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- PDB-7sjo: HtrA1S328A:Fab15H6.v4 complex -

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Basic information

Entry
Database: PDB / ID: 7sjo
TitleHtrA1S328A:Fab15H6.v4 complex
Components
  • Fab15H6.v4 Heavy Chain
  • Fab15H6.v4 Light Chain
  • Serine protease HTRA1
KeywordsHYDROLASE/Immune System / HtrA1 / allosteric inhibition / Age-related macular degeneration / Geographic Atrophy / Antibody complex / HYDROLASE / HYDROLASE-Immune System complex
Function / homology
Function and homology information


chorionic trophoblast cell differentiation / programmed cell death / growth factor binding / negative regulation of BMP signaling pathway / Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases / Degradation of the extracellular matrix / serine-type peptidase activity / molecular function activator activity / negative regulation of transforming growth factor beta receptor signaling pathway / placenta development ...chorionic trophoblast cell differentiation / programmed cell death / growth factor binding / negative regulation of BMP signaling pathway / Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases / Degradation of the extracellular matrix / serine-type peptidase activity / molecular function activator activity / negative regulation of transforming growth factor beta receptor signaling pathway / placenta development / collagen-containing extracellular matrix / positive regulation of apoptotic process / serine-type endopeptidase activity / proteolysis / extracellular space / extracellular exosome / extracellular region / identical protein binding / plasma membrane / cytosol
Similarity search - Function
Insulin-like growth factor binding protein / Insulin-like growth factor-binding protein, IGFBP / Insulin-like growth factor-binding protein (IGFBP) N-terminal domain profile. / Insulin growth factor-binding protein homologues / Kazal-type serine protease inhibitor domain / PDZ domain 6 / Kazal type serine protease inhibitors / PDZ domain / Peptidase S1C / Kazal domain superfamily ...Insulin-like growth factor binding protein / Insulin-like growth factor-binding protein, IGFBP / Insulin-like growth factor-binding protein (IGFBP) N-terminal domain profile. / Insulin growth factor-binding protein homologues / Kazal-type serine protease inhibitor domain / PDZ domain 6 / Kazal type serine protease inhibitors / PDZ domain / Peptidase S1C / Kazal domain superfamily / Trypsin-like peptidase domain / Kazal domain / Kazal domain profile. / Growth factor receptor cysteine-rich domain superfamily / PDZ domain profile. / Domain present in PSD-95, Dlg, and ZO-1/2. / PDZ domain / PDZ superfamily / Peptidase S1, PA clan
Similarity search - Domain/homology
Serine protease HTRA1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsGerhardy, S. / Green, E. / Estevez, A. / Arthur, C.P. / Ultsch, M. / Rohou, A. / Kirchhofer, D.
Funding support United States, 1items
OrganizationGrant numberCountry
Not funded United States
CitationJournal: Nat Commun / Year: 2022
Title: Allosteric inhibition of HTRA1 activity by a conformational lock mechanism to treat age-related macular degeneration.
Authors: Stefan Gerhardy / Mark Ultsch / Wanjian Tang / Evan Green / Jeffrey K Holden / Wei Li / Alberto Estevez / Chris Arthur / Irene Tom / Alexis Rohou / Daniel Kirchhofer /
Abstract: The trimeric serine protease HTRA1 is a genetic risk factor associated with geographic atrophy (GA), a currently untreatable form of age-related macular degeneration. Here, we describe the allosteric ...The trimeric serine protease HTRA1 is a genetic risk factor associated with geographic atrophy (GA), a currently untreatable form of age-related macular degeneration. Here, we describe the allosteric inhibition mechanism of HTRA1 by a clinical Fab fragment, currently being evaluated for GA treatment. Using cryo-EM, X-ray crystallography and biochemical assays we identify the exposed LoopA of HTRA1 as the sole Fab epitope, which is approximately 30 Å away from the active site. The cryo-EM structure of the HTRA1:Fab complex in combination with molecular dynamics simulations revealed that Fab binding to LoopA locks HTRA1 in a non-competent conformational state, incapable of supporting catalysis. Moreover, grafting the HTRA1-LoopA epitope onto HTRA2 and HTRA3 transferred the allosteric inhibition mechanism. This suggests a conserved conformational lock mechanism across the HTRA family and a critical role of LoopA for catalysis, which was supported by the reduced activity of HTRA1-3 upon LoopA deletion or perturbation. This study reveals the long-range inhibition mechanism of the clinical Fab and identifies an essential function of the exposed LoopA for activity of HTRA family proteases.
History
DepositionOct 18, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 7, 2022Provider: repository / Type: Initial release
Revision 1.1Sep 21, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Nov 13, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Serine protease HTRA1
B: Serine protease HTRA1
C: Serine protease HTRA1
E: Fab15H6.v4 Heavy Chain
F: Fab15H6.v4 Light Chain
H: Fab15H6.v4 Heavy Chain
I: Fab15H6.v4 Heavy Chain
J: Fab15H6.v4 Light Chain
L: Fab15H6.v4 Light Chain


Theoretical massNumber of molelcules
Total (without water)234,6039
Polymers234,6039
Non-polymers00
Water00
1


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

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Components

#1: Protein Serine protease HTRA1 / High-temperature requirement A serine peptidase 1 / L56 / Serine protease 11


Mass: 25714.385 Da / Num. of mol.: 3 / Mutation: S328A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HTRA1, HTRA, PRSS11 / Variant: S328A / Production host: Escherichia coli (E. coli)
References: UniProt: Q92743, Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases
#2: Antibody Fab15H6.v4 Heavy Chain


Mass: 26768.996 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#3: Antibody Fab15H6.v4 Light Chain


Mass: 25717.730 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
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: HtrA1PD/SA bound by Fab15H6.v4 at LoopA epitope / Type: COMPLEX / Details: clinical Fab fragment Fab15H6.v4 / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.22 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.2
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMSodium ChlorideNaCl1
250 mMTrisTris1
30.25 PercentCHAPSCHAPS1
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: HtrA1PD/SA:Fab15H6.v4 complex
Specimen supportDetails: SAM: Grids were incubated in 4 mM monothiolalkane(C11)PEG6-OH (11-mercaptoundecyl) hexaethylenglycol (SPT-0011P6, SensoPath Technologies, Inc., Bozeman, MT) for 24h and rinsed in EtOH before sample application
Grid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 3.5s blot time

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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 magnification: 105000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3 sec. / Electron dose: 65 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7336

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Processing

SoftwareName: PHENIX / Version: 1.19.1-4122_final: / Classification: refinement
EM software
IDNameCategory
2SerialEMimage acquisition
13cisTEM3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 505669
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 219676 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 3TJN

3tjn


Pdb chain-ID: A / Accession code: 3TJN / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00314574
ELECTRON MICROSCOPYf_angle_d0.6119809
ELECTRON MICROSCOPYf_dihedral_angle_d4.5641989
ELECTRON MICROSCOPYf_chiral_restr0.0452286
ELECTRON MICROSCOPYf_plane_restr0.0052529

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