BPS2026 – Local residue protonation states govern the conformational switching in mitochondrial pyruvate carrier

Cell energy production relies on cytosolic glycolysis and mitochondrial oxidative phosphorylation to produce ATP. Central to connecting these pathways is the transport of pyruvate, a key respiratory intermediate, from cytosol into mitochondrial matrix. This essential step is mediated by the heterodimeric mitochondrial pyruvate carrier (MPC) located in the inner mitochondrial membrane, operating through a proton-coupled alternating access mechanism. Given its critical metabolic role, MPC dysregulation has been implicated in diabetes and neurodegenerative disorders, making it a promising therapeutic target. However, despite recently resolved cryo-EM structures, the proton-driven transport mechanism remains poorly understood, hindering the development for novel MPC-targeting therapeutics. To address this gap, we leveraged extensive molecular dynamics (MD) simulations to sample MPC’s conformational changes under acidic and basic conditions, examining outward-facing (OF) and inward-facing (IF) states, in both apo and pyruvate-bound forms. Discrete constant pH simulations revealed significantly shifted pKa values for two critical binding-site residues, K49 MPC2 and H84 MPC1 , calculated as 7.0 and 2.0, likely due to their hydrophobic microenvironment. In subsequent unbiased simulations, deprotonation of K49 MPC2 destabilized pyruvate binding in OF conformation; while in IF state, pyruvate dissociation occurred consistently and independently of K49 MPC2 ’s protonation states. Remarkably, a spontaneous OF-to-IF transition was captured at acidic pH, whereas an IF-to-OF transition occurred at basic pH, both driven by the disruption or restoration of cation-π interactions between K49 MPC2 and neighboring aromatic residues. Similar substrate-free transport has also been reported in SemiSWEET sugar transporter, which shares topological similarity with MPC. Complementary DEER experiments on apo MPC validated these findings, confirming stabilization of IF under acidic pH and OF under basic conditions. Together, these results provide atomistic insight into how local residue protonation states govern MPC's conformational changes, offering new understanding to guide future therapeutic development.