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- PDB-7mdf: Full-length S95A ClbP bound to N-acyl-D-asparagine analog -

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Basic information

Entry
Database: PDB / ID: 7mdf
TitleFull-length S95A ClbP bound to N-acyl-D-asparagine analog
ComponentsBeta-lactamase
KeywordsHYDROLASE/INHIBITOR / colibactin peptidase / S12 peptidase / HYDROLASE / HYDROLASE-INHIBITOR complex
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
(2S)-2,3-dihydroxypropyl (9Z)-hexadec-9-enoate / Lauryl Maltose Neopentyl Glycol / Chem-Z9A / N~2~-[4-(4-bromophenyl)butanoyl]-D-asparagine / Beta-lactamase
Similarity search - Component
Biological speciesEscherichia coli CFT073 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.3 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
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 1.4Dec 13, 2023Group: Derived calculations / Structure summary / Category: chem_comp / entity / pdbx_entity_nonpoly
Item: _chem_comp.name / _chem_comp.pdbx_synonyms ..._chem_comp.name / _chem_comp.pdbx_synonyms / _entity.pdbx_description / _pdbx_entity_nonpoly.name

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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)58,04018
Polymers52,9911
Non-polymers5,04917
Water5,567309
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.
  • 116 kDa, 2 polymers
  • Search similar-shape structures of this assembly by Omokage search (details)
Theoretical massNumber of molelcules
Total (without water)116,08036
Polymers105,9822
Non-polymers10,09934
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_565-x,-y+1,z1
Unit cell
Length a, b, c (Å)96.686, 96.686, 182.736
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-989-

HOH

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Beta-lactamase


Mass: 52990.805 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 , 9 types, 326 molecules

#2: Chemical ChemComp-DMS / DIMETHYL SULFOXIDE


Mass: 78.133 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H6OS / Comment: DMSO, precipitant*YM
#3: Chemical ChemComp-Z9A / methyl N~2~-[4-(4-bromophenyl)butanoyl]-D-asparaginyl-L-alaninate


Mass: 442.304 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C18H24BrN3O5
#4: Chemical ChemComp-Z9G / N~2~-[4-(4-bromophenyl)butanoyl]-D-asparagine


Mass: 357.200 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C14H17BrN2O4 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-2PE / NONAETHYLENE GLYCOL


Mass: 414.488 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C18H38O10 / Comment: precipitant*YM
#6: Chemical ChemComp-AV0 / Lauryl Maltose Neopentyl Glycol / 2,2-didecylpropane-1,3-bis-b-D-maltopyranoside / 2-decyl-2-{[(4-O-alpha-D-glucopyranosyl-beta-D-glucopyranosyl)oxy]methyl}dodecyl4-O-alpha-D-glucopyranosyl-beta-D-glucopyranoside


Mass: 1005.188 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C47H88O22
#7: Chemical
ChemComp-97N / (2S)-2,3-dihydroxypropyl (9Z)-hexadec-9-enoate


Mass: 328.487 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C19H36O4
#8: Chemical ChemComp-SO4 / SULFATE ION


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


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


Mass: 18.015 Da / Num. of mol.: 309 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 2

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Sample preparation

Crystal
IDDensity Matthews (Å3/Da)Density % sol (%)Description
13.8868.26Square plates grown in a sponge phase
23.8868.26Square plates grown in a sponge phase
Crystal grow
Temperature (K)Crystal-IDMethodDetailsPH rangeTemp details
2951lipidic cubic phasePrecipitant 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, 1.2% (w/v) myo-inositol7.4-7.8room temperature
2952lipidic cubic phasePrecipitant 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, 1.2% (w/v) myo-inositol7.4-7.8room 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: 0.9202 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Oct 19, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9202 Å / Relative weight: 1
ReflectionResolution: 2.3→46.74 Å / Num. obs: 39268 / % possible obs: 99.81 % / Redundancy: 17.2 % / Biso Wilson estimate: 33.75 Å2 / CC1/2: 0.997 / Rmerge(I) obs: 0.362 / Rpim(I) all: 0.08917 / Rrim(I) all: 0.3731 / Net I/σ(I): 8.89
Reflection shellResolution: 2.3→2.382 Å / Redundancy: 15.9 % / Rmerge(I) obs: 2.781 / Mean I/σ(I) obs: 1.32 / Num. unique obs: 3829 / CC1/2: 0.697 / CC star: 0.907 / Rpim(I) all: 0.7095 / Rrim(I) all: 2.872 / % possible all: 99.63

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
Aimless0.7.4data scaling
XDS20190806data reduction
PHENIX1.19.1-4122-000phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7MDE

