title: 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.affiliation: Department of Medicine, Stony Brook University, Stony Brook, NY 11794, USA; Department of Medicine, Stony Brook Cancer Center, Stony Brook, NY 11794, USA.
authors.forename: Michael V
authors.initials: MV
authors.lastname: Airola
authors.affiliation: Department of Applied Mathematics and Statistics, Stony Brook University, Stony Brook, NY 11794, USA.
authors.forename: William J
authors.initials: WJ
authors.lastname: Allen
authors.affiliation: Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Pulkoski-Gross
authors.affiliation: Department of Medicine, Stony Brook University, Stony Brook, NY 11794, USA; Department of Medicine, Stony Brook Cancer Center, Stony Brook, NY 11794, USA; Department of Medicine, Northport Veterans Affairs Medical Center, Northport, NY 11768, USA.
authors.forename: Lina M
authors.initials: LM
authors.lastname: Obeid
authors.affiliation: Department of Applied Mathematics and Statistics, Stony Brook University, Stony Brook, NY 11794, USA.
authors.forename: Robert C
authors.initials: RC
authors.lastname: Rizzo
authors.affiliation: Department of Medicine, Stony Brook University, Stony Brook, NY 11794, USA; Department of Medicine, Stony Brook Cancer Center, Stony Brook, NY 11794, USA. Electronic address: yusuf.hannun@stonybrookmedicine.edu.
authors.forename: Yusuf A
authors.initials: YA
authors.lastname: Hannun
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D002518:Ceramides / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008024:Ligands; D008246:Lysophospholipids / Q000737:chemistry* / Q000378:metabolism; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D055576:Neutral Ceramidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D061987:Sf9 Cells; D013045:Species Specificity; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D018411:Spodoptera
chemical_list: D002518:Ceramides; D008024:Ligands; D008246:Lysophospholipids; D011994:Recombinant Proteins; C060506:sphingosine 1-phosphate; C526744:ASAH2 protein, human; D055576:Neutral Ceramidase; D013110:Sphingosine