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- PDB-7mde: Full-length S95A ClbP -

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Basic information

Entry
Database: PDB / ID: 7mde
TitleFull-length S95A ClbP
ComponentsBeta-lactamase
KeywordsHYDROLASE / colibactin peptidase / S12 peptidase
Function / homology
Function and homology information


antibiotic catabolic process / beta-lactamase activity / beta-lactamase / outer membrane-bounded periplasmic space / response to antibiotic / membrane
Similarity search - Function
: / Beta-lactamase, class-C active site / Beta-lactamase class-C active site. / Beta-lactamase-related / Beta-lactamase / Beta-lactamase/transpeptidase-like
Similarity search - Domain/homology
IMIDAZOLE / (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate / Beta-lactamase
Similarity search - Component
Biological speciesEscherichia coli CFT073 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.7 Å
AuthorsVelilla, J.A. / Volpe, M.R. / Gaudet, R.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM120996 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01CA208834 United States
CitationJournal: Nat Chem Biol / Year: 2023
Title: Structural basis of colibactin activation by the ClbP peptidase.
Authors: José A Velilla / Matthew R Volpe / Grace E Kenney / Richard M Walsh / Emily P Balskus / Rachelle Gaudet /
Abstract: Colibactin, a DNA cross-linking agent produced by gut bacteria, is implicated in colorectal cancer. Its biosynthesis uses a prodrug resistance mechanism: a non-toxic precursor assembled in the ...Colibactin, a DNA cross-linking agent produced by gut bacteria, is implicated in colorectal cancer. Its biosynthesis uses a prodrug resistance mechanism: a non-toxic precursor assembled in the cytoplasm is activated after export to the periplasm. This activation is mediated by ClbP, an inner-membrane peptidase with an N-terminal periplasmic catalytic domain and a C-terminal three-helix transmembrane domain. Although the transmembrane domain is required for colibactin activation, its role in catalysis is unclear. Our structure of full-length ClbP bound to a product analog reveals an interdomain interface important for substrate binding and enzyme stability and interactions that explain the selectivity of ClbP for the N-acyl-D-asparagine prodrug motif. Based on structural and biochemical evidence, we propose that ClbP dimerizes to form an extended substrate-binding site that can accommodate a pseudodimeric precolibactin with its two terminal prodrug motifs in the two ClbP active sites, thus enabling the coordinated activation of both electrophilic warheads.
History
DepositionApr 4, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 28, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 9, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
Revision 1.2Feb 8, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year
Revision 1.3Oct 25, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 1.4Nov 15, 2023Group: Data collection / Category: chem_comp_atom / chem_comp_bond / Item: _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_2

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Beta-lactamase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)56,22713
Polymers53,5071
Non-polymers2,72112
Water3,207178
1
A: Beta-lactamase
hetero molecules

A: Beta-lactamase
hetero molecules


  • defined by author
  • Evidence: light scattering, SEC-MALS performed on protein solubilized in DDM. Conjugate analysis suggests the average protein molecular weight of the particles in the ClbP peak is approx. 110kDa, ...Evidence: light scattering, SEC-MALS performed on protein solubilized in DDM. Conjugate analysis suggests the average protein molecular weight of the particles in the ClbP peak is approx. 110kDa, which matches the molecular weight of the dimer.
  • 112 kDa, 2 polymers
  • Search similar-shape structures of this assembly by Omokage search (details)
Theoretical massNumber of molelcules
Total (without water)112,45526
Polymers107,0132
Non-polymers5,44124
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_565-x,-y+1,z1
Unit cell
Length a, b, c (Å)97.475, 97.475, 183.973
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number94
Space group name H-MP42212
Space group name HallP4n2n
Components on special symmetry positions
IDModelComponents
11A-758-

HOH

21A-813-

HOH

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Beta-lactamase


Mass: 53506.645 Da / Num. of mol.: 1 / Mutation: S95A, L454M, I478M
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli CFT073 (bacteria) / Strain: CFT073
Gene: clbP, D3C88_24740, E3O05_09835, E4T84_20050, ELT23_23600, ELT33_24260, ELT38_03835, ELY31_20780, EPS76_05775, EQO00_10370, EWK56_23765, FPI65_12330, HMV41_21265, HMW38_10385, IFB95_001925
Plasmid: pET29b / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): C41 / References: UniProt: Q0P7K6, beta-lactamase

