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- PDB-6tek: Structure of siderophore interaction domain of IrtAB -

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Basic information

Entry
Database: PDB / ID: 6tek
TitleStructure of siderophore interaction domain of IrtAB
ComponentsDrug ABC transporter ATP-binding protein
KeywordsMEMBRANE PROTEIN / siderophore interaction domain / IrtAB / ABC transporter
Function / homology
Function and homology information


ABC-type transporter activity / oxidoreductase activity / ATP binding / plasma membrane
Similarity search - Function
Siderophore-interacting protein, C-terminal domain / FAD-binding 9, siderophore-interacting / Siderophore-interacting protein / Siderophore-interacting FAD-binding domain / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / FAD-binding domain, ferredoxin reductase-type / Ferredoxin-NADP reductase (FNR), nucleotide-binding domain ...Siderophore-interacting protein, C-terminal domain / FAD-binding 9, siderophore-interacting / Siderophore-interacting protein / Siderophore-interacting FAD-binding domain / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / FAD-binding domain, ferredoxin reductase-type / Ferredoxin-NADP reductase (FNR), nucleotide-binding domain / Ferredoxin reductase-type FAD binding domain profile. / Riboflavin synthase-like beta-barrel / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
FLAVIN-ADENINE DINUCLEOTIDE / Mycobactin import ATP-binding/permease protein IrtA
Similarity search - Component
Biological speciesMycolicibacterium thermoresistibile (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.8 Å
AuthorsArnold, F.M. / Gonda, I. / Hutter, C.A.J. / Seeger, M.A. / Hurlimann, L.M.
Funding support Switzerland, 2items
OrganizationGrant numberCountry
European Research Council (ERC)772190 Switzerland
Swiss National Science FoundationPP00P3_144823 Switzerland
CitationJournal: Nature / Year: 2020
Title: The ABC exporter IrtAB imports and reduces mycobacterial siderophores.
Authors: Fabian M Arnold / Miriam S Weber / Imre Gonda / Marc J Gallenito / Sophia Adenau / Pascal Egloff / Iwan Zimmermann / Cedric A J Hutter / Lea M Hürlimann / Eike E Peters / Jörn Piel / ...Authors: Fabian M Arnold / Miriam S Weber / Imre Gonda / Marc J Gallenito / Sophia Adenau / Pascal Egloff / Iwan Zimmermann / Cedric A J Hutter / Lea M Hürlimann / Eike E Peters / Jörn Piel / Gabriele Meloni / Ohad Medalia / Markus A Seeger /
Abstract: Intracellular replication of the deadly pathogen Mycobacterium tuberculosis relies on the production of small organic molecules called siderophores that scavenge iron from host proteins. M. ...Intracellular replication of the deadly pathogen Mycobacterium tuberculosis relies on the production of small organic molecules called siderophores that scavenge iron from host proteins. M. tuberculosis produces two classes of siderophore, lipid-bound mycobactin and water-soluble carboxymycobactin. Functional studies have revealed that iron-loaded carboxymycobactin is imported into the cytoplasm by the ATP binding cassette (ABC) transporter IrtAB, which features an additional cytoplasmic siderophore interaction domain. However, the predicted ABC exporter fold of IrtAB is seemingly contradictory to its import function. Here we show that membrane-reconstituted IrtAB is sufficient to import mycobactins, which are then reduced by the siderophore interaction domain to facilitate iron release. Structure determination by X-ray crystallography and cryo-electron microscopy not only confirms that IrtAB has an ABC exporter fold, but also reveals structural peculiarities at the transmembrane region of IrtAB that result in a partially collapsed inward-facing substrate-binding cavity. The siderophore interaction domain is positioned in close proximity to the inner membrane leaflet, enabling the reduction of membrane-inserted mycobactin. Enzymatic ATPase activity and in vivo growth assays show that IrtAB has a preference for mycobactin over carboxymycobactin as its substrate. Our study provides insights into an unusual ABC exporter that evolved as highly specialized siderophore-import machinery in mycobacteria.
History
DepositionNov 12, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 1, 2020Provider: repository / Type: Initial release
Revision 1.1Apr 29, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jan 24, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Drug ABC transporter ATP-binding protein
B: Drug ABC transporter ATP-binding protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)57,3134
Polymers55,7422
Non-polymers1,5712
Water9,368520
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3730 Å2
ΔGint-19 kcal/mol
Surface area22190 Å2
MethodPISA
Unit cell
Length a, b, c (Å)52.070, 111.700, 52.200
Angle α, β, γ (deg.)90.000, 119.760, 90.000
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Drug ABC transporter ATP-binding protein


