Enzyme Inhibition by Allosteric Capture of an Inactive Conformation

Abstract

All members of the human herpesvirus protease 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 μM 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 (HCMV Pr) via a similar mechanism. As all HHV proteases are functionally and structurally homologous, the inhibitor represents a class of compounds that may be developed into broad-spectrum therapeutics which allosterically regulate enzymatic activity by disrupting protein-protein interactions.

Assemblins as maturational proteases in herpesviruses

“…This was predicted earlier and verified by our monomeric structure [75,80,82]. Additionally, there are some models of monomer-like structures of C-terminally truncated KSHVassemblin (D222-256) with inhibitors that bind to the dimerization area [81,84], see the discussion in the Inhibitors section below.…”

“…We assumed that those two C-terminal helices are similarly positioned in other alphaherpesviruses. In contrast, the two C-terminal helices of monomeric assemblins from beta-and gammaherpesviruses are completely disordered according to circular dichroism (CD)-and Nuclear Magnetic resonance (NMR)-spectroscopic data [38,84]. We proposed that this difference results from several residues that are conserved in assemblins from alphaherpesviruses on the one hand and beta-/gammaherpesviruses on the other hand.…”

“…In the dimerization area of assemblins, there is a conserved aromatic residue: Tyr128 in HCMV-assemblin, and Trp as the corresponding residue in EBV-and KSHV-assemblins. It is anticipated that this aromatic residue is a key residue for dimerization [84].…”

“…Crystal structures of truncated KSHV-assemblin (D222-256) that was inhibited with DD2 or DD2 derivatives were determined [81,84]. In this KSHV-assemblin construct, the two C-terminal helices were removed by C-terminally truncating the assemblin.…”

“…In this KSHV-assemblin construct, the two C-terminal helices were removed by C-terminally truncating the assemblin. The rationale behind this is that these helices are disordered in monomeric KSHV-assemblin [84]. In the resulting structural models, the inhibitors are noncovalently bound near the hot spot residue Trp.…”