PB0540 - Investigation of GGCX interaction with VKD proteins

Background: γ-glutamyl carboxylase (GGCX) γ-carboxylates vitamin K dependent (VKD) proteins including the procoagulatory factors FII, FVII, FIX, FX and anti-coagulatory proteins Protein C (PC). This process is essential to maintain blood homeostasis. Interestingly, GGCX has different affinities for the propeptides of the VKD coagulatory proteins.

Aims: The aim of this study is to evaluate the mechanism of differential interaction of GGCX with VKD coagulatory proteins using in silico tools, which will be verified by in vitro assays.

Methods: In silico models of GGCX and FII, FVII, FIX, FX, and PC were downloaded from ITASSER and AlphaFold, where the propeptide and the Gla domain were further considered. These VKD coagulatory proteins were docked on both GGCX model in H-dock server. The models with highest docking score were aligned and protein-protein interaction between GGCX and VKD coagulatory proteins were analyzed in Protein Interaction Calculator server.

Results: The alignment of VKD coagulatory protein-GGCX complex in ITASSER showed that these proteins are binding to GGCX in different clusters.FIX and PC clustered together, whereas FX binds nearby in a different orientation, and FVII and FII formed a third cluster. Interestingly, protein-protein interaction analysis showed different interacting residues between ITASSER and AlphaFold models. Therefore, these substrate specific interacting residues in GGCX were mutated to alanine in silico. GGCX mutations f.e. R498A, E518A (ITASSER model) and E373A, Y379A (AlphaFold model) showed highest change in binding energy that will be further analyzed in vitro to obtain the closest model for the interaction analysis.

Conclusion(s): Our data indicates that VKD coagulatory proteins orient differentially once the propeptide of the VKD protein has bound to the same propeptide binding site in GGCX (495-513). This leads to differential interaction between residues in GGCX and VKD coagulatory proteins. However, there are differences in the models of ITASSER and AlphaFold that will be further validated.