Structures of human plasma β–factor XIIa cocrystallized with potent inhibitors

Abstract

Activated factor XIIa (FXIIa) is a serine protease that has received a great deal of interest in recent years as a potential target for the development of new antithrombotics. Despite the strong interest in obtaining structural information, only the structure of the FXIIa catalytic domain in its zymogen conformation is available. In this work, reproducible experimental conditions found for the crystallization of human plasma β-FXIIa and crystal growth optimization have led to determination of the first structure of the active form of the enzyme. Two crystal structures of human plasma β-FXIIa complexed with small molecule inhibitors are presented herein. The first is the noncovalent inhibitor benzamidine. The second is an aminoisoquinoline containing a boronic acid-reactive group that targets the catalytic serine. Both benzamidine and the aminoisoquinoline bind in a canonical fashion typical of synthetic serine protease inhibitors, and the protease domain adopts a typical chymotrypsin-like serine protease active conformation. This novel structural data explains the basis of the FXII activation, provides insights into the enzymatic properties of β-FXIIa, and is a great aid toward the further design of protease inhibitors for human FXIIa.

Deciphering the canonical blockade of activated Hageman factor (FXIIa) by benzamidine in the coagulation cascade: A thorough dynamical perspective

“…The energy contributions of the respective active site residues toward the binding and stabilization of BEN were further quantified using the MM/PBSA-integrated per-residue decomposition analysis and presented in Figure 10. Taken together, complementary interactions of BEN with these active site residues could account for the high-affinity binding, stability, and inhibitory activity reportedly exhibited by BEN toward FXIIa according to previous reports (Dementiev et al, 2018). Moreover, estimations of the free binding energy revealed that BEN exhibited a favorable binding toward a FXIIa with a ΔG value of −16.64 kcal/mol and a considerably high electrostatic energy of −11.72 kcal/mol as shown in Table 1, indicative of the occurrence of favorable interactions in agreement with results earlier discussed.…”

“…The lowering of the Cα atomistic motions of these loops and residues could possibly be attributed to the restraint effects imposed by the complementary interactions with BEN. This is in contrast to the unbound system that exhibited a considerable motion in its catalytic loops, a feature which facilitates the activation of protease enzymes activation (Dementiev et al., ). The binding of BEN also resulted in an increase in average structural flexibility of the binding pocket residues as showcased by the root‐mean‐square fluctuation (RMSF) analysis shown in Figure .…”

“…The loops that surround the active pocket of most trypsinlike serine proteases have been shown to prominently mediate many interactions between the protease and biomolecules such as cofactors, substrates, and cognate inhibitors (Bode, Turk, & Karshikov, 1992;Dementiev et al, 2018;Horrevoets, Tans, Smilde, van Zonneveld, & Pannekoek, 1993;Madison, Goldsmith, Gerard, Gething, & Sambrook, 1989). As such, the binding of BEN could somewhat interfere with the dynamics of the binding pocket residues in its effort to inactivate β-FXIIa.…”

“…This correlates with the RMSD analysis and could further indicate that the binding of BEN and optimal positioning disoriented the organization of the catalytic pocket and constituent loops. However, while the activation loop of the unbound FXIIa was highly surface exposed as shown in figure 5, the binding of BEN elicited a loop transition into the hydrophobic region thereby blocking canonical protein interactions that exacerbate thrombosis (Dementiev et al, 2018). The dynamics of the autolysis loop was also monitored since it plays a key role in the determination of specificity in coagulation proteases (Yang, Manithody, & Rezaie, 2007).…”

Studies into prekallikrein activation pave the way for new avenues of antithrombotic research

“…By contrast, deficiency of either FXII or FXI protects against thrombosis in murine models . Despite the importance of these proteins in thrombosis, full‐length crystal structures are only available for the zymogen FXI and β ‐FXIIa , a product of activation by PKa .…”