Aaron Ammerman

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^1-4. Modulating γ-carboxylation level provides prevalent treatments for hemorrhagic and thromboembolic diseases⁵. This unique posttranslational modification requires vitamin K hydroquinone (KH₂) to drive highly demanding reactions⁶ 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.

Biogenic monoamines, vital transmitters orchestrating neurological, endocrinal, and immunological functions^1-5, are stored in secretory vesicles by vesicular monoamine transporters (VMATs) for controlled quantal release^6,7. Harnessing proton antiport, VMATs enrich monoamines ~10,000-fold and sequester neurotoxicants to protect neurons^8-10. VMATs are targeted by an arsenal of therapeutic drugs and imaging agents to treat and monitor neurodegenerative disorders, hypertension, and drug addiction^1,8,11-16. However, the structural mechanisms underlying these actions remain elusive. Here, we report eight cryo-electron microscopy structures of human VMAT1 in unbound form and in complex with four monoamines, the Parkinsonism-inducing MPP⁺, the psychostimulant amphetamine, and the antihypertensive drug reserpine. Reserpine binding captures a cytoplasmic-open conformation, while other structures show a lumenal-open conformation stabilized by extensive gating interactions. The favored transition to this lumenal-open state contributes to monoamine accumulation, while protonation facilitates the cytoplasmic-open transition and concurrently prevents monoamine binding to avoid unintended depletion. Monoamines and neurotoxicants share a binding pocket possessing polar sites for specificity and a wrist-and-fist shape for versatility. Variations of this pocket explain substrate preferences across the SLC18 family. Overall, these structural insights and supporting functional studies elucidate the mechanism of vesicular monoamine transport and provide the basis to develop novel therapeutics for neurodegenerative diseases and substance abuse.