7mde


Resolution: 2.3→46.74 Å / Cross valid method: FREE R-VALUE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.219 1964 -
Rwork0.1914 69698 -
obs-39236 99.81 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 57.39 Å2
Refinement stepCycle: LAST / Resolution: 2.3→46.74 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3394 0 195 309 3898
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0073692
X-RAY DIFFRACTIONf_angle_d0.89384999
X-RAY DIFFRACTIONf_chiral_restr0.0514568
X-RAY DIFFRACTIONf_plane_restr0.0079635
X-RAY DIFFRACTIONf_dihedral_angle_d15.7081321
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.3-2.330.36731420.33972608X-RAY DIFFRACTION99.82
2.33-2.360.381430.34152713X-RAY DIFFRACTION99.2
2.36-2.40.33471420.32242664X-RAY DIFFRACTION99.86
2.4-2.430.33191400.30712697X-RAY DIFFRACTION99.86
2.43-2.470.31121410.3012659X-RAY DIFFRACTION99.93
2.47-2.510.31321430.2922698X-RAY DIFFRACTION99.89
2.51-2.550.30411370.27882681X-RAY DIFFRACTION99.93
2.55-2.60.29751410.26672696X-RAY DIFFRACTION99.93
2.6-2.650.31171410.26442662X-RAY DIFFRACTION99.75
2.65-2.70.26891490.25692697X-RAY DIFFRACTION99.82
2.7-2.760.28971470.2512671X-RAY DIFFRACTION99.96
2.76-2.830.26381380.24032708X-RAY DIFFRACTION100
2.83-2.90.25151370.21892648X-RAY DIFFRACTION99.82
2.9-2.980.25431460.18882682X-RAY DIFFRACTION99.86
2.98-3.060.1951450.18542659X-RAY DIFFRACTION99.57
3.06-3.160.21711430.17112692X-RAY DIFFRACTION99.93
3.16-3.280.18561410.16672706X-RAY DIFFRACTION99.93
3.28-3.410.18491430.162672X-RAY DIFFRACTION99.89
3.41-3.560.17391350.14682712X-RAY DIFFRACTION99.75
3.56-3.750.18551500.14572659X-RAY DIFFRACTION100
3.75-3.980.20381390.14492693X-RAY DIFFRACTION100
3.98-4.290.15011450.13412687X-RAY DIFFRACTION100
4.29-4.720.17811440.13582660X-RAY DIFFRACTION99.75
4.72-5.410.15091360.15732711X-RAY DIFFRACTION100
5.41-6.80.20921380.18182677X-RAY DIFFRACTION100
6.81-46.740.19671400.17992686X-RAY DIFFRACTION99.4
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)
14.41372604682-0.1465049969791.505190436135.80939327056-1.800523547175.536117326060.100515062843-0.1097339522930.1061106392290.0274064308222-0.0718187267348-0.373983850951-0.1103700966550.4343072733110.09571562014210.1408310400130.0416877144207-0.002390966385030.254447192446-0.009754179601390.42848711153123.25833.91449.162
22.739067558390.01530977730760.1231500183741.35178804246-0.1579931148730.7319157528-0.0213492991371-0.0668660122548-0.3796973696570.04825017259160.01345627060360.14998847480.150723970594-0.0744343280989-0.00674092259650.235283592712-0.004737192000780.032543700210.2344376735840.008186057968050.48139405316-1.15123.59547.725
33.14671966122-0.441787026404-0.07665129102281.84200003293-0.3093727721041.088077281230.05115187931650.06573840663280.0562611648988-0.05978286986-0.00780661154616-0.157206498539-0.0311124576750.05594913059220.04985666283190.167460352196-0.00819554893491-0.003240925705920.169116015078-0.004941212506830.2781397397747.67440.31745.323
40.535943552263-0.7381585948630.07899993765341.366676711640.7633241439876.980032704370.301618904297-1.22389592327-0.06399549896011.06283382003-0.27199313561-0.31634860535-0.5452335211490.0962124672062-0.004857911100190.997577304275-0.320624256863-0.1054422991831.22060670088-0.05441204327190.47361864962312.10340.91786.572
50.736593334585-0.453114280537-0.08667497881151.631722978430.9863236205043.633288449520.0538790678277-0.876971874288-0.126753796650.751419920354-0.4009681425120.3422945178660.258868631327-0.7434966001990.06488305158920.634093688573-0.1482402640770.1019045873691.016892333350.02431162578030.594963187599-0.76530.00983.277
61.259812765150.1487950393380.8457992697881.882829614680.2339568896690.566116541280.187584809263-1.04165484074-0.1374164771980.5923695324830.0618866482774-0.1759512865140.0726874311165-0.5082332097780.04978389532810.395319098481-0.0112472958636-0.1242967617230.7625023426010.03801631292970.3348846330415.1836.37178.017
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1( CHAIN A AND RESID 36:93 )A36 - 93
2X-RAY DIFFRACTION2( CHAIN A AND RESID 94:295 )A94 - 295
3X-RAY DIFFRACTION3( CHAIN A AND RESID 296:381 )A296 - 381
4X-RAY DIFFRACTION4( CHAIN A AND RESID 382:414 )A382 - 414
5X-RAY DIFFRACTION5( CHAIN A AND RESID 415:468 )A415 - 468
6X-RAY DIFFRACTION6( CHAIN A AND RESID 469:496 )A469 - 496

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