utility of large-scale mutagenesis in relating primary sequence to significant features of protein structure and function.
Chao
S F
SF
Department of Microbiology and Immunology, University of North Carolina at Chapel Hill 27599, USA.
Chan
V L
VL
Juranka
P
P
Kaplan
A H
AH
Swanstrom
R
R
Hutchison
C A
CA
D000595:Amino Acid Sequence; D002384:Catalysis; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase