User:Ashley Steere/Sandbox 1

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The tobacco etch virus (TEV) is a member of the Potyviridae family of RNA viruses. The RNA genome of the TEV is translated into a large polyprotein precursor that is cleaved soon after translation to form independent protein products. The is a 27 kDa 3C-type protease responsible for the processing of the original polyprotein into functional viral proteins. TEV protease resembles well-known serine proteases, such as trypsin and chymotrypsin, except that the TEV protease utilizes the nucleophilic thiol of the active site cysteine residue, as opposed to the serine hydroxyl used in serine proteases. Ultimately, the biological importance of the TEV protease requires that the enzyme have very stringent sequence specificity to ensure proper production of viral proteins, and it is for this reason that the TEV protease has increasingly been used to remove affinity tags from recombinant proteins.

Structure of TEV Protease

TEV protease adopts a two-domain antiparallel β-barrel fold, typical of trypsin-like serine proteases. Located at the interface between the two domains is the , composed of His46, Asp81, and Cys151. A structural comparison with related proteins reveals that the TEV protease fold is most similar to that of the 3C cysteine proteases from hepatitis A virus and rhinovirus, which serve a similar function as the TEV protease in their respective viruses. However, although the overall fold of TEV protease and these related proteins is indeed very similar, the actual atomic coordinates are very different, with the root mean square deviation for Cα carbons between 2.4 to 3.5Å.

Substrate Specificity

The canonical recognition site of the TEV protease is the seven amino acid sequence ENLYFQ/G, with cleavage occurring after the glutamine residue. Although quite specific, TEV protease does have a tendency to undergo self-cleavage at a (KVFM/S) which follows residue 218, yielding a truncated enzyme with diminished catalytic activity. Based on biochemical and structural data, it appears that the C-terminal region of the protein is relatively unstructured and flexible, allowing the scissile bond between Met218 and Ser219 to come dangerously close to the active site, and is readily cleaved. Mutation of Ser 219 to a number of other amino acids (Asp, Val, Pro) functions to limit the mobility of the peptide bond between residues 218 and 219, and has led to the production of TEV protease constructs resistant to this autoproteolytic activity with no affect in the normal catalytic function. Crystallization of the catalytically active TEV protease resistant to autoproteolysis (S219D mutation) in the presence of an artificial substrate revealed that the larger of the two products remain bound within the enzyme active site. Likewise, crystallization of an inactive TEV protease mutant (in which the catalytic Cys151 was mutated to Ala) has been carried out both in the presence and absence of artificial substrate. Interestingly, in the absence of artificial substrate, the of the inactive TEV protease is bound within the active site of the enzyme, suggesting that the C-terminus of binds to the active site with high specificity and may be responsible for the diminished catalytic capacity of the truncated enzyme after autoproteolysis at residue 219.

References