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- PDB-4dk1: Crystal Structure of MacA-MexA chimeric protein, containing the P... -

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Basic information

Entry
Database: PDB / ID: 4dk1
TitleCrystal Structure of MacA-MexA chimeric protein, containing the Pseudomonas aeruginosa MexA alpha-hairpin domain.
ComponentsPutative MacA, Multidrug resistance protein mexA
KeywordsMEMBRANE PROTEIN / alpha-hairpin / lipoyl / beta-barrel domains / PERIPLASMIC PROTEIN
Function / homology
Function and homology information


xenobiotic transmembrane transporter activity / intrinsic component of plasma membrane / transmembrane transporter activity / protein homooligomerization / response to antibiotic / membrane / identical protein binding
Similarity search - Function
N-terminal domain of TfIIb - #140 / Helix hairpin bin / Efflux pump adaptor protein, beta barrel domain / Cation efflux system protein CusB domain 1 / Cation efflux system protein CusB, domain 1 / RND efflux pump, membrane fusion protein, barrel-sandwich domain / RND efflux pump, membrane fusion protein / Barrel-sandwich domain of CusB or HlyD membrane-fusion / N-terminal domain of TfIIb / RNA polymerase II/Efflux pump adaptor protein, barrel-sandwich hybrid domain ...N-terminal domain of TfIIb - #140 / Helix hairpin bin / Efflux pump adaptor protein, beta barrel domain / Cation efflux system protein CusB domain 1 / Cation efflux system protein CusB, domain 1 / RND efflux pump, membrane fusion protein, barrel-sandwich domain / RND efflux pump, membrane fusion protein / Barrel-sandwich domain of CusB or HlyD membrane-fusion / N-terminal domain of TfIIb / RNA polymerase II/Efflux pump adaptor protein, barrel-sandwich hybrid domain / Other non-globular / Prokaryotic membrane lipoprotein lipid attachment site profile. / Elongation Factor Tu (Ef-tu); domain 3 / Special / OB fold (Dihydrolipoamide Acetyltransferase, E2P) / Helix Hairpins / Beta Barrel / Orthogonal Bundle / Mainly Beta / Mainly Alpha
Similarity search - Domain/homology
Multidrug resistance protein MexA / Putative MacA
Similarity search - Component
Biological speciesAggregatibacter actinomycetemcomitans (bacteria)
Pseudomonas aeruginosa (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.499 Å
AuthorsXu, Y. / Ha, N.C.
CitationJournal: J Biol Chem / Year: 2012
Title: Assembly and channel opening of outer membrane protein in tripartite drug efflux pumps of Gram-negative bacteria.
Authors: Yongbin Xu / Arne Moeller / So-Young Jun / Minho Le / Bo-Young Yoon / Jin-Sik Kim / Kangseok Lee / Nam-Chul Ha /
Abstract: Gram-negative bacteria are capable of expelling diverse xenobiotic substances from within the cell by use of three-component efflux pumps in which the energy-activated inner membrane transporter is ...Gram-negative bacteria are capable of expelling diverse xenobiotic substances from within the cell by use of three-component efflux pumps in which the energy-activated inner membrane transporter is connected to the outer membrane channel protein via the membrane fusion protein. In this work, we describe the crystal structure of the membrane fusion protein MexA from the Pseudomonas aeruginosa MexAB-OprM pump in the hexameric ring arrangement. Electron microscopy study on the chimeric complex of MexA and the outer membrane protein OprM reveals that MexA makes a tip-to-tip interaction with OprM, which suggests a docking model for MexA and OprM. This docking model agrees well with genetic results and depicts detailed interactions. Opening of the OprM channel is accompanied by the simultaneous exposure of a protein structure resembling a six-bladed cogwheel, which intermeshes with the complementary cogwheel structure in the MexA hexamer. Taken together, we suggest an assembly and channel opening model for the MexAB-OprM pump. This study provides a better understanding of multidrug resistance in Gram-negative bacteria.
History
DepositionFeb 3, 2012Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Mar 7, 2012Provider: repository / Type: Initial release
Revision 1.1Dec 9, 2015Group: Database references
Revision 1.2Aug 23, 2017Group: Advisory / Refinement description / Source and taxonomy
Category: entity_src_gen / pdbx_unobs_or_zero_occ_atoms / software

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Putative MacA, Multidrug resistance protein mexA
B: Putative MacA, Multidrug resistance protein mexA
C: Putative MacA, Multidrug resistance protein mexA
D: Putative MacA, Multidrug resistance protein mexA


Theoretical massNumber of molelcules
Total (without water)147,3734
Polymers147,3734
Non-polymers00
Water0
1
A: Putative MacA, Multidrug resistance protein mexA
B: Putative MacA, Multidrug resistance protein mexA

A: Putative MacA, Multidrug resistance protein mexA
B: Putative MacA, Multidrug resistance protein mexA

A: Putative MacA, Multidrug resistance protein mexA
B: Putative MacA, Multidrug resistance protein mexA


Theoretical massNumber of molelcules
Total (without water)221,0606
Polymers221,0606
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_665-y+1,x-y+1,z1
crystal symmetry operation3_565-x+y,-x+1,z1
Buried area20570 Å2
ΔGint-81 kcal/mol
Surface area88850 Å2
MethodPISA
2
C: Putative MacA, Multidrug resistance protein mexA
D: Putative MacA, Multidrug resistance protein mexA

C: Putative MacA, Multidrug resistance protein mexA
D: Putative MacA, Multidrug resistance protein mexA

