A Heart Disease-Associated TSPO Variant Alters Transmembrane Helix Dynamics

The 18-kDa translocator protein TSPO is an outer mitochondrial membrane protein involved in cholesterol transport, stress response, and cellular metabolism. Although its five-helix architecture is conserved across species, the structural and dynamic features of human TSPO remain unresolved. Using solution NMR spectroscopy, we characterize the conformational dynamics of human TSPO in complex with a third-generation diagnostic ligand. We identify a dynamic N-terminal segment of the first transmembrane helix that does not form a stably populated helix but instead defines a flexible boundary between the cytosolic region and the transmembrane core. The common disease-associated A14V variant reduces this conformational heterogeneity by introducing short-range contacts and redistributing backbone dynamics across the protein. These changes preserve the overall fold while locally stabilizing the N-terminal transmembrane helix toward the VDAC interaction interface. Our findings reveal a human-specific dynamic architecture of TSPO and link variant-induced stabilization to modulation of transmembrane helix dynamics.