Important note: Information in this article was accurate in 1999. The state of the art may have changed since the publication date.
Auto-inactivation by cleavage within the dimer interface of Kaposi's sarcoma-associated herpesvirus protease.
J Mol Biol. 1999 Jun 4;289(2):197-203. Unique Identifier : AIDSLINE MED/99296622 Pray TR; Nomura AM; Pennington MW; Craik CS; Graduate Group in Biophysics, University of California, San; Francisco, USA.
Abstract:
An autolysis site of functional and structural significance has been mapped within the dimer interface of Kaposi's sarcoma-associated herpesvirus protease. Cleavage 27 residues from the C terminus of the 230 amino acid residue, 25 kDa protein was observed to cause a loss of dimerization and proteolytic activity, even though no active site moieties were lost. Gel-filtration chromatography and analytical ultracentrifugation were used to analyze the changes in oligomerization upon autolysis. The selective auto-disruption of this essential protein-protein interface by proteolytic cleavage resulted in a 60 % loss in mean residue ellipticity by circular dichroism as well as a 20 % weaker, 10 nm red-shifted intrinsic protein fluorescence emission spectrum. These apparent conformational changes induced a strict inhibition of enzymatic activity. An engineered substitution at the P1' position of this cleavage site attenuated autolysis by the enzyme and restored wild-type dimerization. In addition to retaining full proteolytic activity in a continuous fluorescence-based enzyme assay, this protease variant allowed the determination of the enzyme's dimerization dissociation constant of 1.7 (+/-0.9) microM. The structural perturbations observed in this enzyme may play a role in viral maturation, and offer general insight into the allosteric relationship between the dimer interface and active site of herpesviral proteases. The functional coupling between oligomerization and activity presented here may allow for a better understanding of such phenomena, and the design of an enzyme variant stabilized to autolysis should further the structural and mechanistic characterization of this viral protease. Copyright 1999 Academic Press.
Keywords: JOURNAL ARTICLE Amino Acid Sequence Binding Sites Catalytic Domain Dimerization Herpesvirus, Kaposi Sarcoma-Associated/ENZYMOLOGY/GENETICS Human Kinetics Open Reading Frames Protein Conformation Protein Structure, Secondary Recombinant Proteins/CHEMISTRY/METABOLISM Sequence Alignment Serine Proteinases/*CHEMISTRY/GENETICS/*METABOLISM Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S.
990930
A9990588
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Important note: Information in this article was accurate in 1999. The state of the art may have changed since the publication date.
