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- PDB-7sjp: anti-HtrA1 Fab15H6.v4 bound to HtrA1-LoopA peptide -

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Basic information

Entry
Database: PDB / ID: 7sjp
Titleanti-HtrA1 Fab15H6.v4 bound to HtrA1-LoopA peptide
Components
  • Heavy Chain
  • HtrA1-LoopA peptide
  • Light Chain
KeywordsIMMUNE SYSTEM / Fab Htra 1 peptide complex
Function / homologyImmunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Function and homology information
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.1 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
Revision 1.3Nov 6, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
E: HtrA1-LoopA peptide
H: Heavy Chain
L: Light Chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)49,63212
Polymers48,7753
Non-polymers8579
Water7,332407
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6740 Å2
ΔGint-107 kcal/mol
Surface area19050 Å2
MethodPISA
Unit cell
Length a, b, c (Å)60.193, 90.212, 106.310
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

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Protein/peptide , 1 types, 1 molecules E

#1: Protein/peptide HtrA1-LoopA peptide


Mass: 1360.646 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Peptide loop from HtrA 1 / Source: (synth.) synthetic construct (others)

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Antibody , 2 types, 2 molecules HL

#2: 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
#3: 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

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Non-polymers , 3 types, 416 molecules

#4: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: SO4
#5: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


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


Mass: 18.015 Da / Num. of mol.: 407 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
Has protein modificationY

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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: 2.96 Å3/Da / Density % sol: 58.43 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 8 / Details: 12% PEG 4000, 10% Ethanol, 5% glycerol / PH range: 7.0-8.5

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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
RadiationMonochromator: Si filter / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.98 Å / Relative weight: 1
ReflectionResolution: 2.1→36.45 Å / Num. obs: 34460 / % possible obs: 99.67 % / Redundancy: 6.4 % / CC1/2: 0.998 / Rmerge(I) obs: 0.107 / Rpim(I) all: 0.062 / Rrim(I) all: 0.117 / Net I/σ(I): 12.7
Reflection shellResolution: 2.1→2.16 Å / Redundancy: 4.4 % / Rmerge(I) obs: 0.814 / Mean I/σ(I) obs: 1.8 / Num. unique obs: 2691 / CC1/2: 0.727 / Rpim(I) all: 0.547 / % possible all: 96.4

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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: 7SJM

