Conformational Change Coupling the Dimerization and Activation of KSHV Protease

Abstract

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.

One Functional Switch Mediates Reversible and Irreversible Inactivation of a Herpesvirus Protease

“…Previous studies have shown that herpesvirus proteases are regulated by a dimerization-linked conformational change that disrupts the oxyanion loop necessary for transition state stabilization (18,(32)(33)(34). Dimerization induces the folding of helix 5 and helix 6, which stabilize the oxyanion hole and contribute to the surprisingly weak binding affinity of these proteases.…”

“…Comparison of multiple engineered mutations in the KSHV Pr interfacial helix, adjacent to the active site and in the active site, which result in obligate monomers, has shown that loss of dimerization and activity is not linked to any specific point mutant, such as M197D (18; data not shown). The M197D point mutation has been shown to result in obligate monomers, which are structurally indistinct from wild-type monomers isolated at temperatures which allow dimer dissociation (18). In addition, a monomer containing the M197D substitution as well as a cross-link between helix 6 and the oxyanion loop has proteolytic activity (34).…”

“…Additional internal cleavage sites have been identified in multiple herpesvirus proteases; however, KSHV Pr is unique in that autolysis at the D-site (dimer disruption site) leads to inactivation of the protease (17). D-site cleavage occurs in the virus; however, the significance of autolysis to the KSHV life cycle has yet to be clearly elucidated (18).…”

“…Herpesvirus proteases require dimerization for activity (17,18,(27)(28)(29)(30)(31) with binding affinities in the low micromolar to high nanomolar range; however, the dimeric X-ray structures all show the presence of intact active sites in each monomer, which are separate from the dimer interface. Early studies led to the hypothesis that a conformational change connects the active site and dimer interface, linking enzymatic activity to the protein oligomeric state (18,19,21,25,26,32,33). Recently, NMR spectroscopy and engineering of a redox switch, which allowed protease activity to be controlled by the addition of small molecule effectors (34), revealed that dissociation of herpesvirus dimers leads to unfolding of the two carboxyl-terminal R-helices.…”

Kinetics, inhibition and oligomerization of Epstein‐Barr virus protease

“…Examples are the use of 25% glycerol for HSV1 protease [21], 0.5 M Na 2 SO 4 and 10% glycerol for CMV [17], 30% sucrose for CMV [22], 0.8 M potassium phosphate and 25% glycerol for KSHV protease [23]. As most of the enzymological studies have been done in the presence of such agents little kinetic data has been determined in their absence.…”