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- PDB-7nre: Crystal structure of E.coli BamA beta-barrel in complex with daro... -

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Basic information

Entry
Database: PDB / ID: 7nre
TitleCrystal structure of E.coli BamA beta-barrel in complex with darobactin (crystal form 1)
Components
  • Darobactin
  • Outer membrane protein assembly factor BamA
KeywordsMEMBRANE PROTEIN / Beta-Barrel / outer membrane / protein insertion / protein folding / protein maturation / antibiotic / natural product / cyclized peptide
Function / homology
Function and homology information


Bam protein complex / Gram-negative-bacterium-type cell outer membrane assembly / protein insertion into membrane
Similarity search - Function
Outer membrane protein assembly factor BamA / POTRA domain, BamA/TamA-like / Surface antigen variable number repeat / POTRA domain / POTRA domain profile. / Surface antigen D15-like / Bacterial surface antigen (D15) / Omp85 superfamily domain
Similarity search - Domain/homology
Darobactin / Outer membrane protein assembly factor BamA
Similarity search - Component
Biological speciesEscherichia coli O157:H7 (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.3 Å
AuthorsJakob, R.P. / Kaur, H. / Marzinek, J.K. / Green, R. / Imai, Y. / Bolla, J. / Robinson, C. / Bond, P.J. / Lewis, K. / Maier, T. / Hiller, S.
CitationJournal: Nature / Year: 2021
Title: The antibiotic darobactin mimics a β-strand to inhibit outer membrane insertase.
Authors: Hundeep Kaur / Roman P Jakob / Jan K Marzinek / Robert Green / Yu Imai / Jani Reddy Bolla / Elia Agustoni / Carol V Robinson / Peter J Bond / Kim Lewis / Timm Maier / Sebastian Hiller /
Abstract: Antibiotics that target Gram-negative bacteria in new ways are needed to resolve the antimicrobial resistance crisis. Gram-negative bacteria are protected by an additional outer membrane, rendering ...Antibiotics that target Gram-negative bacteria in new ways are needed to resolve the antimicrobial resistance crisis. Gram-negative bacteria are protected by an additional outer membrane, rendering proteins on the cell surface attractive drug targets. The natural compound darobactin targets the bacterial insertase BamA-the central unit of the essential BAM complex, which facilitates the folding and insertion of outer membrane proteins. BamA lacks a typical catalytic centre, and it is not obvious how a small molecule such as darobactin might inhibit its function. Here we resolve the mode of action of darobactin at the atomic level using a combination of cryo-electron microscopy, X-ray crystallography, native mass spectrometry, in vivo experiments and molecular dynamics simulations. Two cyclizations pre-organize the darobactin peptide in a rigid β-strand conformation. This creates a mimic of the recognition signal of native substrates with a superior ability to bind to the lateral gate of BamA. Upon binding, darobactin replaces a lipid molecule from the lateral gate to use the membrane environment as an extended binding pocket. Because the interaction between darobactin and BamA is largely mediated by backbone contacts, it is particularly robust against potential resistance mutations. Our results identify the lateral gate as a functional hotspot in BamA and will allow the rational design of antibiotics that target this bacterial Achilles heel.
History
DepositionMar 3, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 21, 2021Provider: repository / Type: Initial release
Revision 1.1Apr 28, 2021Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2May 19, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Jan 31, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_leaving_atom_flag

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Outer membrane protein assembly factor BamA
C: Darobactin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)48,2688
Polymers46,9942
Non-polymers1,2746
Water3,621201
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2310 Å2
ΔGint-7 kcal/mol
Surface area18920 Å2
Unit cell
Length a, b, c (Å)69.995, 82.563, 94.305
Angle α, β, γ (deg.)90.000, 108.640, 90.000
Int Tables number5
Space group name H-MI121
Components on special symmetry positions
IDModelComponents
11A-1183-

