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- PDB-7uac: Human pro-meprin alpha (zymogen state) -

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Basic information

Entry
Database: PDB / ID: 7uac
TitleHuman pro-meprin alpha (zymogen state)
ComponentsMeprin A subunit alpha
KeywordsONCOPROTEIN / Metalloprotease / complex / helical / extracellular
Function / homology
Function and homology information


meprin A / meprin A complex / epidermal growth factor receptor ligand maturation / metallodipeptidase activity / signaling receptor ligand precursor processing / metalloendopeptidase activity / metallopeptidase activity / extracellular space / extracellular exosome / zinc ion binding / plasma membrane
Similarity search - Function
Meprin alpha/beta subunit / Meprin peptidase domain / Astacin-like domain profile. / Peptidase M12A / Astacin (Peptidase family M12A) / MAM domain signature. / Domain in meprin, A5, receptor protein tyrosine phosphatase mu (and others) / MATH domain / MAM domain, meprin/A5/mu / MAM domain ...Meprin alpha/beta subunit / Meprin peptidase domain / Astacin-like domain profile. / Peptidase M12A / Astacin (Peptidase family M12A) / MAM domain signature. / Domain in meprin, A5, receptor protein tyrosine phosphatase mu (and others) / MATH domain / MAM domain, meprin/A5/mu / MAM domain / MAM domain profile. / MATH/TRAF domain / MATH/TRAF domain profile. / meprin and TRAF homology / TRAF-like / Peptidase, metallopeptidase / Zinc-dependent metalloprotease / EGF-like domain / Metallopeptidase, catalytic domain superfamily / EGF-like domain profile. / EGF-like domain / Neutral zinc metallopeptidases, zinc-binding region signature. / Concanavalin A-like lectin/glucanase domain superfamily
Similarity search - Domain/homology
Meprin A subunit alpha
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsBayly-Jones, C. / Lupton, C.J. / Fritz, C. / Schlenzig, D. / Whisstock, J.C.
Funding support Germany, Australia, 2items
OrganizationGrant numberCountry
German Federal Ministry for Education and Research Germany
Australian Research Council (ARC) Australia
CitationJournal: Nat Commun / Year: 2022
Title: Helical ultrastructure of the metalloprotease meprin α in complex with a small molecule inhibitor.
Authors: Charles Bayly-Jones / Christopher J Lupton / Claudia Fritz / Hariprasad Venugopal / Daniel Ramsbeck / Michael Wermann / Christian Jäger / Alex de Marco / Stephan Schilling / Dagmar ...Authors: Charles Bayly-Jones / Christopher J Lupton / Claudia Fritz / Hariprasad Venugopal / Daniel Ramsbeck / Michael Wermann / Christian Jäger / Alex de Marco / Stephan Schilling / Dagmar Schlenzig / James C Whisstock /
Abstract: The zinc-dependent metalloprotease meprin α is predominantly expressed in the brush border membrane of proximal tubules in the kidney and enterocytes in the small intestine and colon. In normal ...The zinc-dependent metalloprotease meprin α is predominantly expressed in the brush border membrane of proximal tubules in the kidney and enterocytes in the small intestine and colon. In normal tissue homeostasis meprin α performs key roles in inflammation, immunity, and extracellular matrix remodelling. Dysregulated meprin α is associated with acute kidney injury, sepsis, urinary tract infection, metastatic colorectal carcinoma, and inflammatory bowel disease. Accordingly, meprin α is the target of drug discovery programs. In contrast to meprin β, meprin α is secreted into the extracellular space, whereupon it oligomerises to form giant assemblies and is the largest extracellular protease identified to date (~6 MDa). Here, using cryo-electron microscopy, we determine the high-resolution structure of the zymogen and mature form of meprin α, as well as the structure of the active form in complex with a prototype small molecule inhibitor and human fetuin-B. Our data reveal that meprin α forms a giant, flexible, left-handed helical assembly of roughly 22 nm in diameter. We find that oligomerisation improves proteolytic and thermal stability but does not impact substrate specificity or enzymatic activity. Furthermore, structural comparison with meprin β reveal unique features of the active site of meprin α, and helical assembly more broadly.
History
DepositionMar 12, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 2, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
H: Meprin A subunit alpha
hetero molecules


Theoretical massNumber of molelcules
Total (without water)69,4378
Polymers67,2861
Non-polymers2,1517
Water00
1


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

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Components

#1: Protein Meprin A subunit alpha / Endopeptidase-2 / N-benzoyl-L-tyrosyl-P-amino-benzoic acid hydrolase subunit alpha / PABA peptide ...Endopeptidase-2 / N-benzoyl-L-tyrosyl-P-amino-benzoic acid hydrolase subunit alpha / PABA peptide hydrolase / PPH alpha


Mass: 67285.820 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MEP1A / Cell line (production host): Schneider-2 / Production host: Drosophila melanogaster (fruit fly) / References: UniProt: Q16819, meprin A
#2: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#3: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 732.682 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4[LFucpa1-6]DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/3,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1221m-1a_1-5]/1-1-2-3/a4-b1_a6-d1_b4-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}[(6+1)][a-L-Fucp]{}}}LINUCSPDB-CARE
#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Single subunit of helical meprin alpha in the zymogen state
Type: COMPLEX
Details: Focused classification and local refinement reconstruction of single subunit of recombinant, secreted helical pro-meprin alpha
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 85 kDa/nm / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Drosophila (fruit flies) / Cell: Schneider-2 / Plasmid: pMT/BiP/V5
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
130 mMTRIS1
2100 mMSodium chlorideNaCl1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Polydisperse
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: 3 s blot, -3 force

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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: 130000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 44.5 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2648
Image scansMovie frames/image: 50 / Used frames/image: 1-50

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1crYOLO1.8.1particle selectionFilament mode
2EPUimage acquisition
4CTFFIND4.1.13CTF correction
5Warp1.0.7CTF correction
8ISOLDE1.1.0model fitting
9UCSF ChimeraX1.3model fitting
10Coot0.9.2model fitting
12ISOLDE1.1.0model refinement
13PHENIX1.17model refinement
14RELION2.1, 3.1initial Euler assignment
15cryoSPARC3.3.1final Euler assignmentLocal refinement (non-uniform)
16RELION3.1final Euler assignmentGlobal alignment + SIDESPLITTER
Image processingDetails: Compressed to LZW TIFF. Motion corrected by MotionCor.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 905660
Details: Subparticles extracted after particle expansion about pseudo-helical symmetry
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 338429 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 4GWN

4gwn


Accession code: 4GWN / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0124644
ELECTRON MICROSCOPYf_angle_d1.7816292
ELECTRON MICROSCOPYf_dihedral_angle_d21.6331692
ELECTRON MICROSCOPYf_chiral_restr0.109701
ELECTRON MICROSCOPYf_plane_restr0.015805

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