Liu, K.; Li, S.; Tong, J.; Jiang, N.; Hong, M.; Gu, Y.; Chen, L.; Liang, D.; Jin, Y.; Zhao, Y.; Hou, D.; Huang, J.; Tie, J.-K.; Hao, Z.

Gamma-carboxylation, catalyzed by gamma-glutamyl carboxylase (GGCX), is a critical post-translational modification essential for the biological activity of vitamin K-dependent proteins (VKDPs). Mutations in GGCX, depending on their specific location, result in vitamin K-dependent coagulation factor deficiency type 1 (VKCFD1), which encompasses a broad spectrum of clinical manifestations ranging from mild to severe, including bleeding disorders, osteoporosis, and vascular calcification. The limited knowledge of GGCX\'s structure and functional regions hinders our understanding of the consequences of GGCX mutations and the treatment for VKCFD1. This study aimed to identify key functional regions of GGCX and their interactions with VKDPs to better elucidate the molecular mechanisms underlying these diverse clinical symptoms. Using AlphaFold 3 and molecular dynamics simulations, we developed a complex binding model of GGCX, FIX, and reduced vitamin K, which revealed critical regions and residues involved in their interaction. Site-directed mutagenesis and cell-based assays further validated the model, confirming that multisite and regional cooperative binding of FIX to GGCX plays a key role in modulating gamma-carboxylation efficiency. Additionally, novel residues (I296, M303, M401, M402) were identified as essential for GGCX\'s dual enzymatic activities: carboxylation and vitamin K epoxidation. We further demonstrated that the spatial proximity of these active sites supports that GGCX\'s carboxylation and vitamin K epoxidation centers are interconnected, ensuring the efficient coupling of these processes. Our GGCX-FIX binding and carboxylation model aligns with known pathogenic GGCX mutations, providing valuable insights into the molecular basis of coagulation disorders caused by GGCX mutants.