Structural and mutagenic analysis of foot-and-mouth disease virus 3C protease reveals the role of the beta-ribbon in proteolysis

Abstract

The 3C protease (3C(pro)) from foot-and-mouth disease virus (FMDV), the causative agent of a widespread and economically devastating disease of domestic livestock, is a potential target for antiviral drug design. We have determined the structure of a new crystal form of FMDV 3C(pro), a chymotrypsin-like cysteine protease, which reveals features that are important for catalytic activity. In particular, we show that a surface loop which was disordered in previous structures adopts a beta-ribbon structure that is conformationally similar to equivalent regions on other picornaviral 3C proteases and some serine proteases. This beta-ribbon folds over the peptide binding cleft and clearly contributes to substrate recognition. Replacement of Cys142 at the tip of the beta-ribbon with different amino acids has a significant impact on enzyme activity and shows that higher activity is obtained with more hydrophobic side chains. Comparison of the structure of FMDV 3C(pro) with homologous enzyme-peptide complexes suggests that this correlation arises because the side chain of Cys142 contacts the hydrophobic portions of the P2 and P4 residues in the peptide substrate. Collectively, these findings provide compelling evidence for the role of the beta-ribbon in catalytic activity and provide valuable insights for the design of FMDV 3C(pro) inhibitors.

FIG. 1.

Crystal structure of FMDV 3C^pro and comparison with HRV 3C^pro. (A) Structure of molecule A in the new crystal form of FMDV 3C^pro. Secondary structure elements of FMDV 3C^pro are color coded. The strands of the front and back β-sheets of the two β-barrels are green and blue, respectively; helices are pink; and the β-ribbon is orange. β-Strands mentioned in the text are labeled. This and other structural figures were prepared with Pymol (10). (B) Structure of molecule B in the new crystal form of FMDV 3C^pro colored according to the same scheme as in panel A. Note that the B₂-C₂ β-ribbon present in molecule A is absent in FMDV 3C^pro; the visible residues of this loop are orange. Active-site residues are labeled; note that A163 is Cys in the wild-type protein. (C) Electron difference density maps for residues 138 to 150 of FMDV 3C^pro (molecule A) showing the β-ribbon structure. Shaded blue chicken wire represents the initial F_o-F_c difference map, contoured at 2σ, which was generated with model phases calculated prior to incorporation of the loop residues (depicted as sticks and colored by atom type). (D) Structure of HRV 3C^pro (23) colored according to the scheme used in panel A. (E) Close-up view of the β-sheet in molecule A from which the β-ribbon protrudes. β-Strands mentioned in the text are labeled. Residues involved in the stabilization of this conformation are shown as sticks (see text). (F) Close-up view of the β-sheet in molecule B from which the β-ribbon protrudes. Note the reorganization of Y136 and K137 with respect to molecule A (panel E).

FIG. 2.

Comparison of catalytic triads in different FMDV and HAV 3C^pro structures. (A) Close-up view of the active site in molecule A of the present structure. The side chains of key residues are shown as sticks color coded according to atom type. The secondary structure is colored as in Fig. 1A. (B) Equivalent view of the active site of molecule B. Note the interaction between D84 from molecule B and K77′ from a neighboring 3C^pro molecule in the crystal (differentiated by cyan loops and carbon atoms). (C) Active site of HAV 3C^pro showing a similar reorientation of the catalytic Asp (D84) as a result of salt bridge formation with a Lys residue (K202).

FIG. 3.

Assays of FMDV 3C^pro mutant enzyme activities. See Materials and Methods for experimental details and Table 2 for the k_cat/K_m values determined for each mutant.

FIG. 4.

Comparison of β-ribbon structures from FMDV 3C^pro and related proteases. (A) Superposition of FMDV 3C^pro (β-strands, green; α-helices, pink; β-ribbon, orange) and α-lytic protease (yellow). The side chains of active-site residues are shown as sticks. Only residues from FMDV 3C^pro are labeled). (B) Superposition of the β-ribbon and the adjacent E₁-F₁ loop from FMDV 3C^pro (colored as in panel A) and other picornaviral 3C proteases. PV (Protein Database [PDB] identification [ID], 1l1n), HRV (PDB ID, 1cqq), and HAV (PDB ID, 1hav) are dark red; the related serine proteases α-lytic protease (PDB ID, 2alp), S. griseus protease A (PDB ID, 5sga), and S. griseus Glu-specific protease (PDB ID, 1hpg) are yellow; and the TEV NIa protease (PDB ID, 1lvb) is blue. Note that in the TEV NIa protease the β-ribbon is inverted and derives from a C-terminal extension of the protein. Close-up views of the β-ribbon in FMDV 3C^pro (C) and the α-lytic protease (D) show residues that contribute to stabilization of this structural feature. (E) Model of the complex of FMDV 3C^pro with a PAKQ|LL peptide based on homology to the TEV^pro-peptide complex (31). The peptide is depicted with lilac carbon atoms and a semitransparent surface. Key residues discussed in the text are labeled. The position of residue 142 (Ser in the structure), which is inserted between the P2 and P4 positions of the peptide substrate, is highlighted by a dotted line.