Binding of a Noncovalent Inhibitor Exploiting the S′ region Stabilizes the Hepatitis C virus NS3 Protease Conformation in the Absence of Cofactor

We present the first structure of a noncovalent inhibitor bound to the protease domain of hepatitis C virus NS3 protein (NS3p), solved by NMR. The inhibitor exploits interactions with the S′ region of NS3p to form a long-lived complex, although the absence of negative charges strongly reduces the association rate. The inhibitor stabilizes the N-terminal domain of NS3p and the substrate-binding site, and correctly aligns catalytic His-Asp residues. These actions were previously attributed exclusively to the cofactor NS4A, which interacts with the N-terminal domain of the NS3p and functions as an activator in vivo. The structure of the inhibitor/NS3p complex is very similar to that of the NS3p-NS4A complex, showing that binding of the NS4A cofactor is not the only event leading to a stable active-site conformation.