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- PDB-8y2m: Cryo-EM structure of the FB1-bound Lac1-Lip1 complex -

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Basic information

Entry
Database: PDB / ID: 8y2m
TitleCryo-EM structure of the FB1-bound Lac1-Lip1 complex
Components
  • Ceramide synthase LAC1
  • Ceramide synthase subunit LIP1
KeywordsTRANSFERASE / Inhibitor / Complex
Function / homology
Function and homology information


very-long-chain ceramide synthase / acyl-CoA ceramide synthase complex / Sphingolipid de novo biosynthesis / sphingosine N-acyltransferase activity / ceramide biosynthetic process / nuclear periphery / nuclear envelope / endoplasmic reticulum membrane / endoplasmic reticulum
Similarity search - Function
TRAM/LAG1/CLN8 homology domain / Sphingosine N-acyltransferase Lag1/Lac1-like / TLC domain / TLC domain profile. / TRAM, LAG1 and CLN8 homology domains.
Similarity search - Domain/homology
Chem-6PL / hexacosanoic acid / : / Ceramide synthase LAC1 / Ceramide synthase subunit LIP1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.07 Å
AuthorsXie, T. / Zhang, Z. / Fang, Q. / Gong, X.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Structure / Year: 2024
Title: Mechanism of ceramide synthase inhibition by fumonisin B.
Authors: Zike Zhang / Qi Fang / Tian Xie / Xin Gong /
Abstract: Ceramide synthases (CerSs) play crucial roles in sphingolipid metabolism and have emerged as promising drug targets for metabolic diseases, cancers, and antifungal therapy. However, the therapeutic ...Ceramide synthases (CerSs) play crucial roles in sphingolipid metabolism and have emerged as promising drug targets for metabolic diseases, cancers, and antifungal therapy. However, the therapeutic targeting of CerSs has been hindered by a limited understanding of their inhibition mechanisms by small molecules. Fumonisin B (FB) has been extensively studied as a potent inhibitor of eukaryotic CerSs. In this study, we characterize the inhibition mechanism of FB on yeast CerS (yCerS) and determine the structures of both FB-bound and N-acyl-FB-bound yCerS. Through our structural analysis and the observation of N-acylation of FB by yCerS, we propose a potential ping-pong catalytic mechanism for FB N-acylation by yCerS. Lastly, we demonstrate that FB exhibits lower binding affinity for yCerS compared to the C26- coenzyme A (CoA) substrate, suggesting that the potent inhibitory effect of FB on yCerS may primarily result from the N-acyl-FB catalyzed by yCerS, rather than through direct binding of FB.
History
DepositionJan 26, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Nov 27, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Ceramide synthase LAC1
B: Ceramide synthase subunit LIP1
C: Ceramide synthase LAC1
D: Ceramide synthase subunit LIP1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)141,85114
Polymers135,0364
Non-polymers6,81610
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Ceramide synthase LAC1 / Very-long-chain ceramide synthase LAC1


Mass: 50289.082 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: LAC1, DGT1, YKL008C, YKL156 / Production host: Homo sapiens (human)
References: UniProt: P28496, very-long-chain ceramide synthase
#2: Protein Ceramide synthase subunit LIP1 / LAG1/LAC1-interacting protein 1


Mass: 17228.682 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: LIP1, YMR298W / Production host: Homo sapiens (human) / References: UniProt: Q03579
#3: Chemical
ChemComp-6PL / (4S,7R)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSAN-1-AMINIUM 4-OXIDE / 1-PALMITOYL-2-STEAROYL-SN-GLYCERO-3-PHOSPHOCHOLINE


Mass: 763.100 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C42H85NO8P / Comment: phospholipid*YM
#4: Chemical ChemComp-A1D5V / (2~{R})-2-[2-[(5~{R},6~{R},7~{S},9~{S},11~{R},16~{R},18~{S},19~{S})-19-azanyl-6-[(3~{R})-3-carboxy-5-oxidanyl-5-oxidanylidene-pentanoyl]oxy-5,9-dimethyl-11,16,18-tris(oxidanyl)icosan-7-yl]oxy-2-oxidanylidene-ethyl]butanedioic acid / Fumonisin b1 / Macrofusine


Mass: 721.830 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C34H59NO15 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-7PO / hexacosanoic acid


Mass: 396.690 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C26H52O2
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: FB1-bound Lac1-Lip1 complex / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 3.07 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 507891 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0067974
ELECTRON MICROSCOPYf_angle_d0.89510782
ELECTRON MICROSCOPYf_dihedral_angle_d23.4261274
ELECTRON MICROSCOPYf_chiral_restr0.0531160
ELECTRON MICROSCOPYf_plane_restr0.0081298

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