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- PDB-6r72: Crystal structure of BmrA-E504A in an outward-facing conformation -

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Basic information

Entry
Database: PDB / ID: 6r72
TitleCrystal structure of BmrA-E504A in an outward-facing conformation
ComponentsMultidrug exporter ATP-binding cassette
KeywordsTRANSPORT PROTEIN / ABC transporter BmrA Multi-drug transporter
Function / homology
Function and homology information


ATPase-coupled lipid transmembrane transporter activity / Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate / ABC-type transporter activity / transmembrane transport / response to antibiotic / ATP hydrolysis activity / ATP binding / membrane / plasma membrane
Similarity search - Function
Type 1 protein exporter / 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 / 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. ...Type 1 protein exporter / 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 / 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. / P-loop containing nucleotide triphosphate hydrolases / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / Lipid A export ATP-binding/permease protein MsbA / Multidrug resistance ABC transporter ATP-binding/permease protein BmrA
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.95 Å
AuthorsChaptal, V. / Zampieri, V. / Kilburg, A. / Magnard, S. / Falson, P.
Funding support France, 3items
OrganizationGrant numberCountry
French National Research AgencyANR-18-CE11-0002-01 France
French National Research AgencyANR-CE11-0015-03 France
Ligue Regionale Contre le Cancer189393 France
CitationJournal: Sci Adv / Year: 2022
Title: Substrate-bound and substrate-free outward-facing structures of a multidrug ABC exporter.
Authors: Vincent Chaptal / Veronica Zampieri / Benjamin Wiseman / Cédric Orelle / Juliette Martin / Kim-Anh Nguyen / Alexia Gobet / Margot Di Cesare / Sandrine Magnard / Waqas Javed / Jad Eid / ...Authors: Vincent Chaptal / Veronica Zampieri / Benjamin Wiseman / Cédric Orelle / Juliette Martin / Kim-Anh Nguyen / Alexia Gobet / Margot Di Cesare / Sandrine Magnard / Waqas Javed / Jad Eid / Arnaud Kilburg / Marine Peuchmaur / Julien Marcoux / Luca Monticelli / Martin Hogbom / Guy Schoehn / Jean-Michel Jault / Ahcène Boumendjel / Pierre Falson /
Abstract: Multidrug ABC transporters translocate drugs across membranes by a mechanism for which the molecular features of drug release are so far unknown. Here, we resolved three ATP-Mg-bound outward-facing ...Multidrug ABC transporters translocate drugs across membranes by a mechanism for which the molecular features of drug release are so far unknown. Here, we resolved three ATP-Mg-bound outward-facing conformations of the (homodimeric) BmrA by x-ray crystallography and single-particle cryo-electron microscopy (EM) in detergent solution, one of them with rhodamine 6G (R6G), a substrate exported by BmrA when overexpressed in . Two R6G molecules bind to the drug-binding cavity at the level of the outer leaflet, between transmembrane (TM) helices 1-2 of one monomer and TM5'-6' of the other. They induce a rearrangement of TM1-2, highlighting a local flexibility that we confirmed by hydrogen/deuterium exchange and molecular dynamics simulations. In the absence of R6G, simulations show a fast postrelease occlusion of the cavity driven by hydrophobicity, while when present, R6G can move within the cavity, maintaining it open.
History
DepositionMar 28, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 6, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 19, 2022Group: Database references / Derived calculations / Category: database_2 / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_label_asym_id
Revision 1.2Feb 2, 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.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.3Jan 24, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Multidrug exporter ATP-binding cassette
B: Multidrug exporter ATP-binding cassette
C: Multidrug exporter ATP-binding cassette
D: Multidrug exporter ATP-binding cassette
hetero molecules


Theoretical massNumber of molelcules
Total (without water)265,11512
Polymers262,9894
Non-polymers2,1268
Water0
1
A: Multidrug exporter ATP-binding cassette
B: Multidrug exporter ATP-binding cassette
hetero molecules


Theoretical massNumber of molelcules
Total (without water)132,5576
Polymers131,4942
Non-polymers1,0634
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area17150 Å2
ΔGint-105 kcal/mol
Surface area51260 Å2
MethodPISA
2
C: Multidrug exporter ATP-binding cassette
D: Multidrug exporter ATP-binding cassette
hetero molecules


Theoretical massNumber of molelcules
Total (without water)132,5576
Polymers131,4942
Non-polymers1,0634
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area16990 Å2
ΔGint-110 kcal/mol
Surface area51100 Å2
MethodPISA
Unit cell
Length a, b, c (Å)117.805, 110.754, 155.615
Angle α, β, γ (deg.)90.00, 93.23, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein
Multidrug exporter ATP-binding cassette


Mass: 65747.141 Da / Num. of mol.: 4 / Mutation: E504A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria) / Gene: B4417_3307 / Plasmid: pET15b / Production host: Escherichia coli (E. coli) / Variant (production host): C43, Delate-AcrB / References: UniProt: A0A164TVX9, UniProt: O06967*PLUS
#2: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg

