BPS2026 – Conformational dynamics in the mitochondrial pyruvate carrier

Membrane transporters undergo complex conformational transitions that are challenging to capture with static structural methods. The heterodimeric mitochondrial pyruvate carrier (MPC) plays a central role in cellular metabolism by transporting pyruvate into mitochondria, yet its dynamic transport mechanism remains poorly understood. Here, we combined computational modeling with experimental validation to investigate the conformational landscape of MPC and gain mechanistic insight into substrate translocation. Starting from AlphaFold prediction, we systematically varied input sequence information to explore alternative conformations, reducing biases inherent to static models. Predicted structures were evaluated for physical plausibility, and ensemble analysis identified inward-facing, outward-facing, and intermediate states. Docking of pyruvate and derivatives to the predicted occluded state recapitulated key features of drug binding observed in the experimentally determined open structures. Comparison with cryo-EM structures revealed excellent agreement for inward- and outward-facing conformations (≤1.5 Å RMSD). Notably, our predicted occluded conformation closely resembles the occluded apo-state recently reported by independent studies, highlighting the predictive power of computational modeling. Functional temperature-jump assays further validate substrate and drug interactions, supporting a stepwise transport mechanism that involves the coordinated movement of MPC subunits. These findings demonstrate that integrating sequence-based modeling, energy-based validation, docking, and functional assays can accurately generate high-resolution, functionally relevant conformational ensembles. This combined computational-experimental strategy provides mechanistic insight into pyruvate transport and offers a generalizable framework for studying dynamic transitions in membrane transporters essential for mitochondrial function and cellular metabolism.