Mass: 27871.057 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium thermoresistibile (bacteria)
Gene: RMCT_1990 / Plasmid: PBXNH3 / Production host: Escherichia coli MC1061 (bacteria) / References: UniProt: A0A100XEC2
#2: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: FAD*YM
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 520 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: 2.36 Å3/Da / Density % sol: 47.97 %
Crystal growTemperature: 297 K / Method: vapor diffusion, sitting drop / pH: 8 / Details: 400 mM Ammonium-Acetate, 12 % (v/v) PEG 2000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06SA / Wavelength: 1 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Mar 9, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.8→45.32 Å / Num. obs: 45656 / % possible obs: 95 % / Redundancy: 6.197 % / Biso Wilson estimate: 25.718 Å2 / CC1/2: 0.996 / Rmerge(I) obs: 0.101 / Rrim(I) all: 0.111 / Χ2: 0.951 / Net I/σ(I): 11.44 / Num. measured all: 282913 / Scaling rejects: 6895
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured obsNum. possibleNum. unique obsCC1/2Rrim(I) all% possible all
1.8-1.855.9120.6342.920206352234180.8660.69597
1.85-1.96.20.5862.8920670342533340.9030.64197.3
1.9-1.955.5330.4783.5815283335927620.9270.53182.2
1.95-2.016.0990.3545.119431330631860.9440.38796.4
2.01-2.085.7710.2526.517267312929920.9630.27795.6
2.08-2.156.3860.2367.3519170306730020.9730.25797.9
2.15-2.236.4050.2426.4717460292827260.9740.26393.1
2.23-2.325.7920.1948.3714782286825520.960.21789
2.32-2.436.3420.14511.0116653271626260.9860.15896.7
2.43-2.555.8420.1311.3414382259724620.9880.14294.8
2.55-2.686.7980.11713.7116663249124510.990.12798.4
2.68-2.856.7580.09815.6415435233622840.9930.10797.8
2.85-3.046.5540.0818.2514143222421580.9950.08797
3.04-3.295.9820.06620.4311923208919930.9950.07395.4
3.29-3.66.6770.06221.9612365187718520.9960.06798.7
3.6-4.036.3420.06622.9410636174916770.9950.07195.9
4.03-4.656.2150.05824.568950150014400.9960.06396
4.65-5.696.2640.05624.787749128912370.9970.06296
5.69-8.056.5820.05128.14639810069720.9980.05696.6
8.05-45.326.2910.03832.7933475715320.9990.04293.2

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
PHENIX1.17.1_3660refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2GPJ

2gpj


Resolution: 1.8→45.32 Å / SU ML: 0.2 / Cross valid method: THROUGHOUT / σ(F): 1.36 / Phase error: 26.92 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2545 2279 5 %
Rwork0.2226 43278 -
obs0.2242 45557 95.19 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 78.1 Å2 / Biso mean: 23.9367 Å2 / Biso min: 6.78 Å2
Refinement stepCycle: final / Resolution: 1.8→45.32 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3674 0 106 520 4300
Biso mean--15.1 29.6 -
Num. residues----460
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 16

