3 years ago

Structure of human nSMase2 reveals an interdomain allosteric activation mechanism for ceramide generation [Biochemistry]

Structure of human nSMase2 reveals an interdomain allosteric activation mechanism for ceramide generation [Biochemistry]
Can E. Senkal, Miguel Garcia-Diaz, Achraf A. Shamseddine, Lina M. Obeid, Bill X. Wu, Nana Bartke, Rohan Maini, Prajna Shanbhogue, Kip E. Guja, Yusuf A. Hannun, Michael V. Airola

Neutral sphingomyelinase 2 (nSMase2, product of the SMPD3 gene) is a key enzyme for ceramide generation that is involved in regulating cellular stress responses and exosome-mediated intercellular communication. nSMase2 is activated by diverse stimuli, including the anionic phospholipid phosphatidylserine. Phosphatidylserine binds to an integral-membrane N-terminal domain (NTD); however, how the NTD activates the C-terminal catalytic domain is unclear. Here, we identify the complete catalytic domain of nSMase2, which was misannotated because of a large insertion. We find the soluble catalytic domain interacts directly with the membrane-associated NTD, which serves as both a membrane anchor and an allosteric activator. The juxtamembrane region, which links the NTD and the catalytic domain, is necessary and sufficient for activation. Furthermore, we provide a mechanistic basis for this phenomenon using the crystal structure of the human nSMase2 catalytic domain determined at 1.85-Å resolution. The structure reveals a DNase-I–type fold with a hydrophobic track leading to the active site that is blocked by an evolutionarily conserved motif which we term the “DK switch.” Structural analysis of nSMase2 and the extended N-SMase family shows that the DK switch can adopt different conformations to reposition a universally conserved Asp (D) residue involved in catalysis. Mutation of this Asp residue in nSMase2 disrupts catalysis, allosteric activation, stimulation by phosphatidylserine, and pharmacological inhibition by the lipid-competitive inhibitor GW4869. Taken together, these results demonstrate that the DK switch regulates ceramide generation by nSMase2 and is governed by an allosteric interdomain interaction at the membrane interface.

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