One functional switch mediates reversible and irreversible inactivation of a herpesvirus protease.
Distinct mechanisms have evolved to regulate the function of proteolytic enzymes. Viral proteases in particular have developed novel regulatory mechanisms, presumably due to their comparatively rapid life cycles and responses to constant evolutionary pressure. Herpesviruses are a family of human pathogens that require a viral protease with a concentration-dependent zymogen activation involving folding of two alpha-helices and activation of the catalytic machinery, which results in formation of infectious virions. Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) is unique among the herpesvirus proteases in possessing an autolysis site in the dimer interface, which removes the carboxyl-terminal 27 amino acids comprising an alpha-helix adjacent to the active site. Truncation results in the irreversible loss of dimerization and concomitant inactivation. We characterized the conformational and functional differences between the active dimer, inactive monomer, and inactive truncated protease to determine the different protease regulatory mechanisms that control the KSHV lytic cycle. Circular dichroism revealed a loss of 31% alpha-helicity upon dimer dissociation. Comparison of the full-length and truncated monomers by NMR showed differences in 21% of the protein structure, mainly located adjacent to the dimer interface, with little perturbation of the overall protein upon truncation. Fluorescence polarization and active site labeling, with a transition state mimetic, characterized the functional effects of these conformational changes. Substrate turnover is abolished in both the full-length and truncated monomers; however, substrate binding remained intact. Disruption of the helix 6 interaction with the active site oxyanion loop is therefore used in two independent regulatory mechanisms of proteolytic activity.
Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California 94143, USA.
Anson M
AM
Nomura
Alan B
AB
Marnett
Nobuhisa
N
Shimba
Volker
V
Dötsch
Charles S
CS
Craik
D001665:Binding Sites; D002942:Circular Dichroism; D019281:Dimerization; D004789:Enzyme Activation / Q000502:physiology*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000502:physiology*
D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C107889:human herpesvirus 8 protease