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- EMDB-15205: cryoEM structure of the catalytically inactive EndoS from S. pyog... -

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Basic information

Entry
Database: EMDB / ID: EMD-15205
TitlecryoEM structure of the catalytically inactive EndoS from S. pyogenes in complex with the Fc region of immunoglobulin G1
Map dataMain map cryosparc refine
Sample
  • Complex: Complex of the EndoS from S. pyogenes and the Fc region of Immunoglobulin G
    • Protein or peptide: Endo-beta-N-acetylglucosaminidase F2
    • Protein or peptide: Immunoglobulin gamma-1 heavy chain
KeywordsEndoglycosidase S / EndoS / endo-b-N-acetylglucosaminidase / Fc region / antibody / immunoglobulin G1 / Streptococcus pyogenes / N-glycans / HYDROLASE
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsTrastoy B / Cifuente JO / Du JJ / Sundberg EJ / Guerin ME
Funding support United States, 1 items
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, 2022-
Header (metadata) releaseApr 5, 2023-
Map releaseApr 5, 2023-
UpdateJul 26, 2023-
Current statusJul 26, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15205.png

Downloads & links

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Map

FileDownload / File: emd_15205.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain map cryosparc refine
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 180 pix.
= 189.36 Å		1.05 Å/pix.
x 180 pix.
= 189.36 Å		1.05 Å/pix.
x 180 pix.
= 189.36 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.052 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.25330344 - 0.7173578
Average (Standard dev.)0.006468186 (±0.040584818)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 189.36002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15205_msk_1.map
Projections & Slices
AxesZYX

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Additional map: Main map cryosparc refine

Fileemd_15205_additional_1.map
AnnotationMain map cryosparc refine
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: Half map cryosparc refine

Fileemd_15205_half_map_1.map
AnnotationHalf map cryosparc refine
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map cryosparc refine

Fileemd_15205_half_map_2.map
AnnotationHalf map cryosparc refine
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of the EndoS from S. pyogenes and the Fc region of Immuno...

EntireName: Complex of the EndoS from S. pyogenes and the Fc region of Immunoglobulin G
Components
  • Complex: Complex of the EndoS from S. pyogenes and the Fc region of Immunoglobulin G
    • Protein or peptide: Endo-beta-N-acetylglucosaminidase F2
    • Protein or peptide: Immunoglobulin gamma-1 heavy chain

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Supramolecule #1: Complex of the EndoS from S. pyogenes and the Fc region of Immuno...

SupramoleculeName: Complex of the EndoS from S. pyogenes and the Fc region of Immunoglobulin G
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 150 KDa

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

MacromoleculeName: Endo-beta-N-acetylglucosaminidase F2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 108.297039 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EEKTVQVQKG LPSIDSLHYL SENSKKEFKE ELSKAGQESQ KVKEILAKAQ QADKQAQELA KMKIPEKIPM KPLHGPLYGG YFRTWHDKT SDPTEKDKVN SMGELPKEVD LAFIFHDWTK DYSLFWKELA TKHVPKLNKQ GTRVIRTIPW RFLAGGDNSG I AEDTSKYP ...String:
EEKTVQVQKG LPSIDSLHYL SENSKKEFKE ELSKAGQESQ KVKEILAKAQ QADKQAQELA KMKIPEKIPM KPLHGPLYGG YFRTWHDKT SDPTEKDKVN SMGELPKEVD LAFIFHDWTK DYSLFWKELA TKHVPKLNKQ GTRVIRTIPW RFLAGGDNSG I AEDTSKYP NTPEGNKALA KAIVDEYVYK YNLDGLDVDV AHDSIPKVDK KEDTAGVERS IQVFEEIGKL IGPKGVDKSR LF IMDSTYM ADKNPLIERG APYINLLLVQ VYGSQGEKGG WEPVSNRPEK TMEERWQGYS KYIRPEQYMI GFSFYEENAQ EGN LWYDIN SRKDEDKANG INTDITGTRA ERYARWQPKT GGVKGGIFSY AIDRDGVAHQ PKKYAKQKEF KDATDNIFHS DYSV SKALK TVMLKDKSYD LIDEKDFPDK ALREAVMAQV GTRKGDLERF NGTLRLDNPA IQSLEGLNKF KKLAQLDLIG LSRIT KLDR SVLPANMKPG KDTLETVLET YKKDNKEEPA TIPPVSLKVS GLTGLKELDL SGFDRETLAG LDAATLTSLE KVDISG NKL DLAPGTENRQ IFDTMLSTIS NHVGSNEQTV KFDKQKPTGH YPDTYGKTSL RLPVANEKVD LQSQLLFGTV TNQGTLI NS EADYKAYQNH KIAGRSFVDS NYHYNNFKVS YENYTVKVTD STLGTTTDKT LATDKEETYK VDFFSPADKT KAVHTAKV I VGDEKTMMVN LAEGATVIGG SADPVNARKV FDGQLGSETD NISLGWDSKQ SIIFKLKEDG LIKHWRFFND SARNPETTN KPIQEASLQI FNIKDYNLDN LLENPNKFDD EKYWITVDTY SAQGERATAF SNTLNNITSK YWRVVFDTKG DRYSSPVVPE LQILGYPLP NADTIMKTVT TAKELSQQKD KFSQKMLDEL KIKEMALETS LNSKIFDVTA INANAGVLKD CIEKRQLLKK L

UniProtKB: Endo-beta-N-acetylglucosaminidase F2

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Macromolecule #2: Immunoglobulin gamma-1 heavy chain

MacromoleculeName: Immunoglobulin gamma-1 heavy chain / type: protein_or_peptide / ID: 2
Details: TCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL ...Details: TCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK
Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 49.386637 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: QVQLVQSGGG VVQPGRSLRL SCAASGFTFS RYTIHWVRQA PGKGLEWVAV MSYNGNNKHY ADSVNGRFTI SRNDSKNTLY LNMNSLRPE DTAVYYCARI RDTAMFFAHW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS W NSGALTSG ...String:
QVQLVQSGGG VVQPGRSLRL SCAASGFTFS RYTIHWVRQA PGKGLEWVAV MSYNGNNKHY ADSVNGRFTI SRNDSKNTLY LNMNSLRPE DTAVYYCARI RDTAMFFAHW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS W NSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKKVE PKSCDKTHTC PPCPAPELLG GP SVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKE YKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVL DSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5 / Details: Standard PBS
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK II / Details: single blot.
DetailsComplex stabilized by glutaraldehyde crosslinking prepared by ultracentrifugation in a fixation glycerol gradient (Grafix) and size exclusion chromatography purified at approximately 0.2 mg/mL

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-60 / Number grids imaged: 1 / Number real images: 5546 / Average electron dose: 59.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.3 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 797351
Startup modelType of model: OTHER / Details: A negatively stained model obtained in RELION.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.6 Å / Resolution method: FSC 3 SIGMA CUT-OFF / Software - Name: cryoSPARC / Number images used: 339309
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Details: Classified selected particles in RELION were used for reconstruction and CTFrefinement and Polishing
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Details: Imported selected particles CTFrefined and Polished in RELION were imported in cryoSPARC2 and 2D reclassified for final reconstruction
Final 3D classificationNumber classes: 200 / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6en3

chain_id: A

1h3x

chain_id: B

1h3x

chain_id: C
DetailsInitial PDB models were rigid-body refined for individual chains. Later, the model was real-space refined removing side chains that couldn't be assigned.
RefinementProtocol: OTHER
Output model

PDB-8a64:
cryoEM structure of the catalytically inactive EndoS from S. pyogenes in complex with the Fc region of immunoglobulin G1.

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