Figure 1.
Human CypA in complex with CA.
Human CypA is a cytoplasmatic single peptide chain, 165 amino acids long. Top: The X-ray structure shows that it is a β-barrel with eight antiparallel β-strands and two α-helices flanking the β-barrel [13]. These secondary structures are connected by several flexible loops. The β-barrel contains the active site for cis/trans prolyl isomerization. The CA protein is composed by two helices connected by a loop. This contains the G89-P90 target peptide bond (Bottom, right). Bottom, left: Transition state of the cis-trans isomerisation, emerging from this and previous [14],[18],[22] calculations.
Table 1.
Available in vitro experimental and theoretical thermodynamic data (kcal/mol) associated to the cis/trans isomerisation of peptides binding to CypA protein with the values reported for the peptide in water and bound to the protein (bold face)a.
Figure 2.
Dihedral angles in prolyl isomerisation.
The dihedral angles ω and ζ identify the trans (∼±180°) or cis (∼0°) conformation. The angle ψ determines the rotation along Cα(i)-C(i) bond. The dihedral angles χ0, χ1, χ2, χ3 and χ4 measures the puckering of the proline ring.
Figure 3.
Collective variables used in bias exchange metadynamics simulations. The red box encloses the variables used in the PEPT-WAT system, the blue box contains variables used in PEPT-CypA. We define briefly each CV as follows: (A) see Figure 2; (B) Proline nitrogen pyramidalization is defined as the distance between P4N atom and the center of the plane determined by G3C-P4Cα-P4Cδ; (C) P4N interaction with water is defined as P4N coordination with O atoms of all water molecules; (D) P4N intramolecular H-bond was defined as P4N coordination with N(-H) atoms of all H-bonds donor residues of PEPT; (E) P4N@PEPT H-bond with PEPT or CypA was described as the coordination of P4N with N(-H) and O(-H) atoms of all possible H-bond donors of PEPT and CypA; (F) R55 H-bond with PEPT-CypA is illustrated as the coordination between R55@CypA and CypA active site residues; (G) Interaction of L98 and S99 with C-terminal part of the substrate is defined as the coordination between non polar carbons atoms of C-terminal@PEPT and those of L99 S99; (H) Hydrophobic interactions between the N and C terminal of PEPT with the active site were described as the coordination of the non polar carbons of the PEPT N-and C-terminal with non polar carbons of the residues in the active site of CypA; I) Non-polar carbons coordination of G3P4@PEPT with CypA active site residues.
Table 2.
Minima and TSs population in all the CV pairs containing the dihedral angle ζ.
Figure 4.
Free energy (kcal/mol) of the /HAGPIA/ peptide in solution as a function of the dihedral angles ζ and ψ (in degrees, showed in the inset). The plot is divided in transition and minima regions, with representative structures (center of the lower free energy cluster within each minima and TS region) for each region explicitly shown (See Text S1 for details), along with water molecules within a shell of 2 Å (red spheres).
Table 3.
Free energy (kcal/mol) of PEPT-WAT and PEPT-CypA prolyl cis/trans isomerization.
Figure 5.
Free energy (kcal/mol) of the /HAGPIA/ bound to CypA as a function of the dihedral angles ζ and ψ (in degrees). The plot is divided in transition and minima regions (see Text S1 for details). Representative structures of each minima and TS conformation (center of the lower free energy cluster within each minima and TS region) in PDB format are given in Datasets S1, S2, S3, S4, S5, S6, S7, S8.
Table 4.
Number of H-bonds of CypA residues with G3P4@PEPT along prolyl cis/trans isomerization.
Table 5.
Interface coordination numbera of CypA residues with G3P4@PEPT along prolyl cis/trans isomerization.
Figure 6.
Proposed mechanism of action for CypA.
CypA sequesters the most abundant conformation in water, trans0, that is rapidly interconverted into the most abundant conformer trans180. Then, CypA catalyzes the isomerization of the peptide along TS2, producing the mostly populated minimum, cis180. The peptide most probably detaches in the cis180 conformation. This readily interconverts to cis0 once the peptide is in aqueous solution. CypA residues that form important H-bonds (hhb) and hydrophobic interactions (nnb) with the G3P4 moiety are shown. Residues with almost exclusive relevance to each conformation are highlighted in red.