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. 2013 Mar 19;288(18):13068–13081. doi: 10.1074/jbc.M112.386953

FIGURE 3.

FIGURE 3.

The YAV VP4 protease intermolecular (trans) acyl-enzyme complex, product complex, and empty active site. YAV VP4 protease is able to cleave at an internal cleavage site (after Ala716) near its own C terminus (after Ala734), the VP4/VP3 junction. Two crystal structures of YAV VP4 (with and without an active site mutation) reveal the internal cleavage site bound within the active site of a neighboring VP4 molecule. Analysis of the electron density within each molecule of the asymmetric unit reveals three different enzyme states of the protease reaction cycle (empty active site, acyl-enzyme, and product complex). The packing of YAV VP4 protease in the asymmetric unit for each structure is shown along with representative 2FoFc electron density maps (contoured at 1σ) at the active site for representative types of complexes. A, in the monoclinic crystal of native active site YAV VP4, five molecules of VP4 are in the asymmetric unit with the C terminus of each molecule bound in the active site of its neighbor. The C-terminal carbonyl carbons of molecules D and E form an intermolecular acyl-enzyme complex (AE) with the active site nucleophilic serine Oγ of molecules C and D, respectively. An enzyme-product (EP) complex is formed between molecule pairs E/A, A/B, and B/C. B, in the cubic crystal of mutant YAV VP4 (Lys674 general base mutated to alanine), two molecules are in the asymmetric unit. The C-terminal carbonyl carbon of molecule A forms an intermolecular acyl-enzyme complex (AE) with the nucleophilic serine Oγ of molecule B. The active site of molecule A remains in the unbound state (E) because the C terminus of molecule B (shown as red spheres) folds back onto itself instead of being in an extended conformation.