Figure 1.
Computational Protein Design Strategy.
Step 1: Prediction of the structure of the enzyme (PvSUB1) by comparative modelling and of the scaffold for mutational analysis (EETI-II-sub) by replacing one of the loops with a substrate sequence. Step 2: docking of EETI-II-sub to the target protein by ensemble docking procedure with several conformations from molecular dynamics simulations for each protein partner, and refinement of the best solutions. Step 3: mutation of the scaffold, conformational sampling and scoring of the mutants. Step 4: experimental testing by an enzymatic inhibitory assay on the recombinant enzyme of PvSUB1.
Figure 2.
3D model of PvSUB1 catalytic domain.
A: Highlighted in red is the region forming the substrate binding pocket and red sticks correspond to the residues that form the catalytic triad; B: Cartoon representation of secondary structures; C: APBS surface electrostatic representation.
Figure 3.
PvSUB1 molecular dynamics simulations.
A: Average RMSD values for PvSUB1 and the 3D structure of two homologous bacterial subtilisins (1TO2, 1ROR). PvSUB1* shows the RMSD calculated without the regions missing template structural information; B: Fluctuation of the RMSD from the average structure. C: Root mean square fluctuation (RMSF) on a per-residue basis. In orange are highlighted PvSUB1 residues involved in the substrate-binding region.
Table 1.
Catalytic site distances along MD simulations.
Table 2.
Subtilisin catalytic site geometries.
Figure 4.
Docking of PvSUB1 hexapeptide substrate into PvSUB1 catalytic groove.
Blue: P4, Violet: P3, Yellow: P2, Red: P1, Cyan: P1′, Green: P2′.
Figure 5.
Structural alignment of the obtained PvSUB1 model (cyan) with the 3D-structure of Subtilisin E (gray, PDB 1SCJ) that was used as a template in the homology modelling.
The catalytic triads in both proteins are highlighted with a stick representation. PvSUB1 catalytic triad: Asp 316, His 372 and Ser 549. Subtilisin E catalytic triad: Asp 32, His 64, Ser 221.
Figure 6.
The red circles indicate the docking poses that have been selected for refinement.
Figure 7.
Blue: P4, Violet: P3, Yellow: P2, Red: P1, Cyan: P1′.
Table 3.
Sequence and inhibitory activity of EETI-II mutants on PvSUB1.
Figure 8.
Blue: All atoms, Red: Side chain atoms, Green: Backbone atoms. The largest contribution to the free energy of binding comes from the main-chain contacts of residues P4, P3, P2 and P1. The highest contribution comes from the cysteine in P3 and its main-chain, accounting for −4.34 kcal/mol.
Table 4.
SUB1 natural substrates.
Figure 9.
Scoring mutations on P4 and P1.
A: mutants in position P4. The mutational profile of P4 shows that hydrophobic and bulky residues are preferred for this position. B: mutants in position P1. Position P1 instead prefers aromatic residues with polar groups (Tyr, Trp), glutamate and positively charged residues (Lys, Arg).
Figure 10.
Residues forming the S1 and S4 pockets.
The residue P4 (A) and P1 (B) of EETI-II are shown with an orange stick representation.