Abstract
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.
Highlights
Autoproteolysis of the retroviral aspartyl proteases encountered when purifying proteins
Cleavage sites for mutants generated in this study are included, showing that mutations affecting the primary cleavage sites did not affect the identity of the secondary cleavage sites identified in the wild-type enzymes
In this study we show that the identity time, minutes of residues whichwill prevent cleavage a t a given position can represents the ratio of active protein at a given time ( A )to the total Cleavage at the HIV-1proteaseLeu5-Trp6bond in the activeprotein at time = 0 (Ao).Decay curves weregenerated by amino-terminal sequence QITLWQRPoccurs very rapidly assayingremainingprotease activity at varioustime points (0). andissufficienttoinactivatethe enzyme
Summary
0 1993 byThe American Society for Biochemistry and MolecularBiology, Inc. Val. 268, No 16, Issue of June 5,pp. 11939-11945,1993 Printed in U.S.A. Subtilisin has shifted the position of the initial autoproteolytic cleav- recently been stabilized by immobilizing conformationally age from Phes-Ser4to Leu'-Trpa The effects of these flexible loops through calcium binding sites [6].These options mutations demonstrate that small changes in protein were not readily available for HIV-1 protease. We demonstrate here that HIV-1protease and HIV2 protease autoproteolysis may be regulated by appropriately engineered single amino acid substitutions that decrease or increase the probability of hydrolytic cleavage at a particular site These results suggest abroaderapproach to protease stabilization based on the identity of autocleavage sites and the specificity of the protease of interest
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