Mechanism of substrate transport and inhibition of the mitochondrial pyruvate carrier

Abstract

The mitochondrial pyruvate carrier (MPC) imports pyruvate across the mitochondrial inner membrane, linking cytosolic glycolysis to mitochondrial oxidative phosphorylation, increasing the adenosine 5′-triphosphate (ATP) yield by 15-fold. At the outset of this work, MPC was known to be a heterodimer, composed of two small homologous membrane proteins, but its orientation in the membrane, structural fold, transport mechanism, and the molecular basis of its inhibition were unresolved. This thesis defines the molecular basis of pyruvate transport and inhibition of the human MPC1L/2 heterodimer. The disputed membrane orientation of MPC was resolved using protease accessibility and antibody binding assays in mitoplasts and submitochondrial particles. These experiments support a topology in which the N-terminal amphipathic helices of both protomers face the intermembrane space, while the C-termini are oriented toward the matrix, providing a framework for understanding substrate transport and inhibitor accessibility. MPC is a small, pseudo-symmetric membrane complex, which makes single-particle alignment difficult. Therefore, nanobody-based fiducials were developed to stabilise and increase the effective size of the complex for single particle cryogenic electron microscopy. Structures of the MPC1L/2 heterodimer were obtained in outward-open and inward-open conformations, revealing a compact bundle of three transmembrane helices per protomer, each with an N-terminal amphipathic helix. The transmembrane helices form a narrow central cavity, whose accessibility alternates between the intermembrane space and the matrix, demonstrating that MPC operates via an alternating access mechanism. Hydrophobic and aromatic residues gate access from either side of the membrane in each conformation. A positively charged pocket near the centre of the dimer interface, formed by conserved lysine and histidine residues which are shown to be essential in substrate binding, provides a basis for a ΔpH-dependent substrate transport mechanism. Despite marked chemical diversity, inhibitors exploit the same positively charged pocket that coordinates pyruvate. Structures with MPC bound to compound 7, mitoglitazone, and zaprinast in the outward-open conformation show that high affinity binding is achieved by polar or anionic moieties that mimic the carboxylate headgroup, coupled to planar aromatic systems that engage surrounding hydrophobic residues. Together, these findings link the membrane orientation, transport mechanism, and inhibition of MPC, placing decades of biochemical and pharmacological observations on a molecular basis and guiding the development of future chemical modulators.