ABSTRACT
Noroviruses are the major cause of human epidemic nonbacterial gastroenteritis. Viral replication requires a 3C cysteine protease that cleaves a 200 kDa viral polyprotein into its constituent functional proteins. Here we describe the X-ray structure of the Southampton norovirus 3C protease (SV3CP) bound to an active site-directed peptide inhibitor (MAPI) which has been refined at 1.7 Å resolution. The inhibitor, acetyl-Glu-Phe-Gln-Leu-Gln-X, which is based on the most rapidly cleaved recognition sequence in the 200 kDa polyprotein substrate, reacts covalently through its propenyl ethyl ester group (X) with the active site nucleophile, Cys 139. The structure permits, for the first time, the identification of substrate recognition and binding groups in a noroviral 3C protease and thus provides important new information for the development of antiviral prophylactics.
Subject(s)
Antiviral Agents/pharmacology , Cysteine Endopeptidases/chemistry , Norovirus/enzymology , Peptides/pharmacology , Protease Inhibitors/pharmacology , Viral Proteins/antagonists & inhibitors , Viral Proteins/chemistry , 3C Viral Proteases , Amino Acid Sequence , Antiviral Agents/chemistry , Caliciviridae Infections/drug therapy , Caliciviridae Infections/enzymology , Catalytic Domain/drug effects , Crystallography, X-Ray , Cysteine Endopeptidases/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Norovirus/chemistry , Norovirus/drug effects , Peptides/chemistry , Protease Inhibitors/chemistry , Protein Structure, Tertiary , Sequence Alignment , Substrate Specificity , Viral Proteins/metabolismABSTRACT
The X-ray structures of native endothiapepsin and a complex with a hydroxyethylene transition state analog inhibitor (H261) have been determined at atomic resolution. Unrestrained refinement of the carboxyl groups of the enzyme by using the atomic resolution data indicates that both catalytic aspartates in the native enzyme share a single negative charge equally; that is, in the crystal, one half of the active sites have Asp 32 ionized and the other half have Asp 215 ionized. The electron density map of the native enzyme refined at 0.9 A resolution demonstrates that there is a short peptide (probably Ser-Thr) bound noncovalently in the active site cleft. The N-terminal nitrogen of the dipeptide interacts with the aspartate diad of the enzyme by hydrogen bonds involving the carboxyl of Asp 215 and the catalytic water molecule. This is consistent with classical findings that the aspartic proteinases can be inhibited weakly by short peptides and that these enzymes can catalyze transpeptidation reactions. The dipeptide may originate from autolysis of the N-terminal Ser-Thr sequence of the enzyme during crystallization.