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.8-1.840.3111440.26792734287897
1.84-1.880.2721440.26912742288697
1.88-1.930.57831190.47312274239379
1.93-1.980.31131420.27112696283897
1.98-2.040.2741440.23442739288397
2.04-2.110.24921440.22942726287096
2.11-2.180.25231480.22542808295699
2.18-2.270.39591270.32342407253484
2.27-2.370.2551470.22522794294198
2.37-2.50.27381420.22062699284196
2.5-2.650.25211460.21412759290597
2.65-2.860.24151450.22122768291398
2.86-3.140.23621470.2152777292497
3.14-3.60.2081470.18712793294098
3.6-4.530.19311450.17742761290697
4.54-45.320.22751480.19372801294996
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)
10.4630.0922-0.35720.63150.02640.89340.06210.15630.1134-0.2225-0.02920.0771-0.0282-0.18580.11810.08460.034-0.02230.08080.00620.12626.628121.36182.6453
20.89190.1243-0.30961.0452-0.08250.92220.0727-0.07970.0571-0.07050.06680.2222-0.0707-0.03660.00250.08610.0061-0.01170.08490.00150.1167.680819.204610.4559
30.94640.3333-0.32160.23410.21661.0350.19960.20540.4779-0.0738-0.0954-0.0309-0.5572-0.0919-1.07440.23910.00220.04760.15850.10910.401623.294734.1458-5.1635
41.7901-0.2598-0.39410.549-0.10170.90190.0409-0.16630.1741-0.011-0.027-0.1355-0.01870.13570.03880.06810.00570.00530.0907-0.01770.116731.068220.39191.9071
50.245-0.317-0.05440.41480.11910.18960.165-0.2560.33160.1199-0.0052-0.0821-0.1230.0763-0.00010.17010.0064-0.00690.2683-0.03380.284529.498428.17259.2056
60.3672-0.34110.20160.35740.01380.9120.00910.0437-0.21450.12770.02330.0730.089-0.14610.01340.1163-0.02170.00660.10880.0080.1915-19.3584-2.7142-4.3139
71.188-0.07380.26650.7493-0.04550.85510.08520.0276-0.1206-0.0315-0.00220.12850.0819-0.0103-00.10140.0036-0.00950.0907-0.00660.1172-18.4848-0.7547-12.2949
81.61150.1981-0.59990.1119-0.21190.5331-0.077-0.4868-0.806-0.0084-0.0988-0.01210.46190.0773-0.46550.1617-0.00260.00840.11820.08280.2431-1.4623-9.23081.5858
90.41920.34440.01910.43610.39820.60340.0866-0.0933-0.01320.0444-0.0952-0.0626-0.14370.266-0.00130.0788-0.01270.00370.12370.03190.1054.17893.1953-2.6686
101.60430.38710.51850.8217-1.16252.5867-0.0780.0478-0.5117-0.1409-0.2238-0.44060.51040.5468-0.8309-0.01630.0181-0.01760.16120.01790.23469.7083-8.0178-7.2229
110.11820.3023-0.02280.8492-0.07640.0118-0.0360.2097-0.1633-0.0591-0.1265-0.41370.12080.0712-0.19030.1182-0.01050.00470.2761-0.02410.20233.4176-9.4335-11.1138
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 16 through 52 )A16 - 52
2X-RAY DIFFRACTION2chain 'A' and (resid 53 through 112 )A53 - 112
3X-RAY DIFFRACTION3chain 'A' and (resid 113 through 131 )A113 - 131
4X-RAY DIFFRACTION4chain 'A' and (resid 132 through 225 )A132 - 225
5X-RAY DIFFRACTION5chain 'A' and (resid 226 through 245 )A226 - 245
6X-RAY DIFFRACTION6chain 'B' and (resid 16 through 52 )B16 - 52
7X-RAY DIFFRACTION7chain 'B' and (resid 53 through 113 )B53 - 113
8X-RAY DIFFRACTION8chain 'B' and (resid 114 through 151 )B114 - 151
9X-RAY DIFFRACTION9chain 'B' and (resid 152 through 196 )B152 - 196
10X-RAY DIFFRACTION10chain 'B' and (resid 197 through 225 )B197 - 225
11X-RAY DIFFRACTION11chain 'B' and (resid 226 through 245 )B226 - 245

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