The juxtamembrane linker in neutral sphingomyelinase-2 functions as an intramolecular allosteric switch that activates the enzyme.

Neutral sphingomyelinase 2 (nSMase2) produces the bioactive lipid ceramide and has important roles in neurodegeneration, cancer, and exosome formation. Although nSMase2 has low basal activity, it is fully activated by phosphatidylserine (PS). Previous work showed that interdomain interactions within nSMase2 are needed for PS activation. Here, we use multiple approaches, including small angle X-ray scattering, hydrogen-deuterium exchange-MS, circular dichroism and thermal shift assays, and membrane yeast two-hybrid assays, to define the mechanism mediating this interdomain interactions within nSMase2. In contrast to what we previously assumed, we demonstrate that PS binding at the N-terminal and juxtamembrane regions of nSMase2 rather acts as a conformational switch leading to interdomain interactions that are critical to enzyme activation. Our work assigns a unique function for a class of linkers of lipid-activated, membrane-associated proteins. It indicates that the linker actively participates in the activation mechanism via intramolecular interactions, unlike the canonical linkers that typically aid protein dimerization or localization.