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

Abstract

Edited by George M. Carman 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, hydrogendeuterium 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.

The nSMase2/Smpd3 gene modulates the severity of muscular dystrophy and the emotional stress response in mdx mice

“…Neutral sphingomyelinase 2/Sphingomyelin phosphodiesterase 3 (nSMase2/Smpd3) is a membrane-associated enzyme that hydrolyzes sphingomyelin and affects membrane trafficking, receptor clustering, and signal transduction [15,16]. nSMase2/Smpd3 is activated by inflammatory cytokines such as TNF-alpha, IL-1beta, and interferongamma (IFN-gamma), and it in turn activates caspase-12 and calpain in a Ca 2+ -dependent manner [17][18][19]. In addition, nSMase2/Smpd3 deficiency reduces the inflammatory response through decreased macrophage infiltration and lipid deposition [20].…”

Role of Neutral Sphingomyelinase-2 (NSM 2) in the Control of T Cell Plasma Membrane Lipid Composition and Cholesterol Homeostasis

“…The NSM2 protein, encoded by the SMPD3 gene, has two N-terminal hydrophobic segments associated with cytosolic membrane leaflets and a C-terminal catalytic site (Hofmann et al, 2000). In addition to phosphorylation (Filosto et al, 2010, 2012), a conformational switch following binding to phosphatidylserine (PS) has been proposed to be crucial in enzymatic activation (Airola et al, 2017; Shanbhogue et al, 2019). NSM2-catalyzed sphingomyelin breakdown commonly occurs in response to cellular stress and regulates bone mineralization.…”

Neutral sphingomyelinase‐2 and cardiometabolic diseases

“…14,22 It was further elaborated that phosphatidylserine binding at the NTD and juxtamembrane linker region in nSMase2 acts as a conformational switch, leading to interdomain, intramolecular interactions that are critical for nSMase2 activation. 116 It was also revealed that the asparagine residue, ASN130, coordinates the binding of Mg 2+ ion in the active site which contributes to the catalytic activity of nSMase2 by stabilizing the phosphodiester bond of the sphingomyelin in the substrate binding site before its cleavage into phosphocholine and ceramide. 26,117 In addition, the region separating the NTD and CAT domain of nSMase2 harbors five serine residues that can be phosphorylated 115 and also a calcineurin-binding site 113 that are relevant for nSMase2 activation as well as stability as discussed above.…”