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- PDB-8fgx: Cryo-EM structure of the STAR-0215 Fab in complex with active hum... -

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Basic information

Entry
Database: PDB / ID: 8fgx
TitleCryo-EM structure of the STAR-0215 Fab in complex with active human plasma kallikrein
Components
  • Plasma kallikrein light chain
  • STAR-0215 Heavy chain
  • STAR-0215 Light chain
KeywordsHYDROLASE/IMMUNE SYSTEM / plasma kallikrein / Plasma prekallikrein / Fletcher factor / hereditary angioedema / HAE / Kininogenin / C1-INH / Bradykinin / HYDROLASE-IMMUNE SYSTEM complex
Function / homology
Function and homology information


plasma kallikrein / Factor XII activation / Defective SERPING1 causes hereditary angioedema / positive regulation of fibrinolysis / zymogen activation / plasminogen activation / Defective factor XII causes hereditary angioedema / Activation of Matrix Metalloproteinases / fibrinolysis / Intrinsic Pathway of Fibrin Clot Formation ...plasma kallikrein / Factor XII activation / Defective SERPING1 causes hereditary angioedema / positive regulation of fibrinolysis / zymogen activation / plasminogen activation / Defective factor XII causes hereditary angioedema / Activation of Matrix Metalloproteinases / fibrinolysis / Intrinsic Pathway of Fibrin Clot Formation / blood coagulation / serine-type endopeptidase activity / proteolysis / extracellular space / extracellular exosome / extracellular region / plasma membrane
Similarity search - Function
Apple domain. / Apple domain / APPLE domain / PAN/Apple domain profile. / PAN domain / PAN/Apple domain / Serine proteases, trypsin family, histidine active site / Serine proteases, trypsin family, serine active site / Peptidase S1A, chymotrypsin family / Serine proteases, trypsin family, histidine active site. ...Apple domain. / Apple domain / APPLE domain / PAN/Apple domain profile. / PAN domain / PAN/Apple domain / Serine proteases, trypsin family, histidine active site / Serine proteases, trypsin family, serine active site / Peptidase S1A, chymotrypsin family / Serine proteases, trypsin family, histidine active site. / Serine proteases, trypsin domain profile. / Serine proteases, trypsin family, serine active site. / Trypsin-like serine protease / Serine proteases, trypsin domain / Trypsin / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.62 Å
AuthorsFuller, J.R. / Biris, N. / Bista, P.
Funding support United States, 1items
OrganizationGrant numberCountry
Other private United States
CitationJournal: J Pharmacol Exp Ther / Year: 2023
Title: STAR-0215 is a Novel, Long-Acting Monoclonal Antibody Inhibitor of Plasma Kallikrein for the Potential Treatment of Hereditary Angioedema.
Authors: Vahe Bedian / Nikolaos Biris / Charles Omer / Jou-Ku Chung / James Fuller / Rafif Dagher / Sachin Chandran / Peter Harwin / Tomas Kiselak / Jonathan Violin / Andrew Nichols / Pradeep Bista /
Abstract: Hereditary angioedema (HAE) is a rare autosomal dominant disorder caused by a deficiency in functional C1 esterase inhibitor, a serpin family protein that blocks the activity of plasma kallikrein. ...Hereditary angioedema (HAE) is a rare autosomal dominant disorder caused by a deficiency in functional C1 esterase inhibitor, a serpin family protein that blocks the activity of plasma kallikrein. Insufficient inhibition of plasma kallikrein results in the overproduction of bradykinin, a vasoactive inflammatory mediator that produces both pain and unpredictable swelling during HAE attacks, with potentially life-threatening consequences. We describe the generation of STAR-0215, a humanized IgG1 antibody with a long circulating half-life (t) that potently inhibits plasma kallikrein activity, with a >1000-fold lower affinity for prekallikrein and no measurable inhibitory activity against other serine proteases. The high specificity and inhibitory effect of STAR-0215 is demonstrated through a unique allosteric mechanism involving N-terminal catalytic domain binding, destabilization of the activation domain, and reversion of the active site to the inactive zymogen state. The YTE (M252Y/S254T/T256E) modified fragment crystallizable (Fc) domain of STAR-0215 enhances pH-dependent neonatal Fc receptor binding, resulting in a prolonged t in vivo (∼34 days in cynomolgus monkeys) compared with antibodies without this modification. A single subcutaneous dose of STAR-0215 (≥100 mg) was predicted to be active in patients for 3 months or longer, based on simulations using a minimal physiologically based pharmacokinetic model. These data indicate that STAR-0215, a highly potent and specific antibody against plasma kallikrein with extended t, is a potential agent for long-term preventative HAE therapy administered every 3 months or less frequently. SIGNIFICANCE STATEMENT: STAR-0215 is a YTE-modified immunoglobulin G1 monoclonal antibody with a novel binding mechanism that specifically and potently inhibits the enzymatic activity of plasma kallikrein and prevents the generation of bradykinin. It has been designed to be a long-lasting prophylactic treatment to prevent attacks of HAE and to decrease the burden of disease and the burden of treatment for people with HAE.
History
DepositionDec 13, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 6, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 13, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Oct 25, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: STAR-0215 Light chain
B: STAR-0215 Heavy chain
C: Plasma kallikrein light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)117,2406
Polymers116,5773
Non-polymers6643
Water00
1


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

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Components

#1: Antibody STAR-0215 Light chain


Mass: 23540.182 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Cricetulus griseus (Chinese hamster)
#2: Antibody STAR-0215 Heavy chain


Mass: 23750.527 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Cricetulus griseus (Chinese hamster)
#3: Antibody Plasma kallikrein light chain


Mass: 69285.984 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human)
References: UniProt: P03952, Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases
#4: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestN

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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: Complex of STAR-0215 Fab with active plasma kallikrein
Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Cricetulus griseus (Chinese hamster)
Buffer solutionpH: 7
SpecimenConc.: 2.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 291 K
Details: Grids were prepared using a ThermoFisher Vitrobot MkIV with the chamber held at 18C and 100% relative humidity. 3.5 uL of the sample was applied to an UltrAuFoil 1.2/1.3 300 mesh grid that ...Details: Grids were prepared using a ThermoFisher Vitrobot MkIV with the chamber held at 18C and 100% relative humidity. 3.5 uL of the sample was applied to an UltrAuFoil 1.2/1.3 300 mesh grid that had been freshly glow discharged, blotted for 4 seconds, and plunge frozen in liquid ethane.

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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: 2100 nm / Nominal defocus min: 700 nm / Calibrated defocus min: 400 nm / Calibrated defocus max: 2700 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4.88 sec. / Electron dose: 70 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3094
Image scansWidth: 5760 / Height: 4092

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameVersionCategory
1Topaz0.2.5particle selection
2EPU3image acquisition
4CTFFIND4.1.14CTF correction
5RELION4CTF correction
8PHENIX1.20.1model fitting
9UCSF Chimera1.15model fitting
11RELION4initial Euler assignment
12RELION4final Euler assignment
13RELION4classification
14RELION43D reconstruction
15PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 718169
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.62 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 299175 / Algorithm: EXACT BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 74.25 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00255148
ELECTRON MICROSCOPYf_angle_d0.41767002
ELECTRON MICROSCOPYf_chiral_restr0.0437788
ELECTRON MICROSCOPYf_plane_restr0.0028883
ELECTRON MICROSCOPYf_dihedral_angle_d9.73511834

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