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- PDB-7sjm: anti-HtrA1 Fab15H6.v4 -

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Basic information

Entry
Database: PDB / ID: 7sjm
Titleanti-HtrA1 Fab15H6.v4
Components
  • Heavy Chain
  • Light Chain
KeywordsIMMUNE SYSTEM / Fab
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.8 Å
AuthorsUltsch, M.H. / Gerhardy, S.
Funding support1items
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, 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 1.2Oct 18, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
H: Heavy Chain
L: Light Chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)47,9838
Polymers47,4152
Non-polymers5686
Water8,449469
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4970 Å2
ΔGint-75 kcal/mol
Surface area19160 Å2
MethodPISA
Unit cell
Length a, b, c (Å)154.118, 60.338, 53.444
Angle α, β, γ (deg.)90.000, 97.320, 90.000
Int Tables number5
Space group name H-MC121

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Components

#1: Antibody Heavy Chain


Mass: 24232.924 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli) / Strain (production host): 64B4
#2: Antibody Light Chain


Mass: 23181.654 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli) / Strain (production host): 64B4
#3: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: SO4
#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 469 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.6 Å3/Da / Density % sol: 52.68 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 5.1
Details: 2.1M ammonium sulfate, 0.1M Tri-sodium citrate pH 5.1
PH range: 5.0-6.0

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Data collection

DiffractionMean temperature: 93 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL12-2 / Wavelength: 0.98 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jan 10, 2018 / Details: Liquid nitrogen-cooled double crystal Si(111)
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.98 Å / Relative weight: 1
ReflectionResolution: 1.8→39.47 Å / Num. obs: 44014 / % possible obs: 97.27 % / Redundancy: 2 % / CC1/2: 0.998 / Rmerge(I) obs: 0.047 / Rpim(I) all: 0.07 / Rrim(I) all: 0.101 / Net I/σ(I): 11.2
Reflection shellResolution: 1.8→1.83 Å / Redundancy: 1.9 % / Rmerge(I) obs: 0.4061 / Mean I/σ(I) obs: 2.09 / Num. unique obs: 4284 / CC1/2: 0.674 / Rpim(I) all: 0.639 / Rrim(I) all: 0.919 / % possible all: 95.37

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Processing

Software
NameVersionClassification
BUSTER2.11.5refinement
PDB_EXTRACT3.27data extraction
XDSdata reduction
autoPROCdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1FVD

1fvd


Resolution: 1.8→39.47 Å / Cor.coef. Fo:Fc: 0.956 / Cor.coef. Fo:Fc free: 0.9425 / SU R Cruickshank DPI: 0.114 / Cross valid method: THROUGHOUT / σ(F): 0 / SU R Blow DPI: 0.12 / SU Rfree Blow DPI: 0.111 / SU Rfree Cruickshank DPI: 0.108
RfactorNum. reflection% reflectionSelection details
Rfree0.202 2220 5.04 %RANDOM
Rwork0.1714 ---
obs0.1729 44014 97.26 %-
Displacement parametersBiso max: 113.44 Å2 / Biso mean: 27.38 Å2 / Biso min: 10.15 Å2
Baniso -1Baniso -2Baniso -3
1-0.458 Å20 Å20.2342 Å2
2---0.4298 Å20 Å2
3----0.0282 Å2
Refine analyzeLuzzati coordinate error obs: 0.196 Å
Refinement stepCycle: final / Resolution: 1.8→39.47 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3279 0 48 469 3796
Biso mean--61.34 40.63 -
Num. residues----432
Refine LS restraints
Refine-IDTypeNumberRestraint functionWeightDev ideal
X-RAY DIFFRACTIONt_dihedral_angle_d1146SINUSOIDAL2
X-RAY DIFFRACTIONt_trig_c_planes72HARMONIC2
X-RAY DIFFRACTIONt_gen_planes507HARMONIC5
X-RAY DIFFRACTIONt_it3458HARMONIC20
X-RAY DIFFRACTIONt_nbd
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_chiral_improper_torsion458SEMIHARMONIC5
X-RAY DIFFRACTIONt_sum_occupancies
X-RAY DIFFRACTIONt_utility_distance
X-RAY DIFFRACTIONt_utility_angle
X-RAY DIFFRACTIONt_utility_torsion
X-RAY DIFFRACTIONt_ideal_dist_contact4337SEMIHARMONIC4
X-RAY DIFFRACTIONt_bond_d3458HARMONIC20.01
X-RAY DIFFRACTIONt_angle_deg4731HARMONIC21.08
X-RAY DIFFRACTIONt_omega_torsion4.07
X-RAY DIFFRACTIONt_other_torsion15.92
LS refinement shellResolution: 1.8→1.85 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.2668 154 4.9 %
Rwork0.2131 2990 -
all0.2158 3144 -
obs--97.26 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.6022-0.16830.47430.4104-0.1880.947-0.02560.09360.0231-0.0529-0.0356-0.08220.00370.00450.0612-0.001-0.01980.0266-0.0474-0.001-0.0374181.835427.42710.6233
21.2084-0.25230.01080.7408-0.10570.5428-0.0506-0.06880.02220.08040.0193-0.1108-0.04280.00010.0312-0.0158-0.00580.0056-0.0662-0.0025-0.0742178.787130.003128.4043
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1{H|1 - 217}H1 - 217
2X-RAY DIFFRACTION2{L|1 - 211}L1 - 211

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