HIV-1 protease: mutagenesis of asparagine 88 indicates a domain required for dimer formation.
Considerable interest exists in the HIV-1 protease for biochemical studies as a potential therapeutic target of acquired immunodeficiency syndrome. We have produced the retroviral enzyme in E. coli from a synthetic gene encoding the protease that was constructed by assembling six overlapping and complementary oligonucleotides into the vector pKK223-3. When expressed in E. coli, the recombinant protease was able to correctly process the HIV-1 core protein p24 from a beta-galactosidase-gag fusion protein and to use a heptapeptide as a substrate for proteolytic cleavage. A single base pair mutation was identified in a recombinant that resulted in the substitution of lysine for asparagine at position 88 and a significant loss of enzyme activity. Through site-directed mutagenesis, the Asn88 was changed to five other residues representative of all classes of amino acids. The correlation between enzyme activity and amino acid substitution suggests that the protease domain surrounding position 88 affects the protein's potential for forming an active homodimeric protein and hence, indicates a biochemical interaction that could be inhibited by novel antiviral compounds.
Guenet
C
C
Merrell Dow Research Institute, Strasbourg, France.
Leppik
R A
RA
Pelton
J T
JT
Moelling
K
K
Lovenberg
W
W
Harris
B A
BA
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001216:Asparagine / Q000235:genetics*; D001483:Base Sequence; D010450:Endopeptidases / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D005813:Genes, Synthetic; D005814:Genes, Viral; D016333:HIV Protease; D015497:HIV-1 / Q000201:enzymology*; D008969:Molecular Sequence Data; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics
D000911:Antibodies, Monoclonal; D011993:Recombinant Fusion Proteins; D001216:Asparagine; D010450:Endopeptidases; D016333:HIV Protease