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Non-polymers , 7 types, 190 molecules

#2: Chemical ChemComp-OLC / (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate / 1-Oleoyl-R-glycerol


Mass: 356.540 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C21H40O4
#3: Chemical
ChemComp-2PE / NONAETHYLENE GLYCOL


Mass: 414.488 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C18H38O10 / Comment: precipitant*YM
#4: Chemical ChemComp-IMD / IMIDAZOLE


Mass: 69.085 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H5N2
#5: Chemical ChemComp-DMS / DIMETHYL SULFOXIDE


Mass: 78.133 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6OS / Comment: DMSO, precipitant*YM
#6: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#7: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Cl
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 178 / Source method: isolated from a natural source / Formula: H2O

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Details

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: 3.88 Å3/Da / Density % sol: 68.27 % / Description: Square plates grown in the sponge phase
Crystal growTemperature: 295 K / Method: lipidic cubic phase
Details: Precipitant composition: 1 part 0.1M imidazole pH 7.8, 10% (v/v) PEG 400, 150 mM Li2SO4, 5.5 mM (4-(4-bromophenyl)butanoyl)-D-asparagine plus 3.5 parts 0.1M tris pH 7.4, 28%(v/v) PEG400, 100 ...Details: Precipitant composition: 1 part 0.1M imidazole pH 7.8, 10% (v/v) PEG 400, 150 mM Li2SO4, 5.5 mM (4-(4-bromophenyl)butanoyl)-D-asparagine plus 3.5 parts 0.1M tris pH 7.4, 28%(v/v) PEG400, 100 mM Li2SO4, 4% (v/v) PPG
PH range: 7.4-7.8 / Temp details: room temperature

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Data collection

DiffractionMean temperature: 100 K / Ambient temp details: Cryojet cryocooler / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.9792 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Jun 14, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9792 Å / Relative weight: 1
ReflectionResolution: 2.7→48.74 Å / Num. obs: 25099 / % possible obs: 99.79 % / Redundancy: 6.5 % / Biso Wilson estimate: 48.7 Å2 / CC1/2: 0.994 / CC star: 0.998 / Rmerge(I) obs: 0.211 / Rpim(I) all: 0.0886 / Rrim(I) all: 0.2294 / Net I/σ(I): 7.74
Reflection shellResolution: 2.7→2.797 Å / Redundancy: 6.8 % / Rmerge(I) obs: 1.338 / Mean I/σ(I) obs: 1.22 / Num. unique obs: 2449 / CC1/2: 0.67 / CC star: 0.896 / Rpim(I) all: 0.5474 / Rrim(I) all: 1.447 / % possible all: 99.96

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
Aimless0.7.1data scaling
PDB_EXTRACT3.27data extraction
XDS20190806data reduction
PHENIX1.19.1-4122-000phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3O3V

