A computational modeling and molecular dynamics study of the Michaelis complex of human protein Z-dependent protease inhibitor (ZPI) and factor Xa (FXa)

Abstract

Protein Z-dependent protease inhibitor (ZPI) and antithrombin III (AT3) are members of the serpin superfamily of protease inhibitors that inhibit factor Xa (FXa) and other proteases in the coagulation pathway. While experimental structural information is available for the interaction of AT3 with FXa, at present there is no structural data regarding the interaction of ZPI with FXa, and the precise role of this interaction in the blood coagulation pathway is poorly understood. In an effort to gain a structural understanding of this system, we have built a solvent equilibrated three-dimensional structural model of the Michaelis complex of human ZPI/FXa using homology modeling, protein-protein docking and molecular dynamics simulation methods. Preliminary analysis of interactions at the complex interface from our simulations suggests that the interactions of the reactive center loop (RCL) and the exosite surface of ZPI with FXa are similar to those observed from X-ray crystal structure-based simulations of AT3/FXa. However, detailed comparison of our modeled structure of ZPI/FXa with that of AT3/FXa points to differences in interaction specificity at the reactive center and in the stability of the inhibitory complex, due to the presence of a tyrosine residue at the P1 position in ZPI, instead of the P1 arginine residue in AT3. The modeled structure also shows specific structural differences between AT3 and ZPI in the heparin-binding and flexible N-terminal tail regions. Our structural model of ZPI/FXa is also compatible with available experimental information regarding the importance for the inhibitory action of certain basic residues in FXa.

Fig. 1

Multiple sequence alignment of protein Z-dependent protease inhibitor (ZPI) with human antithrombin III (AT3), α1-antitrypsin (A1AT) and heparin cofactor II (HCII) using ClustalW. The N-terminal regions of the serpins share low homology and are not included in the figure. β-sheets (arrows) and α-helices (cylinders) are indicated above the alignment. Asterisks denote identity, colons strong similarity, and periods weak similarity. The reactive center loop (RCL) and the P1 residue at the reactive center are indicated

Fig. 2

Plot of backbone root mean square deviation (RMSD) of a ZPI/factor Xa (FXa) over 25 ns molecular dynamics (MD) simulation using the best homology model as a reference, and b AT3/FXa over 17 ns simulation using the X-ray crystal structure (2GD4) as a reference. The crystal structure of AT3 was modified to include missing regions and to mutate the active site Ala195 of FXa back to serine

Fig. 3

Root mean square fluctuation (RMSF) of the backbone atoms averaged over each residue for a the FXa regions bound to ZPI and AT3, and b the serpins, ZPI and AT3

Fig. 4

Cartoon representation of the solvent-equilibrated structural model of ZPI/FXa complex. Structural elements colored in blue (also denoted by asterisks) highlight the regions of ZPI that are comparatively more flexible than in AT3, as indicated by RMSF values obtained from the simulation. The key secondary structural elements are labeled; P1 and the S1 residues are shown in stick representation

Fig. 5

a,b Heparin binding region. a Residues involved in binding to heparin in AT3. b Residues in the corresponding region of ZPI

Fig. 6

Distance between P1 and S1 residues in a ZPI/FXa and b AT3/FXa. ZPI and AT3 are shown in cartoon representation with the portion of RCL region directly interacting with FXa colored orange. The P1 and P1′ residues are shown in stick representation. The catalytic triad residues and the S1 residue in FXa are also shown in stick representation (cyan) and colored by atom type. The right-hand panel shows a plot of the distances between P1 and S1 and P1 and Ser195 over the simulation time for c ZPI/FXa, d AT3/FXa, and e R393A AT3/FXa. The plots show that the P1–Ser195 distance is comparable between all three complexes, irrespective of their P1–S1 distance

Fig. 7

Mapping of FXa basic residues predicted to be important for ZPI inhibition [43]. FXa is shown in cartoon representation, with ZPI residues in stick representation and colored red. The residues in FXa (stick representation and colored blue) make direct contacts with ZPI residues (red), while the FXa residues colored gray are on a surface distant from ZPI