The γ-carboxylation of glutamate residues enables Ca2+-mediated membrane assembly of protein complexes that support broad physiological functions, including haemostasis, calcium homeostasis, immune response and endocrine regulation1–4. Modulating γ-carboxylation levels provides prevalent treatments for haemorrhagic and thromboembolic diseases5. This unique post-translational modification requires vitamin K hydroquinone (KH2) to drive highly demanding reactions6 catalysed by the membrane-integrated γ-carboxylase (VKGC). Here, to decipher the underlying mechanisms, we determined cryo-electron microscopy structures of human VKGC in unbound form, with KH2 and four haemostatic and non-haemostatic proteins possessing propeptides and glutamate-rich domains in different carboxylation states. VKGC recognizes substrate proteins through knob-and-hole interactions with propeptides, thereby bringing tethered glutamate-containing segments for processive carboxylation within a large chamber that provides steric control. Propeptide binding also triggers a global conformational change to signal VKGC activation. Through sequential deprotonation and KH2 epoxidation, VKGC generates a free hydroxide ion as an exceptionally strong base that is required to deprotonate the γ-carbon of glutamate for CO2 addition. The diffusion of this superbase—protected and guided by a sealed hydrophobic tunnel—elegantly resolves the challenge of coupling KH2 epoxidation to γ-carboxylation across the membrane interface. These structural insights and extensive functional experiments advance membrane enzymology and propel the development of treatments for γ-carboxylation disorders.
