OC 74.5 - Factor XI and Plasma Kallikrein apple domain structures reveal alternate kininogen bound complexes

Background: High molecular weight kininogen (HK) is a key cofactor for appropriate assembly and activation of the contact system. How it interacts with prekallikrein (PK) and Factor XI (FXI) is unknown. The PK-HK interaction serves as a critical exosite to facilitate efficient HK cleavage and bradykinin generation.

Aims: To understand the structure of the complex formed between HK and PK, and to compare this with the FXI-HK complex.

Methods: Key residues involved in the HK interaction with PK and FXI were analysed by protein crystallography using a 31 amino acid HK D6 domain peptide to determine the complex structure. Interactions of the full length FXI-HK complex were studied in solution by Hydrogen Deuterium exchange Mass Spectrometry (HDX-MS).

Results: We determined a 2.3Å PK-HK fragment crystal structure revealing that the HK sequence WIPDIQ (W569-Q574) binds to the A1 domain and FNPISDFPDT (F582-T591) to the A2 domain. The intervening HK-peptide residues 575-581 are not observed in the electron density and are assumed to be flexible. A 3.2Å FXI-HK fragment crystal structure reveals an identical interaction in the A2 domain but an alternate, straightened conformation of HK with no flexible linker that extends into the A3 domain. The FXI-HK interaction at A3 was unexpected so we analysed the full length FXI-HK complex in solution using HDX which detected FXI interactions with A2 and A3. Unlike the PK-HK interaction no interactions were observed between HK and the FXI A1 domain.

Conclusion(s): Although PK and FXI both bind to HK, they do so via interactions with different apple domains. Apple 2 is involved in HK binding to both PK and FXI, but an important exosite of the HK F582-T591 sequence shifts from the A1 domain in PK to the A3 domain in FXI.