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- PDB-7nna: Toxin Import Through the Antibiotic Efflux Channel TolC -

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Basic information

Entry
Database: PDB / ID: 7nna
TitleToxin Import Through the Antibiotic Efflux Channel TolC
ComponentsKlebicin C activity
KeywordsANTIBIOTIC / TOXIN / IMPORT
Function / homology
Function and homology information


cytolysis / hydrolase activity, acting on ester bonds / : / ribosome binding / defense response to bacterium / RNA binding
Similarity search - Function
Colicin E3-like ribonuclease domain / Colicin E3-like ribonuclease domain superfamily / Cytotoxic / S-type Pyocin / Pyosin/cloacin translocation domain / Pyosin/cloacin translocation domain superfamily
Similarity search - Domain/homology
Biological speciesKlebsiella pneumoniae (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 1.901 Å
AuthorsLowe, E.D. / Kleanthous, C. / Housden, N.G.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust201505/Z/16/Z United Kingdom
CitationJournal: Nat Commun / Year: 2021
Title: Toxin import through the antibiotic efflux channel TolC.
Authors: Nicholas G Housden / Melissa N Webby / Edward D Lowe / Tarick J El-Baba / Renata Kaminska / Christina Redfield / Carol V Robinson / Colin Kleanthous /
Abstract: Bacteria often secrete diffusible protein toxins (bacteriocins) to kill bystander cells during interbacterial competition. Here, we use biochemical, biophysical and structural analyses to show how a ...Bacteria often secrete diffusible protein toxins (bacteriocins) to kill bystander cells during interbacterial competition. Here, we use biochemical, biophysical and structural analyses to show how a bacteriocin exploits TolC, a major outer-membrane antibiotic efflux channel in Gram-negative bacteria, to transport itself across the outer membrane of target cells. Klebicin C (KlebC), a rRNase toxin produced by Klebsiella pneumoniae, binds TolC of a related species (K. quasipneumoniae) with high affinity through an N-terminal, elongated helical hairpin domain common amongst bacteriocins. The KlebC helical hairpin opens like a switchblade to bind TolC. A cryo-EM structure of this partially translocated state, at 3.1 Å resolution, reveals that KlebC associates along the length of the TolC channel. Thereafter, the unstructured N-terminus of KlebC protrudes beyond the TolC iris, presenting a TonB-box sequence to the periplasm. Association with proton-motive force-linked TonB in the inner membrane drives toxin import through the channel. Finally, we demonstrate that KlebC binding to TolC blocks drug efflux from bacteria. Our results indicate that TolC, in addition to its known role in antibiotic export, can function as a protein import channel for bacteriocins.
History
DepositionFeb 24, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 7, 2021Provider: repository / Type: Initial release
Revision 2.0Oct 13, 2021Group: Atomic model / Data collection / Database references
Category: atom_site / citation ...atom_site / citation / citation_author / database_2 / diffrn_source / pdbx_database_proc
Item: _atom_site.pdbx_tls_group_id / _citation.country ..._atom_site.pdbx_tls_group_id / _citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _diffrn_source.pdbx_synchrotron_site

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Klebicin C activity


Theoretical massNumber of molelcules
Total (without water)23,7961
Polymers23,7961
Non-polymers00
Water6,485360
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area14610 Å2
MethodPISA
Unit cell
Length a, b, c (Å)47.914, 47.914, 200.488
Angle α, β, γ (deg.)90, 90, 120
Int Tables number154
Space group name H-MP3221

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Components

#1: Protein Klebicin C activity / KlebC Tol binding domain


Mass: 23796.283 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Gene: kca / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): T7 Express Crystal / References: UniProt: Q5V9K0
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 360 / Source method: isolated from a natural source / Formula: H2O
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: 2.79 Å3/Da / Density % sol: 55.94 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: Condition G1 from Morpheus screen (0.1M Morpheus Carboxylic acids, 0.1M Morpheus Buffer 1 pH 6.5, 30% v/v Morpheus Precipitant Mix 1)

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: DIAMOND / Beamline: I03 / Wavelength: 0.97932 Å
DetectorType: DECTRIS EIGER2 XE 16M / Detector: PIXEL / Date: Oct 17, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97932 Å / Relative weight: 1
ReflectionResolution: 1.9→40.63 Å / Num. obs: 22045 / % possible obs: 99.9 % / Redundancy: 4.8 % / CC1/2: 0.997 / Rmerge(I) obs: 0.05 / Rpim(I) all: 0.036 / Rrim(I) all: 0.062 / Χ2: 0.96 / Net I/σ(I): 14.3
Reflection shellResolution: 1.9→1.94 Å / Redundancy: 4.9 % / Rmerge(I) obs: 0.433 / Mean I/σ(I) obs: 3.1 / Num. unique obs: 1386 / CC1/2: 0.913 / Rpim(I) all: 0.318 / Rrim(I) all: 0.54 / Χ2: 0.99 / % possible all: 99.9

