Structural Basis for Ceramide Recognition and Hydrolysis by Human Neutral Ceramidase.

Abstract

Neutral ceramidase (nCDase) catalyzes conversion of the apoptosis-associated lipid ceramide to sphingosine, the precursor for the proliferative factor sphingosine-1-phosphate. As an enzyme regulating the balance of ceramide and sphingosine-1-phosphate, nCDase is emerging as a therapeutic target for cancer. Here, we present the 2.6-Å crystal structure of human nCDase in complex with phosphate that reveals a striking, 20-Å deep, hydrophobic active site pocket stabilized by a eukaryotic-specific subdomain not present in bacterial ceramidases. Utilizing flexible ligand docking, we predict a likely binding mode for ceramide that superimposes closely with the crystallographically observed transition state analog phosphate. Our results suggest that nCDase uses a new catalytic strategy for Zn(2+)-dependent amidases, and generates ceramide specificity by sterically excluding sphingolipids with bulky headgroups and specifically recognizing the small hydroxyl head group of ceramide. Together, these data provide a foundation to aid drug development and establish common themes for how proteins recognize the bioactive lipid ceramide.

• Authors: Michael V Airola, William J Allen, Michael J Pulkoski-Gross, Lina M Obeid, Robert C Rizzo, Yusuf A Hannun
• Journal: Structure (London, England : 1993) (Structure) Vol. 23 Issue 8 Pg. 1482-1491 (Aug 04 2015) ISSN: 1878-4186 [Electronic] United States
• PMID: 26190575 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)
• Copyright: Copyright © 2015 Elsevier Ltd. All rights reserved.

Chemical References

• Ceramides
• Ligands
• Lysophospholipids
• Recombinant Proteins
• sphingosine 1-phosphate
• ASAH2 protein, human
• Neutral Ceramidase
• Sphingosine

Topics

• Amino Acid Sequence
• Animals
• Catalytic Domain
• Ceramides (chemistry, metabolism)
• Crystallography, X-Ray
• Escherichia coli (chemistry)
• Humans
• Hydrolysis
• Hydrophobic and Hydrophilic Interactions
• Kinetics
• Ligands
• Lysophospholipids (chemistry, metabolism)
• Molecular Docking Simulation
• Molecular Sequence Data
• Neutral Ceramidase (chemistry, genetics, metabolism)
• Protein Binding
• Protein Structure, Secondary
• Protein Structure, Tertiary
• Recombinant Proteins (chemistry, genetics, metabolism)
• Sequence Alignment
• Sf9 Cells
• Species Specificity
• Sphingosine (analogs & derivatives, chemistry, metabolism)
• Spodoptera