Figure 1.
The structures of the P1 amino acids being studied here.
Figure 2.
X-ray crystal structure analysis of huPA-H99Y in complex with peptidic inhibitors.
(A) Overall structure of the complexes between huPA-H99Y and mupain-1 (cyan), mupain-1-12 (salmon), mupain-1-16 (grey), and mupain-1-16-D9A (red). (B) A zoom on interactions of mupain-1's Arg6 in the S1 pocket; polar interactions are indicated by stippled lines. (C) A zoom on the polar interactions (stippled lines) between huPA-H99Y residue Arg35 and mupain-1 residues Tyr7 and Asp9. (D) A zoom on the polar interaction (stippled lines) between huPA-H99Y residue Tyr99 and mupain-1 residue Ser5. (E) An overlay of the active site areas of the huPA-H99Y – mupain-1 complex and the huPA S195A – PAI-1 Michaëlis complex (pdb entry 3pb1; [29]); the P2, P1, and P1′ residues are indicated, those of PAI-1 in salmon. (F) A zoom on the Lys143 – Gln192 area of the huPA-H99Y – mupain-1 complex; distances, in Å, between different residues are indicated. In all parts of the figure, huPA-H99Y are shown in wheat cartoon presentation. In Fig. 1E, huPA S195A is shown in grey cartoon presentation. The peptides are shown in stick representation. huPA-H99Y residues are labelled with black letters, peptide residues with dark red letters.
Table 1.
Inhibition of huPA-H99Y and huPA-H99Y exosite mutants by mupain-1 variants.
Table 2.
Surface plasmon resonance analysis of the binding of peptides to muPA or huPA-H99Y.
Table 3.
Isothermal titration calorimetry for binding of peptides to huPA-H99Y.
Figure 3.
Structural model of mupain-1 in complex with muPA.
(A) Overall model of the complex between muPA (teal ribbon presentation) and mupain-1 (red stick representation). The enzyme is shown in teal cartoon presentation. (B) A zoom on the Lys143 area of the model, showing distances from Lys143 to the closest atoms in the peptide. (C) A zoom on the Lys41 area of the model, showing the distances between Lys41 and peptide residues Tyr7and Asp9. (D) A zoom on the entrance to the S1 pocket of the model, with Val213 at the entrance indicated; the enzyme is represented as teal surface. huPA-H99Y residues are labelled with black letters, peptide residues with dark red letters.
Table 4.
Inhibition of muPA wt and muPA exosite mutants by mupain-1 variants.
Table 5.
Inhibition of muPA wt and muPA S1 pocket mutants by mupain-1 variants.
Figure 4.
The relationship between the Ki values for inhibition of muPA S1 mutants and the Ki values for inhibition of muPA wt.
The figure is based on the Ki values presented in Table 6. The x-axis shows the Ki values for inhibition of muPA wt by the indicated peptides. The y-axis shows the corresponding Ki values for inhibition of muPA S190A (filled dots) or muPA V213T (open dots). The lines resulted from simple linear regression analysis. The slopes of the lines are 0.68 (muPA-S190A) and 1.27 (V123T). The stippled line is the one which would have resulted if the Ki values for inhibition of the muPA S1 mutants had been identical to those for inhibition of muPA wt (y = x).
Figure 5.
The relationship between the Ki values for inhibition of muPA exosite mutants and the Ki value for inhibition of muPA wt.
The figure is based on the Ki values presented in Table 5. The x-axes show the Ki values for inhibition of muPA wt by the indicated peptides. The y-axes shows the corresponding Ki values for inhibition of muPA K41A, Y99A, or K143A. The lines resulted from simple linear regression analysis. The slope of the lines are 1.04 (muPA K41A with Asp9 peptides); 1.15 (muPA K41A with Ala9 peptides); 1.15 (muPA Y99A); 1.40 (muPA K143A). The stippled line shows the line which would have resulted if the Ki values for exosite mutants and muPA wt had been identical (y = x).
Figure 6.
TOCSY (orange) and NOESY (green) of mupain-1-16.
Assignments of the two isomeric forms is illustrated by connecting TOCSY Hα(i)-HN(i) and NOESY Hα(i)-HN(i+1) cross peaks with lines representing a shared reference commonly referred to as “backbone walks”.
Figure 7.
Predicted order parameter S2 (top) calculated by TALOS+.
The dots (bottom) represent classification of the residue from TALOS+, as based on the mobility of the backbone, the certainty of the angles of the reference triplets and whether the angles fall into allowed regions in the Ramachandran plot [34]. The colour codes represent good (green), dynamic (yellow), ambiguous (red), and no prediction (blue) for the two different peptides in cis and trans conformations.