Structural bases of the conformational transitions of the mitochondrial choline carrier SLC25A48 captured by...

The mitochondrial choline carrier SLC25A48 mediates choline import into the mitochondrial matrix, supporting one‑carbon metabolism and epigenetic regulation. Despite its physiological relevance, the structural determinants of substrate recognition and conformational transitions remain poorly characterized. Here, the conformational dynamics of human SLC25A48 was investigated using deep-learning-based structure prediction combined with multiple extensive molecular dynamics simulations in a realistic mitochondrial membrane environment. Structural models representing the cytoplasmic-open (c-state), occluded and matrix-open (m-state) conformations were generated and used as starting points for MD simulations, for a total of 21 μs. Unbiased simulations captured spontaneous, bidirectional transitions between the occluded and m-state, confirmed by structural convergence and comparison with available crystal structures, and shown to remain well separated in a distinct descriptor space by a supervised classifier (LDA). Transitions were driven by an asymmetric rearrangement primarily involving Domain I, while the remaining helices behaved as a rigid scaffold, consistent with mechanistic features described for other SLC25 carriers. A matrix-side loop, whose dynamics correlated with this asymmetry, adopted distinct capping and opening conformations across states, suggesting a complementary gating role. Choline remained stably coordinated throughout the transition, indicating that the conformational change corresponds to the opening step of the transport cycle rather than substrate release. Context-dependent lipid interactions were also observed, affecting ligand position or carrier conformation. These results provide mechanistic insight into SLC25A48 conformational plasticity and a structural framework, with candidate collective variables, to guide future investigation of mitochondrial choline transport.