title: Auto-inactivation by cleavage within the dimer interface of Kaposi's sarcoma-associated herpesvirus protease.
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.
authors.affiliation: Graduate Group in Biophysics, University of California, San Francisco, USA.
authors.forename: T R
authors.initials: TR
authors.lastname: Pray
authors.forename: A M
authors.initials: AM
authors.lastname: Nomura
authors.forename: M W
authors.initials: MW
authors.lastname: Pennington
authors.forename: C S
authors.initials: CS
authors.lastname: Craik
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D019281:Dimerization; D019288:Herpesvirus 8, Human / Q000201:enzymology / Q000235:genetics; D006801:Humans; D007700:Kinetics; D016366:Open Reading Frames; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C107889:human herpesvirus 8 protease