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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: 3.9 Å3/Da / Density % sol: 68.5 % / Description: rods
Crystal growTemperature: 295.15 K / Method: vapor diffusion, hanging drop / pH: 8.5 / Details: 23-27% PEG 1000, 0.1M Tris pH 8.5 / PH range: 8-8.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SOLEIL / Beamline: PROXIMA 2 / Wavelength: 0.98 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Jul 5, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.98 Å / Relative weight: 1
ReflectionResolution: 3.92→80.6 Å / Num. obs: 20484 / % possible obs: 92 % / Observed criterion σ(F): 1.25 / Redundancy: 3.5 % / Biso Wilson estimate: 178.75 Å2 / Rmerge(I) obs: 0.068 / Net I/σ(I): 11.65
Reflection shellResolution: 3.92→4.3 Å / Rmerge(I) obs: 3.7 / Mean I/σ(I) obs: 1.39 / Num. unique obs: 959

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Processing

Software
NameVersionClassification
BUSTER2.10.3refinement
XDSdata reduction
STARANISOdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3b60, 2hyd

3b60

2hyd


Resolution: 3.95→28.42 Å / Cor.coef. Fo:Fc: 0.708 / Cor.coef. Fo:Fc free: 0.644 / Cross valid method: THROUGHOUT / SU Rfree Blow DPI: 1.354
Details: 9 big cycles of 500 small cycles each. Refinement R values go up in the first cycles before going down and converging to a stable value in the later stages. Use of TLS refinement. Use of ...Details: 9 big cycles of 500 small cycles each. Refinement R values go up in the first cycles before going down and converging to a stable value in the later stages. Use of TLS refinement. Use of NCS, strong initially, only loosened in the last stages of refinement Data displaying very strong anisotropy.
RfactorNum. reflection% reflectionSelection details
Rfree0.321 1019 5 %RANDOM
Rwork0.26 ---
obs0.263 20388 92 %-
Displacement parametersBiso mean: 113.9 Å2
Baniso -1Baniso -2Baniso -3
1--1.5522 Å20 Å213.8816 Å2
2--15.746 Å20 Å2
3----14.1937 Å2
Refine analyzeLuzzati coordinate error obs: 0.79 Å
Refinement stepCycle: 1 / Resolution: 3.95→28.42 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms17552 0 128 0 17680
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.00917948HARMONIC2
X-RAY DIFFRACTIONt_angle_deg1.0924315HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d6379SINUSOIDAL2
X-RAY DIFFRACTIONt_incorr_chiral_ct
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_trig_c_planes
X-RAY DIFFRACTIONt_gen_planes3017HARMONIC5
X-RAY DIFFRACTIONt_it17948HARMONIC20
X-RAY DIFFRACTIONt_nbd8SEMIHARMONIC5
X-RAY DIFFRACTIONt_omega_torsion2.02
X-RAY DIFFRACTIONt_other_torsion24.29
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_chiral_improper_torsion2462SEMIHARMONIC5
X-RAY DIFFRACTIONt_sum_occupancies
X-RAY DIFFRACTIONt_utility_distance
X-RAY DIFFRACTIONt_utility_angle
X-RAY DIFFRACTIONt_utility_torsion
X-RAY DIFFRACTIONt_ideal_dist_contact20789SEMIHARMONIC4
LS refinement shellResolution: 3.95→4.17 Å / Total num. of bins used: 50
RfactorNum. reflection% reflection
Rfree0.2263 -5.39 %
Rwork0.2249 386 -
all0.225 408 -
obs--7.68 %
Refinement TLS params.

T12: -0.1432 Å2 / T23: -0.1603 Å2 / 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)T13 (Å2)T22 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
15.00182.750.33778.31552.92463.4011-0.0201-0.5740.53530.5551-0.1183-0.1766-0.55320.51270.13840.04740.00350.15080.017635.284724.21923.7964
23.57292.79710.96468.31552.08923.40760.01660.0982-0.52790.05330.25630.54810.5606-0.5396-0.27290.19710.1790.304-0.2632-39.9322-22.453353.7758
36.94512.8170.51647.71271.73785.41050.2449-0.2884-0.5334-0.3813-0.29870.52360.5551-0.56140.05380.0685-0.1653-0.14650.28618.0749-18.18178.5268
44.18472.37820.09986.44992.35176.2369-0.1052-0.57110.53260.5740.0041-0.5127-0.55590.46070.1011-0.0232-0.17040.10330.2858-13.106519.859869.8005
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1{ A|10 - A|323 B|9 - B|323 }
2X-RAY DIFFRACTION2{ C|10 - C|323 D|10 - D|323 }
3X-RAY DIFFRACTION3{ A|324 - A|701 B|324 - B|701 }
4X-RAY DIFFRACTION4{ C|324 - C|701 D|324 - D|701 }

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