3o3v


Resolution: 2.7→48.74 Å / Cross valid method: FREE R-VALUE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.235 751 3 %
Rwork0.1948 45001 -
obs-25074 99.79 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 67.17 Å2
Refinement stepCycle: LAST / Resolution: 2.7→48.74 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3341 0 86 178 3605
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00713489
X-RAY DIFFRACTIONf_angle_d0.9094730
X-RAY DIFFRACTIONf_chiral_restr0.0506542
X-RAY DIFFRACTIONf_plane_restr0.0076603
X-RAY DIFFRACTIONf_dihedral_angle_d15.15541233
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.7-2.80.39131410.31874494X-RAY DIFFRACTION99.89
2.8-2.910.34821330.28884522X-RAY DIFFRACTION99.94
2.91-3.040.29461410.26664485X-RAY DIFFRACTION99.94
3.04-3.20.28681410.23754509X-RAY DIFFRACTION99.68
3.2-3.40.2541420.21644458X-RAY DIFFRACTION99.63
3.4-3.660.20951440.17794519X-RAY DIFFRACTION99.91
3.66-4.030.22291420.15984525X-RAY DIFFRACTION99.96
4.03-4.620.18761420.1334485X-RAY DIFFRACTION99.61
4.62-5.810.18081290.1564506X-RAY DIFFRACTION99.85
5.81-48.740.2091360.16444498X-RAY DIFFRACTION99.46
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)
15.01598609803-0.1405263775440.4387823822972.7156986172-0.6888114520354.750377001440.220080573299-0.0511126112780.4772125817710.0328093854419-0.197117902649-0.586290988757-0.135637251160.32088629281-0.06824216986570.2004075191230.03329064869210.07821199004160.3639318808670.004599569485070.75323488381824.417333639635.779828388448.948647483
23.728748384980.1210154898370.01939535063470.7742629891180.007450819695250.877674840556-0.0683410905132-0.14087629842-0.3328317206150.1139826509990.03030888453120.1998584941010.147384202962-0.1062869900450.05250361285840.29852396371-0.001521016823490.02619262327310.2873378876870.01106010209230.82159718051-3.1528357249223.180199443649.6445037763
32.70663616512-0.09799544596480.392089787281.446135292660.1673223304080.923455967486-0.0336068394940.152441048394-0.0931666912325-0.1144196996990.0202262983607-0.04722657246110.0520937545186-0.02360315861450.05432749741440.195936755192-0.03039800691780.03092655941990.3217150041170.00392829324720.6544377325375.0995968225732.26037227243.238546427
42.358768595110.301437763897-1.258384277182.71642091508-3.53333354275.144393722970.194255942385-0.176075521626-0.1023715690080.323048710655-0.01221359813740.0646552524074-0.3030456381770.1309358579220.06158209558730.2304413474390.01424586860920.007240617666050.338203035112-0.05824376630850.78040500299115.191320191342.174307053348.9148248117
53.55322682144-1.05459203751-2.285835997232.243799606560.2875865304087.086326862620.356912200113-1.28863894732-0.05721739032441.20858570436-0.179430418366-0.52896774928-0.9943675465990.445368373568-0.1458932822340.961665137374-0.217387909191-0.1312048268381.06610890228-0.01897004177830.60710150595211.534658396641.381717446583.1332846515
62.23181380233-1.989888872131.053141565135.89559380211-4.903547735234.33483542212-0.471605301891-0.517832388263-0.0546942732113-0.1762832743571.2491385959-0.888303620781-0.128839300999-2.25343033395-0.8200148115310.772637257-0.1896959008430.1487159897511.28515496544-0.09421605656960.894349040197-2.4563176714929.549817127889.6622753053
74.762947370974.18556526605-6.441695516397.42592803959-4.138588536559.761378592570.435184479408-0.681932561393-0.2761566213570.559715715759-0.7136297140951.82105040228-0.345764616551-1.256338322680.2956175141110.5065980388180.03536494378320.1195522926450.909870045866-0.02164312035321.09841796069-2.0885242569330.665328739470.9253688421
82.015040677490.3182069890770.4442591149162.602076069390.4139161886622.708460104520.0841078600855-0.9546498121010.09408823335421.255518941160.2104577160580.2760547175451.25319161956-2.94914278151-0.245527528570.842209899983-0.0424000988173-0.1880936824961.399650708350.03633872519360.5060575834165.9704605509833.824712617687.2788193694
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION / Auth asym-ID: A / Label asym-ID: A

IDRefine TLS-IDSelection detailsAuth seq-IDLabel seq-ID
11chain 'A' and (resid 35 through 85 )35 - 851 - 51
22chain 'A' and (resid 86 through 245 )86 - 24552 - 211
33chain 'A' and (resid 246 through 344 )246 - 344212 - 310
44chain 'A' and (resid 345 through 381 )345 - 381311 - 347
55chain 'A' and (resid 382 through 408 )382 - 408348 - 374
66chain 'A' and (resid 433 through 448 )433 - 448384 - 399
77chain 'A' and (resid 449 through 468 )449 - 468400 - 419
88chain 'A' and (resid 469 through 489 )469 - 489420 - 440

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