Molecular basis of vitamin K driven γ-carboxylation at membrane interface

The γ-carboxylation of glutamate residues enables Ca2+-mediated membrane assembly of protein complexes that support broad physiological functions including hemostasis, calcium homeostasis, immune response, and endocrine regulation. Modulating γ-carboxylation level provides prevalent treatments for hemorrhagic and thromboembolic diseases. This unique posttranslational modification requires vitamin K hydroquinone (KH2) to drive highly demanding reactions catalyzed by the membrane-integrated γ-carboxylase (VKGC). To decipher underlying mechanisms, we determined cryo-electron microscopy structures of human VKGC in unbound form, with KH2 and four hemostatic and non-hemostatic proteins possessing propeptides and glutamate-rich domains in different carboxylation states. VKGC recognizes substrate proteins via 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 free hydroxide ion as an exceptionally strong base 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 novel treatments for γ-carboxylation disorders.