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- PDB-8a64: cryoEM structure of the catalytically inactive EndoS from S. pyog... -

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Basic information

Entry
Database: PDB / ID: 8a64
TitlecryoEM structure of the catalytically inactive EndoS from S. pyogenes in complex with the Fc region of immunoglobulin G1.
Components
  • Endo-beta-N-acetylglucosaminidase F2
  • Immunoglobulin gamma-1 heavy chain
KeywordsHYDROLASE / Endoglycosidase S / EndoS / endo-b-N-acetylglucosaminidase / Fc region / antibody / immunoglobulin G1 / Streptococcus pyogenes / N-glycans
Function / homology: / Endo-beta-N-acetylglucosaminidase F2, Ig-like domain / Glycosyl hydrolases family 18 (GH18) active site / Glycosyl hydrolases family 18 (GH18) active site signature. / hydrolase activity, hydrolyzing O-glycosyl compounds / Leucine-rich repeat domain superfamily / Glycoside hydrolase superfamily / carbohydrate metabolic process / Endo-beta-N-acetylglucosaminidase F2
Function and homology information
Biological speciesStreptococcus pyogenes (bacteria)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsTrastoy, B. / Cifuente, J.O. / Du, J.J. / Sundberg, E.J. / Guerin, M.E.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)1R01AI149297-01 United States
CitationJournal: Nat Commun / Year: 2023
Title: Mechanism of antibody-specific deglycosylation and immune evasion by Streptococcal IgG-specific endoglycosidases.
Authors: Beatriz Trastoy / Jonathan J Du / Javier O Cifuente / Lorena Rudolph / Mikel García-Alija / Erik H Klontz / Daniel Deredge / Nazneen Sultana / Chau G Huynh / Maria W Flowers / Chao Li / ...Authors: Beatriz Trastoy / Jonathan J Du / Javier O Cifuente / Lorena Rudolph / Mikel García-Alija / Erik H Klontz / Daniel Deredge / Nazneen Sultana / Chau G Huynh / Maria W Flowers / Chao Li / Diego E Sastre / Lai-Xi Wang / Francisco Corzana / Alvaro Mallagaray / Eric J Sundberg / Marcelo E Guerin /
Abstract: Bacterial pathogens have evolved intricate mechanisms to evade the human immune system, including the production of immunomodulatory enzymes. Streptococcus pyogenes serotypes secrete two multi- ...Bacterial pathogens have evolved intricate mechanisms to evade the human immune system, including the production of immunomodulatory enzymes. Streptococcus pyogenes serotypes secrete two multi-modular endo-β-N-acetylglucosaminidases, EndoS and EndoS2, that specifically deglycosylate the conserved N-glycan at Asn297 on IgG Fc, disabling antibody-mediated effector functions. Amongst thousands of known carbohydrate-active enzymes, EndoS and EndoS2 represent just a handful of enzymes that are specific to the protein portion of the glycoprotein substrate, not just the glycan component. Here, we present the cryoEM structure of EndoS in complex with the IgG1 Fc fragment. In combination with small-angle X-ray scattering, alanine scanning mutagenesis, hydrolytic activity measurements, enzyme kinetics, nuclear magnetic resonance and molecular dynamics analyses, we establish the mechanisms of recognition and specific deglycosylation of IgG antibodies by EndoS and EndoS2. Our results provide a rational basis from which to engineer novel enzymes with antibody and glycan selectivity for clinical and biotechnological applications.
History
DepositionJun 16, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 5, 2023Provider: repository / Type: Initial release
Revision 1.1May 24, 2023Group: Structure summary / Category: struct / Item: _struct.title
Revision 1.2Jul 26, 2023Group: Other / Category: pdbx_database_status / Item: _pdbx_database_status.pdb_format_compatible

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Endo-beta-N-acetylglucosaminidase F2
B: Immunoglobulin gamma-1 heavy chain
C: Immunoglobulin gamma-1 heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)207,6574
Polymers207,0703
Non-polymers5871
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: SAXS
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Endo-beta-N-acetylglucosaminidase F2


Mass: 108297.039 Da / Num. of mol.: 1 / Mutation: E235A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes (bacteria) / Gene: endoS, M1GAS476_1618 / Production host: Escherichia coli (E. coli) / References: UniProt: J7M8R4
#2: Antibody Immunoglobulin gamma-1 heavy chain


Mass: 49386.637 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: TCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL ...Details: TCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK
Source: (gene. exp.) Homo sapiens (human) / Production host: Cricetulus griseus (Chinese hamster)
#3: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
Has ligand of interestY

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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: Complex of the EndoS from S. pyogenes and the Fc region of Immunoglobulin G
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.150 MDa / Experimental value: YES
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5 / Details: Standard PBS
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Complex stabilized by glutaraldehyde crosslinking prepared by ultracentrifugation in a fixation glycerol gradient (Grafix) and size exclusion chromatography purified at approximately 0.2 mg/mL
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 283 K / Details: single blot

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 1300 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 59.4 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5546
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 60 / Used frames/image: 1-60

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameCategoryDetails
1RELIONparticle selection
2EPUimage acquisition
4RELIONCTF correction
7UCSF Chimeramodel fittingInitial rigid body fitting
8PHENIXmodel fittingRigid body fitting
10RELIONinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
14PHENIXmodel refinementReal space refinement
CTF correctionDetails: RELION CTFfind4 implementation / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 797351
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.6 Å / Resolution method: FSC 3 SIGMA CUT-OFF / Num. of particles: 339309 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER
Details: Initial PDB models were rigid-body refined for individual chains. Later, the model was real-space refined removing side chains that couldn't be assigned.
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
16EN3

6en3

A1
21H3X

1h3x

B1
31H3X

1h3x

C1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0086734
ELECTRON MICROSCOPYf_angle_d0.8489375
ELECTRON MICROSCOPYf_dihedral_angle_d5.9171376
ELECTRON MICROSCOPYf_chiral_restr0.0561293
ELECTRON MICROSCOPYf_plane_restr0.0061354

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