Communication between the active sites and dimer interface of a herpesvirus protease revealed by a transition-state inhibitor

Abstract

Structurally diverse organophosphonate inhibitors targeting the active site of the enzyme were used to investigate the relationship of the active site and the dimer interface of wild-type protease in solution. Positional scanning synthetic combinatorial libraries revealed Kaposi's sarcoma-associated herpesvirus protease to be highly specific, even at sites distal to the peptide bond undergoing hydrolysis. Specificity results were used to synthesize a hexapeptide diphenylphosphonate inhibitor of Kaposi's sarcoma-associated herpesvirus protease. The transition state analog inhibitors covalently phosphonylate the active site serine, freezing the enzyme structure during catalysis. 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. NMR, circular dichroism, and size exclusion chromatography analysis showed that active site inhibition strongly regulates the binding affinity of the monomer-dimer equilibrium at the spatially separate dimer interface of the protease, shifting the equilibrium to the dimeric form of the enzyme. Furthermore, inhibitor studies revealed that the catalytic cycles of the spatially separate active sites are independent. These results (i) provide direct evidence that peptide bond hydrolysis is integrally linked to the quaternary structure of the enzyme, (ii) establish a molecular mechanism of protease activation and stabilization during catalysis, and (iii) highlight potential implications of substoichiometric inhibition of the viral protease in developing herpesviral therapeutics.

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

“…Samples incorporating isotopically labeled Ala, Cys, His, Pro, or Arg were recombinantly expressed in E. coli strain BL21(DE3) in M9 media supplemented with all amino acids, nucleosides, and vitamins (35) 13 C, and 15 N. Protease concentrations were determined using a calculated 280 ) 0.931 mg/mL for wild-type and M197D proteins, 280 ) 1.000 mg/mL for ∆, and 280 ) 0.957 mg/mL for inhibited wild-type protease covalently modified with the inhibitor acetyl-Pro-Val-TyrtBug-Gln-AlaP-(OPh) 2 after hydrolysis of the two phenoxy moieties. The inhibitor was synthesized both with and without a biotin moiety on the amino terminus as previously described (33).…”

“…The Tyr-Val-tBug-Ala-ACC substrate was synthesized and purified as described (36), and substrate turnover was monitored for 1 h with an excitation wavelength of 380 nm and an emission wavelength of 460 nm. Protease samples were diluted into assay buffer and allowed to equilibrate for at least 1 h. The concentration of dimeric protease was calculated using the previously determined dissociation constant of 1.8 µM and the total concentration of protease using the equation previously derived (33).…”

“…Use of the inhibitor, which stabilizes the dimeric conformation of KSHV Pr (33), has allowed the direct comparison of the R-helical content between the monomeric and dimeric conformations of a herpesvirus protease, revealing a loss of 31% R-helicity upon dissociation.…”

“…A direct comparison between the monomer and dimer has been conducted through the use of an M197D mutation, which yields inactive monomers, and a hexapeptide diphenyl phosphonate covalent inhibitor, which stabilizes the protease dimer (33). 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).…”

“…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.…”