Transmembrane domain interactions underlie NSG1 regulation of sortilin ectodomain shedding

Sortilin is a single-pass transmembrane receptor involved in intracellular trafficking, neurotrophic signaling, and protein clearance pathways relevant to neurodegenerative disease. We recently identified the neuron-specific protein NSG1 as a selective modulator of sortilin function, promoting its ectodomain shedding via ADAM10. However, the molecular basis of this interaction remains unresolved. Here, we present a structural framework for NSG1-mediated regulation of sortilin shedding. Using mutagenesis, biochemical assays, and structural modeling, we mapped the interaction interface of NSG1 to the helical transmembrane domain (TMD) of sortilin. We show that NSG1 binds a specific interface within the sortilin TMD, modulating its susceptibility to ectodomain shedding. Mutational analysis revealed that substitutions in the central region of the sortilin TMD, particularly T770W and A773W, significantly reduce NSG1-dependent shedding without disrupting complex formation. Coarse-grained molecular dynamics simulations identified two potential binding interfaces on the sortilin TMD and demonstrated that the T770W mutation shifts the preferred interface, thereby diminishing the ability of NSG1 to promote proteolytic processing. Notably, the closely related protein NSG2 has a different preferred binding mode on the sortilin TMD and does not induce shedding, highlighting the functional specificity of NSG1. Our findings establish the TMD-TMD interaction as an important basis for NSG1-mediated regulation of sortilin shedding. This study advances our understanding of how transmembrane interactions govern substrate-specific a disintegrin and metalloproteinase proteolysis and provides new insight into the molecular control of sortilin function. Given the emerging role of sortilin in Alzheimer's disease, these insights may help clarify how its processing is regulated in the diseased brain.