7sjm


Resolution: 2.1→36.45 Å / Cor.coef. Fo:Fc: 0.9513 / Cor.coef. Fo:Fc free: 0.9417 / SU R Cruickshank DPI: 0.159 / Cross valid method: THROUGHOUT / σ(F): 0 / SU R Blow DPI: 0.174 / SU Rfree Blow DPI: 0.145 / SU Rfree Cruickshank DPI: 0.14
RfactorNum. reflection% reflectionSelection details
Rfree0.1988 1733 5.04 %RANDOM
Rwork0.1682 ---
obs0.1698 34399 99.66 %-
Displacement parametersBiso max: 130.9 Å2 / Biso mean: 39.17 Å2 / Biso min: 17.32 Å2
Baniso -1Baniso -2Baniso -3
1-7.208 Å20 Å20 Å2
2---2.1279 Å20 Å2
3----5.0801 Å2
Refine analyzeLuzzati coordinate error obs: 0.241 Å
Refinement stepCycle: final / Resolution: 2.1→36.45 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3373 0 55 407 3835
Biso mean--87.18 49 -
Num. residues----444
Refine LS restraints
Refine-IDTypeNumberRestraint functionWeightDev ideal
X-RAY DIFFRACTIONt_dihedral_angle_d1167SINUSOIDAL2
X-RAY DIFFRACTIONt_trig_c_planes72HARMONIC2
X-RAY DIFFRACTIONt_gen_planes511HARMONIC5
X-RAY DIFFRACTIONt_it3535HARMONIC20
X-RAY DIFFRACTIONt_nbd
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_chiral_improper_torsion466SEMIHARMONIC5
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_contact4329SEMIHARMONIC4
X-RAY DIFFRACTIONt_bond_d3535HARMONIC20.01
X-RAY DIFFRACTIONt_angle_deg4822HARMONIC21.14
X-RAY DIFFRACTIONt_omega_torsion4.12
X-RAY DIFFRACTIONt_other_torsion16.32
LS refinement shellResolution: 2.1→2.17 Å / Rfactor Rfree error: 0 / Total num. of bins used: 17
RfactorNum. reflection% reflection
Rfree0.2389 144 5 %
Rwork0.2042 2734 -
all0.2058 2878 -
obs--99.66 %
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)
11.2226-1.02150.20833.3678-0.51842.5180.06030.2080.1089-0.1563-0.0574-0.1217-0.09560.1209-0.0029-0.0515-0.03040.0278-0.11360.0598-0.00512.4651-3.5274-23.9962
21.4672-0.57210.62881.0935-1.13852.0788-0.0617-0.03730.24140.04020.0403-0.1018-0.14250.00170.0214-0.0598-0.01940.0196-0.11690.02970.00924.7943-8.2301-14.8269
34.5778-1.61241.31260.9822-0.40781.0162-0.1304-0.23410.29470.12580.0755-0.1195-0.1753-0.05610.0548-0.0367-0.00060.0156-0.0199-0.0045-0.009711.7421-15.722.5514
40.05941.927-1.0770.78990.62890.3338-0.04110.0811-0.0426-0.21980.0520.36450.0295-0.1103-0.0110.058-0.0048-0.0448-0.0169-0.08150.0568-0.8735-30.4493-27.3621
52.43531.6583-1.5455.71620.2280.60950.08670.19870.0232-0.2936-0.1412-0.0752-0.10050.06010.0546-0.02240.05490.0111-0.0795-0.0047-0.06614.8758-20.3626-28.6927
62.7879-2.0270.47433.4794-1.04821.35820.16170.35370.0111-0.5442-0.1345-0.01430.08480.0925-0.02720.0270.0078-0.017-0.0754-0.0525-0.05145.4741-24.7447-29.9981
70.9402-0.0115-0.03010.5950.82131.2634-0.0099-0.073-0.23460.10250.0505-0.03670.1382-0.1271-0.04060.00040.0228-0.0269-0.1027-0.02320.0596.2885-36.0348-11.5749
82.7817-0.5450.09010.0036-0.52422.33770.03-0.44790.06570.1242-0.0998-0.0934-0.08350.01880.0698-0.01780.018-0.01590.1401-0.0199-0.07838.5218-20.042314.363
92.9377-0.59190.01741.49740.8833.04190.0279-0.3239-0.30550.13260.04920.14120.1185-0.0478-0.077-0.1243-0.00980.0061-0.09210.0702-0.07883.2492-31.08785.1741
101.9413-0.5075-0.73181.04510.87744.1496-0.0767-0.5442-0.26670.25790.09920.11540.28-0.0012-0.0224-0.02870.01360.01160.03840.0742-0.02363.3355-29.51879.8862
110.31391.42840.497300.12810.53380.01560.08390.0213-0.1414-0.0297-0.17060.01610.07580.01410.10860.04570.1111-0.00510.0912-0.07118.7631-5.2941-40.5987
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1{ H|1 - H|67 }H1 - 67
2X-RAY DIFFRACTION2{ H|68 - H|140 }H68 - 140
3X-RAY DIFFRACTION3{ H|141 - H|221 }H141 - 221
4X-RAY DIFFRACTION4{ L|1 - L|25 }L1 - 25
5X-RAY DIFFRACTION5{ L|26 - L|47 }L26 - 47
6X-RAY DIFFRACTION6{ L|48 - L|100 }L48 - 100
7X-RAY DIFFRACTION7{ L|101 - L|112 }L101 - 112
8X-RAY DIFFRACTION8{ L|113 - L|127 }L113 - 127
9X-RAY DIFFRACTION9{ L|128 - L|149 }L128 - 149
10X-RAY DIFFRACTION10{ L|150 - L|212 }L150 - 212
11X-RAY DIFFRACTION11{ E|190 - E|200 }E190 - 200

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