Regulation of autoproteolysis of the HIV-1 and HIV-2 proteases with engineered amino acid substitutions.
Autoproteolysis of the retroviral aspartyl proteases is a major obstacle to purification and analysis of these enzymes. A mutagenic approach to rendering autolytic cleavage sites less labile was applied to the primary cleavage site between Leu5 and Trp6 in human immunodeficiency virus-1 (HIV-1) protease. From predictions based on known substrates it was concluded that amino acids Lys or Ser in place of Gln at position 7 would prevent cleavage at the Leu5-Trp6 peptide bond, therefore stabilizing the protein. Autoproteolytic stability was enhanced at least 100-fold by these mutations. At longer time points the protease was degraded at secondary sites which contained adequate substrate sequences but were conformationally restricted. Conversely, a mutation in HIV-2 protease which changed Lys7 to Gln rendered the protein 3-fold less stable and shifted the position of the initial autoproteolytic cleavage from Phe3-Ser4 to Leu5-Trp6. The effects of these mutations demonstrate that small changes in protein sequence can have a major impact on their autoproteolytic stability. The work described here suggests a general method for stabilizing proteases and perhaps other recombinantly produced proteins to autolysis.
Department of Pharmacology, University of California, San Francisco 94143-0446.
J R
JR
Rosé
R
R
Salto
C S
CS
Craik
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D016333:HIV Protease / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D015498:HIV-2 / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D007930:Leucine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000378:metabolism; D015302:Simian Immunodeficiency Virus / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity; D014364:Tryptophan
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014364:Tryptophan; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; D016333:HIV Protease; C065184:p16 protease, Human immunodeficiency virus 2; D007930:Leucine