Localization of Ligand-Binding Exosites In the Catalytic Domain of Factor XIa.
Authors: Su Ya-Chi , Sinha Dipali , Walsh Peter N. 
Published: October 2019

Abstract
Abstract 1148
Coagulation factor XI (FXI) is a plasma zymogen that is activated to FXIa, the catalytic domain of which contains exosites that interact with its normal macromolecular substrate (FIX), and its major regulatory inhibitor (protease nexin-2 kunitz protease inhibitor, PN2KPI). To localize the catalytic domain residues involved in active site architecture and in various ligand-binding exosites, we aligned the sequence of the FXI catalytic domain with that of the prekallikrein (PK) catalytic domain which is highly homologous (64% identity) in sequence, but functionally very different from FXI. Six distinct regions (R1-R6) of dissimilarity between the two proteins were identified as possible candidates for FXIa-specific ligand binding exosites. FXI/PK chimeric proteins (FXI-R1, FXI-R2, FXI-R3, FXI-R4, FXI-R5, and FXI-R6) containing substitutions with PK residues within the six regions were prepared and characterized. FXIa-R1, R2, R3 displayed enhanced proteolysis after activation suggesting that the residues within R1, R2 and R3 regions may be important to maintain proper folding of the enzyme. Comparisons of amidolytic assays vs. activated partial thromboplastin time assays showed similar activities for all chimeras except FXI-R6, which displayed 60% of the normal amidolytic activity but only 28% of clotting activity suggesting the possibility that the R6 region (autolysis loop) of FXIa may comprise an exosite involved in the interaction with its macromolecular substrate FIX. This hypothesis was further confirmed experiments showing that the proteolytic activation of FIX by FXIa-R6 was significantly impaired compared with that achieved by FXIawt. Although FXIa-R5 and FXIa-R6 were defective (50-60%) in amidolytic assays, these chimeras were very similar to FXIawt in heparin and high molecular weight kininogen binding assays, suggesting that residues within the R5 and R6 regions are involved in active-site architecture. These chimeras were further investigated to determine whether any of them had acquired kallikrein activity. After activation all except FXIa-R4 showed insignificant activity using a kallikrein-specific substrate. FXIa-R4 displayed 87% of the activity of kallikrein using the kallikrein-specific substrate but only 3% of the activity of FXIawt using the FXIa chromogenic substrate. Moreover the cleavage pattern and cleavage rate of high molecular weight kininogen by FXIa-R4 were similar to those achieved by kallikrein but not by FXIawt. Therefore substitutions in the R4 region of FXI with the corresponding residues of PK resulted in loss of activity for the FXIa substrates and gain of activity for the kallikrein substrates suggesting that the R4 region (99-loop) of FXIa plays a role in determining the substrate specificity. From the co-crystal structure of the FXIa catalytic domain with PN2KPI, the residues R3704, Y5901, E98, Y143, I151, and K192 (chymotrypsin numbering) in the FXIa catalytic domain have been identified to be possibly involved in the interactions with its inhibitors. A single mutation comprising Y5901A in the R2 region of FXIa does not affect folding however this mutant displayed resistance to inhibition by PN2KPI indicating that Y5901 is involved in the interaction of FXIa with PN2KPI. In conclusion, these studies of FXI/PK chimeric and mutant proteins implicate residues within the R4 region (99-loop) of FXIa in the determination of amidolytic substrate specificity; residues within the R6 region (autolysis loop) of FXIa in the interaction with the macromolecular substrate, FIX; and the residue Y5901 in the R2 region of FXIa in the interaction of FXIa with PN2KPI.

Disclosures:
No relevant conflicts of interest to declare.