+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-25163 | |||||||||
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Title | HtrA1S328A:Fab15H6.v4 complex | |||||||||
Map data | Main Map without enforced symmetry (C1). Used for structure determination | |||||||||
Sample |
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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 | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.3 Å | |||||||||
Authors | Gerhardy S / Green E / Estevez A / Arthur CP / Ultsch M / Rohou A / Kirchhofer D | |||||||||
Funding support | United States, 1 items
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Citation | Journal: 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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_25163.map.gz | 7.4 MB | EMDB map data format | |
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Header (meta data) | emd-25163-v30.xml emd-25163.xml | 26.5 KB 26.5 KB | Display Display | EMDB header |
Images | emd_25163.png | 59.6 KB | ||
Others | emd_25163_additional_1.map.gz emd_25163_additional_2.map.gz emd_25163_additional_3.map.gz emd_25163_half_map_1.map.gz emd_25163_half_map_2.map.gz | 9.2 MB 7.4 MB 9.2 MB 9.2 MB 9.2 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-25163 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-25163 | HTTPS FTP |
-Validation report
Summary document | emd_25163_validation.pdf.gz | 644.8 KB | Display | EMDB validaton report |
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Full document | emd_25163_full_validation.pdf.gz | 644.4 KB | Display | |
Data in XML | emd_25163_validation.xml.gz | 13.1 KB | Display | |
Data in CIF | emd_25163_validation.cif.gz | 14.7 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-25163 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-25163 | HTTPS FTP |
-Related structure data
Related structure data | 7sjoMC 7sjmC 7sjnC 7sjpC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_25163.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | Main Map without enforced symmetry (C1). Used for structure determination | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.2 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: Half-maps 2/2 for additional map (C3)
File | emd_25163_additional_1.map | ||||||||||||
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Annotation | Half-maps 2/2 for additional map (C3) | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: Additional Map with enforced symmetry (C3).
File | emd_25163_additional_2.map | ||||||||||||
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Annotation | Additional Map with enforced symmetry (C3). | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: Half-maps 1/2 for additional map (C3)
File | emd_25163_additional_3.map | ||||||||||||
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Annotation | Half-maps 1/2 for additional map (C3) | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half-maps 2/2 for main map (C1)
File | emd_25163_half_map_1.map | ||||||||||||
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Annotation | Half-maps 2/2 for main map (C1) | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half-maps 1/2 for main map (C1)
File | emd_25163_half_map_2.map | ||||||||||||
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Annotation | Half-maps 1/2 for main map (C1) | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : HtrA1PD/SA bound by Fab15H6.v4 at LoopA epitope
Entire | Name: HtrA1PD/SA bound by Fab15H6.v4 at LoopA epitope |
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Components |
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-Supramolecule #1: HtrA1PD/SA bound by Fab15H6.v4 at LoopA epitope
Supramolecule | Name: HtrA1PD/SA bound by Fab15H6.v4 at LoopA epitope / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all / Details: clinical Fab fragment Fab15H6.v4 |
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Source (natural) | Organism: Homo sapiens (human) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 220 KDa |
-Macromolecule #1: Serine protease HTRA1
Macromolecule | Name: 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 |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 25.714385 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MHHHHHHGEN LYFQGSDPNS LRHKYNFIAD VVEKIAPAVV HIELFRKLPF SKREVPVASG SGFIVSEDGL IVTNAHVVTN KHRVKVELK NGATYEAKIK DVDEKADIAL IKIDHQGKLP VLLLGRSSEL RPGEFVVAIG SPFSLQNTVT TGIVSTTQRG G KELGLRNS ...String: MHHHHHHGEN LYFQGSDPNS LRHKYNFIAD VVEKIAPAVV HIELFRKLPF SKREVPVASG SGFIVSEDGL IVTNAHVVTN KHRVKVELK NGATYEAKIK DVDEKADIAL IKIDHQGKLP VLLLGRSSEL RPGEFVVAIG SPFSLQNTVT TGIVSTTQRG G KELGLRNS DMDYIQTDAI INYGNAGGPL VNLDGEVIGI NTLKVTAGIS FAIPSDKIKK FLTESHDRQA KGKAITK |
-Macromolecule #2: Fab15H6.v4 Heavy Chain
Macromolecule | Name: Fab15H6.v4 Heavy Chain / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 26.768996 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MKKNIAFLLA SMFVFSIATN AYAEVQLVQS GAEVKKPGAS VKVSCKASGY KFTDSEMHWV RQAPGQGLEW IGGVDPETEG AAYNQKFKG RATITRDTST STAYLELSSL RSEDTAVYYC TRGYDYDYAL DYWGQGTLVT VSSASTKGPS VFPLAPSSKS T SGGTAALG ...String: MKKNIAFLLA SMFVFSIATN AYAEVQLVQS GAEVKKPGAS VKVSCKASGY KFTDSEMHWV RQAPGQGLEW IGGVDPETEG AAYNQKFKG RATITRDTST STAYLELSSL RSEDTAVYYC TRGYDYDYAL DYWGQGTLVT VSSASTKGPS VFPLAPSSKS T SGGTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GTQTYICNVN HKPSNTKVDK KV EPKSCDK THT |
-Macromolecule #3: Fab15H6.v4 Light Chain
Macromolecule | Name: Fab15H6.v4 Light Chain / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 25.71773 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MKKNIAFLLA SMFVFSIATN AYADIQMTQS PSSLSASVGD RVTITCRASS SVEFIHWYQQ KPGKAPKPLI SATSNLASGV PSRFSGSGS GTDFTLTISS LQPEDFATYY CQQWSSAPWT FGQGTKVEIK RTVAAPSVFI FPPSDEQLKS GTASVVCLLN N FYPREAKV ...String: MKKNIAFLLA SMFVFSIATN AYADIQMTQS PSSLSASVGD RVTITCRASS SVEFIHWYQQ KPGKAPKPLI SATSNLASGV PSRFSGSGS GTDFTLTISS LQPEDFATYY CQQWSSAPWT FGQGTKVEIK RTVAAPSVFI FPPSDEQLKS GTASVVCLLN N FYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 4 mg/mL | ||||||||||||
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Buffer | pH: 7.2 Component:
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Grid | Model: Quantifoil R0.6/1 / Material: GOLD / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / 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 | ||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: LEICA EM GP / Details: 3.5s blot time. | ||||||||||||
Details | HtrA1PD/SA:Fab15H6.v4 complex |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 7336 / Average exposure time: 3.0 sec. / Average electron dose: 65.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 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 stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |