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

• 成果类型: Article
• 署名作者: Cao, Qing; Ammerman, Aaron; Saimi, Mierxiati; Lin, Zongtao; Shen, Guomin; Chen, Huaping; Sun, Jie; Chai, Mengqi; Liu, Shixuan; Hsu, Fong-Fu; Krezel, Andrzej M.; Gross, Michael L.; Xu, Jinbin; Garcia, Benjamin A.; Liu, Bin; Li, Weikai
• 署名单位: Washington University (WUSTL); Washington University (WUSTL); Washington University (WUSTL); Washington University (WUSTL); University of Minnesota System; Harbin Medical University
• 刊物名称: Nature
• ISSN/ISSBN: 0028-1101
• DOI: 10.1038/s41586-025-08648-1
• 发表日期: 2025-03-20
• 关键词: glutamate substrate-binding; dependent carboxylase; factor-ix; missense mutations; recognition site; signal peptide; propeptide; protein; deficiency; mechanism

摘要

The gamma-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, 2, 3-4. Modulating gamma-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 gamma-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 gamma-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 gamma-carboxylation across the membrane interface. These structural insights and extensive functional experiments advance membrane enzymology and propel the development of treatments for gamma-carboxylation disorders.