Conformational change coupling the dimerization and activation of KSHV protease.
The mechanism of herpesviral protease activation upon dimerization was studied using two independent spectroscopic assays augmented by directed mutagenesis. Spectroscopic changes, attributable to dimer interface conformational plasticity, were observed upon dimerization of Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr). KSHV Pr's dissociation constant of 585 +/- 135 nM at 37 degrees C was measured by a concentration-dependent, 100-fold increase in specific activity to a value of 0.275 +/- 0.023 microM product min(-1) (microM enzyme)(-1). A 4 nm blue-shifted fluorescence emission spectrum and a 25% increase in ellipticity at 222 nm were detected by circular dichroism upon dimer association. This suggested enhanced hydrophobic packing within the dimer interface and/or core, as well as altered secondary structures. To better understand the structure-activity relationship between the monomer and the dimer, KSHV Pr molecules were engineered to remain monomeric via substitution of two separate residues within the dimer interface, L196 and M197. These mutants were proteolytically inactive while exhibiting the spectroscopic signature and thermal stability of wild type, dissociated monomers (T(M) = 75 degrees C). KSHV Pr conformational changes were found to be relevant in vivo, as the autoproteolytic inactivation of KSHV Pr at its dimer disruption site [Pray et al. (1999) J. Mol. Biol. 289, 197-203] was detected in viral particles from KSHV-infected cells. This characterization of structural plasticity suggests that the structure of the KSHV Pr monomer is stable and significantly different from its structure in the dimer. This structural uniqueness should be considered in the development of compounds targeting the dimer interface of KSHV Pr monomers.
Pray
Todd R
TR
Graduate Group in Biophysics, Department of Pharmaceutical Chemistry and of Biochemistry and Biophysics, The University of California, San Francisco, California 94143, USA.
Reiling
K Kinkead
KK
Demirjian
Berj G
BG
Craik
Charles S
CS
D002460:Cell Line; D002942:Circular Dichroism; D019281:Dimerization; D004789:Enzyme Activation / Q000235:genetics; D019288:Herpesvirus 8, Human / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012514:Sarcoma, Kaposi / Q000201:enzymology* / Q000821:virology*; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014779:Virus Replication
D012697:Serine Endopeptidases; C107889:human herpesvirus 8 protease