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- EMDB-25162: HtrA1:Fab15H6.v4 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-25162
TitleHtrA1:Fab15H6.v4 complex
Map dataMain map with enforced C3 symmetry. Used for structure determination.
Sample
  • Complex: HtrA1PD bound by Fab15H6.v4 at LoopA epitope
    • Protein or peptide: Serine protease HTRA1
    • Protein or peptide: Fab15H6.v4 Heavy Chain
    • Protein or peptide: Fab15H6.v4 Light Chain
KeywordsHtrA1 / 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 / Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases / negative regulation of BMP signaling pathway / 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 / Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases / negative regulation of BMP signaling pathway / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsGerhardy S / Green E
Funding support1 items
OrganizationGrant numberCountry
Not funded
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, 2021-
Header (metadata) releaseSep 7, 2022-
Map releaseSep 7, 2022-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25162.png

Downloads & links

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Map

FileDownload / File: emd_25162.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain map with enforced C3 symmetry. Used for structure determination.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.2 Å/pix.
x 288 pix.
= 345.6 Å		1.2 Å/pix.
x 288 pix.
= 345.6 Å		1.2 Å/pix.
x 288 pix.
= 345.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.2 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.64
Minimum - Maximum-4.0580316 - 6.4571915
Average (Standard dev.)0.0053271637 (±0.07798276)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 345.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Additional map without enforced symmetry (C1).

Fileemd_25162_additional_1.map
AnnotationAdditional map without enforced symmetry (C1).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Half-map 1/2 for C1 additional map

Fileemd_25162_additional_2.map
AnnotationHalf-map 1/2 for C1 additional map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: Half-map 2/2 for C1 additional map

Fileemd_25162_additional_3.map
AnnotationHalf-map 2/2 for C1 additional map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 1/2 for C3 main map

Fileemd_25162_half_map_1.map
AnnotationHalf-map 1/2 for C3 main map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 2/2 for C3 main map

Fileemd_25162_half_map_2.map
AnnotationHalf-map 2/2 for C3 main map
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 : HtrA1PD bound by Fab15H6.v4 at LoopA epitope

EntireName: HtrA1PD bound by Fab15H6.v4 at LoopA epitope
Components
  • Complex: HtrA1PD bound by Fab15H6.v4 at LoopA epitope
    • Protein or peptide: Serine protease HTRA1
    • Protein or peptide: Fab15H6.v4 Heavy Chain
    • Protein or peptide: Fab15H6.v4 Light Chain

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Supramolecule #1: HtrA1PD bound by Fab15H6.v4 at LoopA epitope

SupramoleculeName: HtrA1PD bound by Fab15H6.v4 at LoopA epitope / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: HtrA1PD bound by clinical Fab15H6.v4 at LoopA epitope
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 220 KDa

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Macromolecule #1: Serine protease HTRA1

MacromoleculeName: Serine protease HTRA1 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
EC number: Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 22.434699 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: DPNSLRHKYN FIADVVEKIA PAVVHIELFR KLPFSKREVP VASGSGFIVS EDGLIVTNAH VVTNKHRVKV ELKNGATYEA KIKDVDEKA DIALIKIDHQ GKLPVLLLGR SSELRPGEFV VAIGSPFSLQ NTVTTGIVST TQRGGKELGL RNSDMDYIQT D AIINYGNS ...String:
DPNSLRHKYN FIADVVEKIA PAVVHIELFR KLPFSKREVP VASGSGFIVS EDGLIVTNAH VVTNKHRVKV ELKNGATYEA KIKDVDEKA DIALIKIDHQ GKLPVLLLGR SSELRPGEFV VAIGSPFSLQ NTVTTGIVST TQRGGKELGL RNSDMDYIQT D AIINYGNS GGPLVNLDGE VIGINTLKVT AGISFAIPSD KIKKFLTES

UniProtKB: Serine protease HTRA1

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Macromolecule #2: Fab15H6.v4 Heavy Chain

MacromoleculeName: Fab15H6.v4 Heavy Chain / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 12.906205 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
EVQLVQSGAE VKKPGASVKV SCKASGYKFT DSEMHWVRQA PGQGLEWIGG VDPETEGAAY NQKFKGRATI TRDTSTSTAY LELSSLRSE DTAVYYCTRG YDYDYALDYW GQGTLVTV

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Macromolecule #3: Fab15H6.v4 Light Chain

MacromoleculeName: Fab15H6.v4 Light Chain / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.421634 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
DIQMTQSPSS LSASVGDRVT ITCRASSSVE FIHWYQQKPG KAPKPLISAT SNLASGVPSR FSGSGSGTDF TLTISSLQPE DFATYYCQQ WSSAPWTFGQ GTKVEIK

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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

Concentration4 mg/mL
BufferpH: 7.2
Component:
ConcentrationFormulaName
200.0 mMNaClSodium Chloride
50.0 mMTristris(hydroxymethyl)aminomethane
0.25 PercentCHAPS3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate
GridModel: Quantifoil R0.6/1 / Material: GOLD / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.067 kPa
Details: 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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: LEICA EM GP / Details: 3.5s blot time.
DetailsHtrA1PD:Fab15H6.v4 complex

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Number grids imaged: 1 / Number real images: 5345 / Average exposure time: 3.0 sec. / Average electron dose: 64.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
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: 566088
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3tjn


Details: apo HtrA1
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1) / Number images used: 184782
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final 3D classificationSoftware - Name: cryoSPARC (ver. 3.1)

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Atomic model buiding 1

Initial modelPDB ID:

3tjn


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: RIGID BODY FIT
Output model

PDB-7sjn:
HtrA1:Fab15H6.v4 complex

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