biostudies-literatureWu KNational Natural Science Foundation of China (National Science Foundation of China)10480https://www.ebi.ac.uk/biostudies/studies/S-EPMC12647623biostudies-literatureUnknown16(1)Vitamin K-dependent (VKD) carboxylation, mediated by γ-glutamyl carboxylase (GGCX), is essential for the maturation of VKD proteins involved in critical physiological processes such as blood clotting, vascular calcification and bone metabolism. Here, we present cryo-electron microscopic structures of human GGCX alone and in complex with VKD proteins, vitamin K, and inhibitor anisindione. GGCX specifically recognizes diverse VKD substrates through high-affinity propeptide binding, while substrates like osteocalcin utilize a secondary exosite to enhance interaction. GGCX employs a conserved dipeptide anchoring mechanism that ensures processive carboxylation of glutamate residues. GGCX undergoes allosteric conformational changes that enable coordinated binding of vitamin K and glutamate substNature communicationsStructural insight into bicarbonate-mediated carboxylation by human vitamin K-dependent carboxylase.PMC12647623317702593247134431570235Wu KWu JCao MLei MLi SLan PZhang YYao DXing SWang XShen YWang ZfalseStructural insight into bicarbonate-mediated carboxylation by human vitamin K-dependent carboxylase.Vitamin K-dependent (VKD) carboxylation, mediated by γ-glutamyl carboxylase (GGCX), is essential for the maturation of VKD proteins involved in critical physiological processes such as blood clotting, vascular calcification and bone metabolism. Here, we present cryo-electron microscopic structures of human GGCX alone and in complex with VKD proteins, vitamin K, and inhibitor anisindione. GGCX specifically recognizes diverse VKD substrates through high-affinity propeptide binding, while substrates like osteocalcin utilize a secondary exosite to enhance interaction. GGCX employs a conserved dipeptide anchoring mechanism that ensures processive carboxylation of glutamate residues. GGCX undergoes allosteric conformational changes that enable coordinated binding of vitamin K and glutamate subst2025-01-01T00:00:00Z2025 Nov2026-06-05T19:54:17.19Z2026-05-21T03:11:40.269ZS-EPMC126476234129065010.1038/s41467-025-65488-3