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Processing

Software
NameVersionClassification
BUSTER2.10.3refinement
DIALSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: SAD / Resolution: 1.901→40.63 Å / Cor.coef. Fo:Fc: 0.917 / Cor.coef. Fo:Fc free: 0.929 / SU R Cruickshank DPI: 0.138 / Cross valid method: THROUGHOUT / SU R Blow DPI: 0.171 / SU Rfree Blow DPI: 0.131 / SU Rfree Cruickshank DPI: 0.117
RfactorNum. reflection% reflectionSelection details
Rfree0.2017 1073 -RANDOM
Rwork0.1884 ---
obs0.1891 21983 99.7 %-
Displacement parametersBiso mean: 38.5 Å2
Baniso -1Baniso -2Baniso -3
1--7.6082 Å20 Å20 Å2
2---7.6082 Å20 Å2
3---15.2163 Å2
Refine analyzeLuzzati coordinate error obs: 0.21 Å
Refinement stepCycle: LAST / Resolution: 1.901→40.63 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1509 0 0 360 1869
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.0151708HARMONIC2
X-RAY DIFFRACTIONt_angle_deg1.062296HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d697SINUSOIDAL2
X-RAY DIFFRACTIONt_gen_planes316HARMONIC5
X-RAY DIFFRACTIONt_it1708HARMONIC10
X-RAY DIFFRACTIONt_chiral_improper_torsion229SEMIHARMONIC5
X-RAY DIFFRACTIONt_ideal_dist_contact2262SEMIHARMONIC4
X-RAY DIFFRACTIONt_omega_torsion2.58
X-RAY DIFFRACTIONt_other_torsion16.55
LS refinement shellResolution: 1.901→1.91 Å
RfactorNum. reflection% reflection
Rfree0.2272 29 -
Rwork0.1962 --
obs--99.53 %
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)
13.5441-1.09910.46985.7962-3.04287.3265-0.22550.5061-0.00830.5061-0.0819-0.3288-0.0083-0.32880.3075-0.04430.00470.11240.1186-0.0195-0.0562-23.78710.814629.0542
21.4106-0.3001-2.37040.37270.61084.20010.0018-0.04210.1211-0.0421-0.027-0.14520.1211-0.14520.0252-0.0445-0.00220.02770.0069-0.00390.0347-10.36777.81956.3365
33.4445-0.6395-3.1583-1.8198-0.35047.9979-0.1717-0.23960.5645-0.2396-0.027-0.0890.5645-0.0890.19880.10880.01370.1101-0.0648-0.0249-0.00047.45486.0135-20.9022
41.02251.0345-0.96950.8537-3.8770-0.029-0.27940.1119-0.27940.1771-0.25730.1119-0.2573-0.14810.18180.01950.1696-0.06680.0102-0.059911.94597.2401-31.4465
5-2.52051.4552-2.15942.5205-2.08078.3154-0.23020.23150.53730.2315-0.17890.15780.53730.15780.40910.12880.03530.141-0.07610.0597-0.034116.90958.7543-38.9003
62.12810.244-1.3111.70312.35472.5765-0.0148-0.3820.653-0.382-0.1202-0.06030.653-0.06030.13490.1205-0.00470.0907-0.08350.0624-0.000720.922110.1302-46.4628
73.1811-1.0889-2.6778-1.653-1.18277.0347-0.17590.03690.42170.03690.00260.35810.42170.35810.17330.04750.08020.0618-0.04850.01570.024526.867513.9409-55.6932
81.99834.06413.97574.0267-1.06531.6532-0.0165-0.3437-0.555-0.3437-0.43680.0661-0.5550.06610.4533-0.02050.07480.16810.09690.01290.104528.255524.3512-71.0166
93.46114.21321.18235.3174-0.00953.14320.0075-0.32810.1406-0.3281-0.15470.70180.14060.70180.14720.01270.14210.04580.08850.0065-0.053223.920616.4852-72.1161
101.0597-0.8598-0.0221-0.20060.15483.84640.0877-0.04340.3189-0.0434-0.058-0.24510.3189-0.2451-0.02970.0205-0.01360.01210.0026-0.0104-0.013718.746616.0723-58.4