C: Putative MacA, Multidrug resistance protein mexA
D: Putative MacA, Multidrug resistance protein mexA


Theoretical massNumber of molelcules
Total (without water)221,0606
Polymers221,0606
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_665-y+1,x-y+1,z1
crystal symmetry operation3_565-x+y,-x+1,z1
Buried area20440 Å2
ΔGint-77 kcal/mol
Surface area89450 Å2
MethodPISA
Unit cell
γ
α
β
Length a, b, c (Å)151.708, 151.708, 216.953
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number150
Space group name H-MP321
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-ID
11
21
31
41

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection details
111CHAIN A AND (RESSEQ 42:74 OR RESSEQ 76:238 OR RESSEQ 242:255 OR RESSEQ 257:282 )
211CHAIN B AND (RESSEQ 42:74 OR RESSEQ 76:238 OR RESSEQ 242:255 OR RESSEQ 257:282 )
311CHAIN C AND (RESSEQ 42:74 OR RESSEQ 76:238 OR RESSEQ 242:255 OR RESSEQ 257:282 )
411CHAIN D AND (RESSEQ 42:74 OR RESSEQ 76:238 OR RESSEQ 242:255 OR RESSEQ 257:282 )

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Components

#1: Protein
Putative MacA, Multidrug resistance protein mexA


Mass: 36843.281 Da / Num. of mol.: 4 / Fragment: UNP RESIDUES 30-88, 95-158, 181-394
Source method: isolated from a genetically manipulated source
Details: The chimeric protein of MacA (RESIDUES 30-88), mexA (RESIDUES 95-158), and MacA (RESIDUES 181-394)
Source: (gene. exp.) Aggregatibacter actinomycetemcomitans (bacteria), (gene. exp.) Pseudomonas aeruginosa (bacteria)
Strain: PAO1 / Gene: mexA, PA0425 / Production host: Escherichia coli (E. coli) / References: UniProt: Q2EHL9, UniProt: P52477
Sequence detailsTHESE RESIDUES DERIVE FROM A VARIANT OF THE STRAIN OF THE BACTERIA

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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: 4.89 Å3/Da / Density % sol: 74.85 %
Crystal growTemperature: 287 K / Method: evaporation / pH: 7.5
Details: 0.16M CaCl2, 0.1M Hepes (pH 7.5) 23.5% PEG 400, EVAPORATION, temperature 287K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Photon Factory / Beamline: AR-NE3A / Wavelength: 1 Å
DetectorType: ADSC QUANTUM 270 / Detector: CCD / Date: Oct 10, 2011
RadiationMonochromator: Double mirror / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 3.499→20 Å / Num. all: 36819 / Num. obs: 33690 / % possible obs: 91.5 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0
Reflection shellResolution: 3.5→3.56 Å / % possible all: 95

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Processing

Software
NameVersionClassification
HKL-2000data collection
MOLREPphasing
PHENIX(phenix.refine: 1.7.2_869)refinement
HKL-2000data reduction
HKL-2000data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.499→19.951 Å / SU ML: 1.47 / σ(F): 1.68 / Phase error: 34.93 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.368 1709 5.08 %Random
Rwork0.3141 ---
all0.3168 33685 --
obs0.3168 33667 91.35 %-
Solvent computationShrinkage radii: 0.47 Å / VDW probe radii: 0.8 Å / Solvent model: FLAT BULK SOLVENT MODEL / Bsol: 0 Å2 / ksol: 0.129 e/Å3
Displacement parameters
Baniso -1Baniso -2Baniso -3
1--11.013 Å20 Å20 Å2
2---11.013 Å2-0 Å2
3---22.026 Å2
Refinement stepCycle: LAST / Resolution: 3.499→19.951 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8568 0 0 0 8568
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0258652
X-RAY DIFFRACTIONf_angle_d2.97311739
X-RAY DIFFRACTIONf_dihedral_angle_d26.0383219
X-RAY DIFFRACTIONf_chiral_restr0.2071442
X-RAY DIFFRACTIONf_plane_restr0.0151514
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDTypeRms dev position (Å)
11A1802X-RAY DIFFRACTIONPOSITIONAL
12B1802X-RAY DIFFRACTIONPOSITIONAL0.119
13C1802X-RAY DIFFRACTIONPOSITIONAL0.11
14D1802X-RAY DIFFRACTIONPOSITIONAL0.111
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 12

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
3.4989-3.60130.41721400.4286269394
3.6013-3.71680.43621530.4087282198
3.7168-3.84870.36711490.3552270995
3.8487-4.00170.36511330.3183236481
4.0017-4.18220.38731130.3079228379
4.1822-4.40050.38311420.2786258790
4.4005-4.67280.35551410.283247485
4.6728-5.02830.30251540.2503263891
5.0283-5.52450.35211470.2687268892
5.5245-6.30180.33771400.2819281895
6.3018-7.85810.34611600.2686285097
7.8581-19.95110.37741370.3261303398
Refinement TLS params.Method: refined / Origin x: -21.0638 Å / Origin y: 78.0611 Å / Origin z: 137.541 Å
111213212223313233
T-0.0137 Å2-0.0182 Å2-0.0663 Å2-0.0058 Å20.0701 Å2---0.044 Å2
L0.0049 °2-0.0025 °2-0.0265 °2-0.0152 °20.0089 °2--0.0229 °2
S0.0273 Å °0.2699 Å °0.0049 Å °-0.2777 Å °-0.0144 Å °0.0799 Å °0.0036 Å °-0.0131 Å °-0.003 Å °
Refinement TLS groupSelection details: ALL

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