Enzyme Inhibition by Allosteric Capture of an Inactive Conformation

All members of the human herpesvirus protease (HHV Pr) family are active as weakly associating dimers but inactive as monomers. A small-molecule allosteric inhibitor of Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) traps the enzyme in an inactive monomeric state where the C-terminal helices are unfolded and the hydrophobic dimer interface is exposed. NMR titration studies demonstrate that the inhibitor binds to KSHV Pr monomers with low micromolar affinity. A 2.0-Å-resolution X-ray crystal structure of a C-terminal truncated KSHV Pr–inhibitor complex locates the binding pocket at the dimer interface and displays significant conformational perturbations at the active site, 15 Å from the allosteric site. NMR and CD data suggest that the small molecule inhibits human cytomegalovirus protease via a similar mechanism. As all HHV Prs are functionally and structurally homologous, the inhibitor represents a class of compounds that may be developed into broad-spectrum therapeutics that allosterically regulate enzymatic activity by disrupting protein–protein interactions.

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► First presentation of an allosterically inhibited HHV Pr monomer crystal structure.
► Inhibitor binds to monomeric protease in the absence of the interfacial helices.
► Trapping an inactivate KSHV Pr monomeric state subsequently inhibits dimerization.
► Loss of helices at the dimer interface disrupts enzyme active-site conformation.
► Small-molecule binding pocket is conserved among HHV Prs.