Because of its stringent sequence specificity, the 3C-type protease from tobacco etch virus (TEV) is frequently used to remove affinity tags from recombinant proteins. It is unclear, however, exactly how TEV protease recognizes its substrates with such high selectivity. The crystal structures of two TEV protease mutants, inactive C151A and autolysis-resistant S219D, have now been solved at 2.2-and 1.8-Å resolution as complexes with a substrate and product peptide, respectively. The enzyme does not appear to have been perturbed by the mutations in either structure, and the modes of binding of the product and substrate are virtually identical. Analysis of the protein-ligand interactions helps to delineate the structural determinants of substrate specificity and provides guidance for reengineering the enzyme to further improve its utility for biotechnological applications.The Picornaviridae are a large superfamily of (ϩ)-strand RNA viruses that are responsible for a variety of plant and animal pathologies (1). Their RNA genomes are translated into polyprotein precursors that are co-translationally cleaved by viral proteases to generate the mature proteins (2). The majority of these processing events are mediated by the picornavirus 3C-type proteases, which are structurally similar to serine proteases like trypsin and chymotrypsin, but utilize a cysteine thiol instead of a serine hydroxyl as the active-site nucleophile (1, 3). Because they play an essential role in viral replication, 3C proteases are viewed as attractive molecular targets for antiviral therapeutics (4).The stringent sequence specificity of rhinovirus 3C protease and the 3C-like nuclear inclusion protease encoded by TEV 1 has also led to their widespread application in the biotechnology sector as reagents for endoproteolytic removal of affinity tags from recombinant proteins (5). In contrast to Factor Xa, enterokinase, and thrombin, neither of these viral proteases has ever been reported to cleave genetically engineered fusion proteins at unintended locations. All 3C-type proteases exhibit a strong preference for glutamine in the P1 position of their substrates and for small aliphatic residues in the P1Ј subsite, but these are clearly not the only specificity determinants (3, 6). Studies with oligopeptide substrates have established that the P6 and P3 subsites are also important specificity determinants for TEV protease (7), whereas it is the P4 and P2Ј positions that appear to make the greatest contribution to the unique specificity of rhinovirus 3C protease (8).Despite the fact that 3C-type proteases have been the subject of considerable interest, the structural basis of their substrate specificity remains obscure. Although the crystal structures of 3C proteases from hepatitis A virus (9), rhinovirus-14 (10), and poliovirus (11, 12) have been determined, none of them have cognate peptides in the active site. Consequently, efforts to explain the substrate specificity of these enzymes have relied on modeling or, in a few cases, on the structures of e...