A genetic approach for identifying critical residues in the fingers and palm subdomains of HIV-1 reverse transcriptase.
By using oligonucleotide-directed saturation mutagenesis, we collected 366 different single amino acid substitutions in a 109-aa segment (residues 95-203) in the fingers and palm subdomains of the HIV-1 reverse transcriptase (RT), the enzyme that replicates the viral genome. After expression in Escherichia coli, two phenotypic assays were performed. The first assay tested for RNA-dependent DNA polymerase activity. The other assay used Western blot analysis to estimate the stability of each mutant protein by measuring the processing of the RT into its mature heterodimeric form, consisting of a 66-kDa subunit and a 51-kDa subunit. The resulting phenotypic data provided a "genetic" means to identify amino acid side chains that are important for protein function or stability, as well as side chains located on the protein surface. Several HIV-1 RT crystal structures were used to evaluate the mutational analysis. Our genetic map correlates well with the crystal structures. Combining our phenotype data with crystallographic data allowed us to study the genetically defined critical residues. The important functional residues are found near the enzyme active site. Many residues important for the stability of the RT participate in potential hydrogen bonding or hydrophobic interactions in the protein interior. In addition to providing a better understanding of the HIV-1 RT, this work demonstrates the utility of saturation mutagenesis to study the function, structure, and stability of proteins in general. This strategy should be useful for studying proteins for which no crystallographic data are available.
Wrobel
J A
JA
Department of Microbiology and Immunology, University of North Carolina, Chapel Hill 27599, USA.
Chao
S F
SF
Conrad
M J
MJ
Merker
J D
JD
Swanstrom
R
R
Pielak
G J
GJ
Hutchison
C A
CA
D000595:Amino Acid Sequence; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004789:Enzyme Activation; D054303:HIV Reverse Transcriptase / Q000235:genetics* / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data
D054303:HIV Reverse Transcriptase