PB0591 - Alphafold AI based applications explored as tools for predicting contact proteins PK, FXI and HK structure and evolutionary relationships

Background: High molecular weight kininogen (HK) is a key cofactor for appropriate assembly and activation of the contact system which binds prekallikrein (PK) and Factor XI (FXI). The FXI and PK-HK interactions mediate critical binding interactions that facilitate contact activation and bradykinin generation.

Aims: To understand the utility of Alphafold to accurately predict coagulation factor PK-HK or FXI-HK structures and to probe evolutionary relationships.

Methods: AlphaFold and AlphaFold-Multimer were utilised to investigate the 3D structure interactions of PK/FXI with HK and other ligands for sequences from human and a variety of other species including examples from reptile, amphibian and fish contact proteins.

Results: Alphafold and evolutionary relationships. The HK interaction with PK and FXI was analysed by Alphafold-Multimer using the human sequences and a variety of other species including examples from reptile, amphibian and fish contact proteins. Previous studies using contact factor genes and protein sequences have predicted that the contact system first appeared in lobe-finned fish, the ancestors of all land vertebrates. FXI subsequently arose from a duplication of the PK gene in mammals. Using Alphafold the 3D structure is predicted for a variety of species for FXI, PK, HK to trace ancestral protein:protein interactions.

Conclusion(s): Alphafold is able to accurately predict structures for the PK and FXI A1-A4 apple domain disk and isolated protease domain. The whole structure of the full length FXI and PK was not correctly predicted, esablishing limitations of protein prediction for larger multi-domain proteins. Alphafold Multimer was able to predict the experimentally observed interactions in PK and FXI-HKD6 peptide complexes. The accuracy and utility of these Alphafold predictions for sequences from reptile, amphibian and fish contact proteins facilitates analyses of the evolutionary relationships of the contact system.