Abstract
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.
Highlights
Neutral sphingomyelinase 2 produces the bioactive lipid ceramide and has important roles in neurodegeneration, cancer, and exosome formation
Discussion Neutral sphingomyelinase 2 (nSMase2) is emerging as a complex enzyme because it is regulated at multiple levels through layers of modular switches embedded into its structure
The results show that the juxtamembrane region (JX) region of nSMase2, which links the N-terminal domain (NTD) and the catalytic domain (CAT), plays a critical role in allosteric activation of the CAT domain
Summary
Neutral sphingomyelinase 2 (nSMase2) produces the bioactive lipid ceramide and has important roles in neurodegeneration, cancer, and exosome formation. In contrast to what we previously assumed, we demonstrate that PS binding at the N-terminal and juxtamembrane regions of nSMase 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. SMases are phosphodiesterases that generate ceramide by hydrolyzing sphingomyelin (SM) and releasing a phosphocholine moiety as byproduct They are broadly classified, based on their pH optima, into acid sphingomyelinases, neutral sphingomyelinases (nSMases), and alkaline sphingomyelinases. We elucidate the importance of these interdomain interactions in nSMase function
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