Structural basis of calcium-mediated conformational dynamics and cargo binding in vacuolar Sorting Receptor 1 (VSR1)

Vacuolar sorting receptors (VSRs) are type I membrane proteins crucial for seed germination and plant development. Although VSR trafficking has been extensively studied, the mechanism of cargo recognition and release by the N-terminal lumenal region remains poorly understood. Here, we solved the crystal structures of both unloaded and cargo-loaded forms of the VSR1 lumenal region comprising the protease-associated (PA) domain, the Central domain and epidermal growth factor-like repeats. Two cargo binding sites were identified: one previously reported site found in the PA and Central domains and one uncharacterised site located within the Central domain. The large conformational differences observed between the two crystal structures are confirmed in solution by Small-angle X-ray scattering (SAXS) experiments. Their combination revealed that calcium coordination remodels inter-domain linkers, triggering large conformational changes that expose and remodel the two cargo binding sites. Complementary X-ray footprinting mass spectrometry (XFMS) mapped the residues undergoing solvent accessibility changes induced by cargo binding and confirmed residues affected in both cargo binding sites and across all three domains in solution. Together, these findings reveal a dynamic calcium-dependent mechanism in which high calcium concentrations promote cargo binding, while cargo release is favoured by lower calcium concentrations and pH. This work provides a structural framework for understanding VSR-mediated vacuolar trafficking and cargo sorting in plants.