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- PDB-31mr: CryoEM structure of a catalytically inactive CXC Chemokine-degrad... -

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Basic information

Entry
Database: PDB / ID: 31mr
TitleCryoEM structure of a catalytically inactive CXC Chemokine-degrading protease SpyCEP from Streptococcus pyogenes complexed with an anti-PA-domain monoclonal antibody
Components
  • (Cell envelope proteinase A) x 2
  • Anti-PA-domain monoclonal antibody (3F2G10) Heavy chain variable region
  • Anti-PA-domain monoclonal antibody (3F2G10) Light chain variable region
KeywordsPROTEIN BINDING / Bacterial / protease / complex / antibody / immune evasion / chemokine
Function / homology
Function and homology information


serine-type endopeptidase activity / proteolysis / membrane
Similarity search - Function
Fn3-like domain / C5a peptidase-like domain / Fn3-like domain / : / PA domain / PA domain superfamily / PA domain / YSIRK type signal peptide / YSIRK Gram-positive signal peptide / Peptidase S8, subtilisin, His-active site ...Fn3-like domain / C5a peptidase-like domain / Fn3-like domain / : / PA domain / PA domain superfamily / PA domain / YSIRK type signal peptide / YSIRK Gram-positive signal peptide / Peptidase S8, subtilisin, His-active site / Serine proteases, subtilase family, histidine active site. / Serine proteases, subtilase family, aspartic acid active site. / Peptidase S8, subtilisin, Asp-active site / Serine proteases, subtilase family, serine active site. / Peptidase S8, subtilisin, Ser-active site / Gram-positive cocci surface proteins LPxTG motif profile. / LPXTG cell wall anchor domain / Peptidase S8, subtilisin-related / Serine proteases, subtilase domain profile. / Peptidase S8/S53 domain superfamily / Subtilase family / Peptidase S8/S53 domain / Immunoglobulin-like fold
Similarity search - Domain/homology
Cell envelope proteinase A
Similarity search - Component
Biological speciesStreptococcus pyogenes (bacteria)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.07 Å
AuthorsLau, R.J. / Barritt, J.D. / Wu, G.H.Y. / Huemer, C.B. / Matthews, S.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)UKRI682 United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2026
Title: SpyCEP dismantles neutrophil immunity via disorder-driven chemokine remodeling and GAG targeting.
Authors: Rikin J Lau / Sean P Giblin / Andra Sugar / Antonio Di Maio / Giulio Tassini / Kristin Huse / Dror Chorev / Yuan Chen / Grace Ho-Yan Wu / Camilla Berg Huemer / Seung Yon Kim / Jayden ...Authors: Rikin J Lau / Sean P Giblin / Andra Sugar / Antonio Di Maio / Giulio Tassini / Kristin Huse / Dror Chorev / Yuan Chen / Grace Ho-Yan Wu / Camilla Berg Huemer / Seung Yon Kim / Jayden Matthews / Bel Muloud / Lu Chen / Sophie McKenna / Yingqi Xu / Luisa Massai / Chiara Muzzi / Xhenti Ferhati / Francesca Necchi / Danilo Gomes Moriel / Ten Feizi / Yan Liu / James E Pease / Shiranee Sriskandan / Steve Matthews /
Abstract: (Group A ) employs sophisticated virulence strategies to evade human immunity, including secretion of the cell envelope protease SpyCEP, which cleaves and inactivates key neutrophil-attracting ... (Group A ) employs sophisticated virulence strategies to evade human immunity, including secretion of the cell envelope protease SpyCEP, which cleaves and inactivates key neutrophil-attracting chemokines such as CXCL8. Here, we integrate cryo-electron microscopy, NMR spectroscopy, and native mass spectrometry to investigate how SpyCEP disrupts CXCL8 function. We demonstrate that a disordered aromatic and acidic region within the cleaved autocatalytic maturation loop (CAML) of SpyCEP mimics receptor N-domains and binds an allosteric site on CXCL8. The resulting interaction forms a dynamic fuzzy complex and is coupled to dimer dissociation, consistent with enhanced access to the cleavage site. This disorder-mediated substrate engagement differs from classical protease mechanisms that rely on rigid recognition interfaces. Additionally, glycan microarray and NMR analyses show that the CAML region mediates glycosaminoglycan (GAG) binding, suggesting a means for SpyCEP to maximize encounters with GAG-enriched CXCL8 reservoirs. Together, these findings provide a structural and biophysical framework for understanding how SpyCEP combines substrate engagement with GAG targeting to dismantle chemokine gradients and inhibit neutrophil recruitment. More broadly, this work highlights the role of intrinsic disorder in protease recognition and suggests avenues for anti-virulence therapies and vaccine strategies targeting SpyCEP.
History
DepositionJun 12, 2026Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 1, 2026Provider: repository / Type: Initial release
Revision 1.0Jul 1, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jul 1, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jul 1, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 1, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 1, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 1, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jul 22, 2026Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 1.1Jul 22, 2026Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / EM metadata / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cell envelope proteinase A
B: Cell envelope proteinase A
H: Anti-PA-domain monoclonal antibody (3F2G10) Heavy chain variable region
L: Anti-PA-domain monoclonal antibody (3F2G10) Light chain variable region


Theoretical massNumber of molelcules
Total (without water)198,7634
Polymers198,7634
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Cell envelope proteinase A / Peptidase S8 PrtS/CepA protein


Mass: 24609.822 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes (bacteria) / Gene: cepA, SP119_0326 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q201Z7
#2: Protein Cell envelope proteinase A / Peptidase S8 PrtS/CepA protein


Mass: 148494.438 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes (bacteria) / Gene: cepA, SP119_0326 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q201Z7
#3: Antibody Anti-PA-domain monoclonal antibody (3F2G10) Heavy chain variable region


Mass: 13333.943 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse)
#4: Antibody Anti-PA-domain monoclonal antibody (3F2G10) Light chain variable region


Mass: 12324.799 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse)
Has protein modificationN

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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: SpyCEP complexed with 3F2G10 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.325 MDa / Experimental value: YES
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8 / Details: 20 mM Tris pH 8.0, 200 mM NaCl
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium chlorideNaCl1
220 mMtris hydrochlorideTris-HCL1
SpecimenConc.: 1.625 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 294.15 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 79000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1959
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
4cryoSPARCCTF correction
7ISOLDEmodel fitting
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13ISOLDEmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1351301
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.07 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 660420 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 129.9 / Protocol: FLEXIBLE FIT / Space: REAL / Details: Flexible fitting all performed in ISOLDE
Atomic model buildingSource name: AlphaFold / Type: in silico model

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