Background:  All human herpesviruses use a homologous virally encoded maturational protease for the formation of infectious virions.  Protease activity is regulated by dimerization, with each active dimer containing two spatially separate active sites. Circumstantial evidence supporting a relationship between activity and dimerization in herpesvirus proteases exists but, no direct link between the active site and the dimer interface or between the two active sites has been shown.

Methods:  Positional scanning-synthetic combinatorial libraries were used to define KSHV Pr substrate specificity.  A tetrapeptide diphenylphosphonate inhibitor was then synthesized targeting the active site of the enzyme. In addition to circular dichroism and size exclusion chromatography, an NMR-based assay was developed to monitor the native monomer-dimer equilibrium in solution and was used to demonstrate the effect of protease inhibition on the quaternary structure of the enzyme.  Activity assays were also employed to monitor the relationship between the two active sites.

Results:  Substrate library results revealed KSHV Pr to be a highly selective protease.  HSQC analysis of KSHV Pr labeled with ¹³C Met, revealed an interfacial methionine to be in distinct chemical environments in the dimer versus the monomer. Upon modification of the active site Ser with organophosphonate transition-state inhibitors, the equilibrium was found to be dramtically shifted towards dimer.  Inhibition with structurally diverse organophosphonates defined formation of the oxyanion as the minimum structural requirement for dimer stabilization.  Enzyme activity assays showed addition of substoiciometric amounts of inhibitor initially increased protease activity as a result of dimer stabilization.

Conclusions: The results provide the first indisputable link between the active site Ser and the dimer interface and elucidate the importance of substrate structural features on enzymatic catalysis. This highlights the possibility of inhibiting activity by preventing association of monomers. It is possible that the dimer interface will provide a target more amenable to inhibitor development. 

Keywords: protease; inhibitor; design