115.93560.15761.81836.27950.22722.0346-0.0110.1685-0.16060.16850.134-0.7211-0.1606-0.7211-0.123-0.0149-0.02940.06730.12120.0424-0.079311.357215.9005-46.0304
128.9165-0.43430.9036.33013.41425.2841-0.40510.0581-0.2410.05810.4731-0.121-0.241-0.121-0.0680.01150.01410.09910.07570.0818-0.09598.804415.4033-37.1158
13-2.39431.2945-2.61512.8322-0.00946.25520.15730.198-0.29840.1980.06050.3678-0.29840.3678-0.21790.1122-0.00240.15150.01630.0415-0.09858.754315.5641-24.0456
141.18810.0089-1.76010.03190.0056.85070.1694-0.1336-0.294-0.1336-0.03060.1153-0.2940.1153-0.13880.01680.02770.0523-0.05650.00050.04351.851613.7529-5.1768
15-1.83060.423-0.77064.0264-0.10725.781-0.01820.0191-0.26210.01910.1893-0.0188-0.2621-0.0188-0.1711-0.03170.0235-0.01190.0001-0.03440.0437-2.809212.29199.6694
161.15070.2453-3.1620.6223-0.34411.8110.00770.080.19320.08-0.03670.25450.19320.25450.029-0.03630.01270.00250.09220.025-0.0107-10.2887.912622.5166
17-1.5745-0.18330.88925.3584-2.84733.57320.06040.26880.16940.2688-0.27810.12420.16940.12420.21770.03110.03140.09450.10680.0871-0.1106-16.45315.261134.6709
186.3689-4.03433.97574.3059-1.06530.14660.13990.1471-0.32150.1471-0.11370.2475-0.32150.2475-0.02630.09230.03430.1536-0.00360.038-0.0833-20.28874.83442.3141
196.59910.74181.227-0.58423.69575.6652-0.0793-0.16020.3512-0.1602-0.110.48010.35120.48010.18930.1730.00920.1119-0.05970.0595-0.0821-23.93522.112850.3459
204.47531.363-3.97570.08211.06538.3095-0.04630.0162-0.0920.0162-0.09390.5753-0.0920.57530.14020.0186-0.00160.08430.00910.0255-0.0182-24.8013.004657.9468
211.81240.00542.8925-0.59790.30237.2286-0.1471-0.1084-0.247-0.1084-0.08150.0204-0.2470.02040.2287-0.0103-0.008-0.0155-0.0074-0.01560.0296-27.9111.14366.6268
225.7679-4.3812-2.59984.10561.85971.8638-0.25630.3774-0.02180.37740.155-0.641-0.0218-0.6410.10130.0615-0.00450.034-0.0469-0.0806-0.0169-27.0978-11.76367.8908
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1{ A|62 - A|65 }A62 - 65
2X-RAY DIFFRACTION2{ A|66 - A|98 }A66 - 98
3X-RAY DIFFRACTION3{ A|99 - A|108 }A99 - 108
4X-RAY DIFFRACTION4{ A|109 - A|113 }A109 - 113
5X-RAY DIFFRACTION5{ A|114 - A|120 }A114 - 120
6X-RAY DIFFRACTION6{ A|121 - A|125 }A121 - 125
7X-RAY DIFFRACTION7{ A|126 - A|134 }A126 - 134
8X-RAY DIFFRACTION8{ A|135 - A|142 }A135 - 142
9X-RAY DIFFRACTION9{ A|143 - A|145 A|146 - A|146 }A143 - 145
10X-RAY DIFFRACTION9{ A|143 - A|145 A|146 - A|146 }A146
11X-RAY DIFFRACTION10{ A|147 - A|160 }A147 - 160
12X-RAY DIFFRACTION11{ A|161 - A|165 }A161 - 165
13X-RAY DIFFRACTION12{ A|166 - A|173 }A166 - 173
14X-RAY DIFFRACTION13{ A|174 - A|182 }A174 - 182
15X-RAY DIFFRACTION14{ A|183 - A|199 }A183 - 199
16X-RAY DIFFRACTION15{ A|200 - A|204 }A200 - 204
17X-RAY DIFFRACTION16{ A|205 - A|218 }A205 - 218
18X-RAY DIFFRACTION17{ A|219 - A|224 }A219 - 224
19X-RAY DIFFRACTION18{ A|225 - A|229 }A225 - 229
20X-RAY DIFFRACTION19{ A|230 - A|235 }A230 - 235
21X-RAY DIFFRACTION20{ A|236 - A|240 }A236 - 240
22X-RAY DIFFRACTION21{ A|241 - A|250 }A241 - 250
23X-RAY DIFFRACTION22{ A|251 - A|255 }A251 - 255

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