HOH

21A-1265-

HOH

31A-1295-

HOH

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Components

#1: Protein Outer membrane protein assembly factor BamA


Mass: 46022.789 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli O157:H7 (bacteria) / Gene: bamA, yaeT, Z0188, ECs0179 / Plasmid: pET15a / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A942
#2: Protein/peptide Darobactin


Type: Peptide-like / Class: Antibiotic / Mass: 971.047 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) / References: Darobactin
#3: Chemical
ChemComp-C8E / (HYDROXYETHYLOXY)TRI(ETHYLOXY)OCTANE


Mass: 306.438 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C16H34O5 / Comment: C8E, detergent*YM
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 201 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Nonpolymer detailsOriginal Refinement was done with Darobactin defined as one ligand.

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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: 2.75 Å3/Da / Density % sol: 55.23 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop
Details: 0.06 M Magnesium chloride/Calcium Chloride, 0.1 M imidazole/2-(N-morpholino)ethanesulfonic acid pH 6.5, 12.5% v/v 2-Methyl-2,4-pentanediol, 12.5% w/v Polyethylene glycol 1,000, and 12.5% w/v ...Details: 0.06 M Magnesium chloride/Calcium Chloride, 0.1 M imidazole/2-(N-morpholino)ethanesulfonic acid pH 6.5, 12.5% v/v 2-Methyl-2,4-pentanediol, 12.5% w/v Polyethylene glycol 1,000, and 12.5% w/v Polyethylene glycol 3,350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06SA / Wavelength: 1.000009 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Jul 19, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.000009 Å / Relative weight: 1
ReflectionResolution: 2.3→46.602 Å / Num. obs: 22630 / % possible obs: 99.7 % / Redundancy: 11.7 % / CC1/2: 0.997 / Rmerge(I) obs: 0.156 / Rpim(I) all: 0.072 / Rrim(I) all: 0.172 / Net I/σ(I): 7.1
Reflection shellResolution: 2.3→2.38 Å / Redundancy: 8.7 % / Rmerge(I) obs: 1.456 / Mean I/σ(I) obs: 1.3 / Num. unique obs: 2230 / CC1/2: 0.535 / Rpim(I) all: 0.66 / Rrim(I) all: 1.602 / % possible all: 98.8

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Processing

Software
NameVersionClassification
PHENIX1.13_2998refinement
PDB_EXTRACT3.25data extraction
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6QGW

6qgw


Resolution: 2.3→46.602 Å / SU ML: 0.33 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 25.92
RfactorNum. reflection% reflection
Rfree0.2315 1129 4.99 %
Rwork0.2045 --
obs0.2059 22630 99.74 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 158.33 Å2 / Biso mean: 49.6919 Å2 / Biso min: 19.08 Å2
Refinement stepCycle: final / Resolution: 2.3→46.602 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2916 0 258 201 3375
Biso mean--55.29 47.9 -
Num. residues----369
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.3001-2.40480.36421410.2868265699
2.4048-2.53160.29531410.24932661100
2.5316-2.69020.22821460.21862669100
2.6902-2.89780.25811450.20142694100
2.8978-3.18940.22761370.19842686100
3.1894-3.65080.20981420.17762662100
3.6508-4.59890.18651450.1742725100
4.5989-46.6020.24731320.2232748100
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)
10.9110.56380.34470.35050.20360.307-0.20520.06460.44060.23020.23010.3303-0.35870.15080.03270.3504-0.03640.01090.3841-0.0170.346830.4876-0.72133.6636
20.6228-0.41010.22840.3656-0.32080.357-0.01710.02440.0705-0.0040.0394-0.09950.03670.10020.00170.28610.01260.02250.24270.00810.270817.439-12.99770.1423
30.3390.03620.09540.00170.02960.0306-0.0858-0.07850.0018-0.0376-0.010.0764-0.0538-0.0762-0.00130.25010.0364-0.00320.2879-0.01560.238115.8597-15.08914.6153
41.16410.19820.05970.24910.35351.19920.0062-0.2498-0.05480.10170.0631-0.05630.04370.07230.00210.23660.03110.02220.31240.03230.26327.5427-18.862320.8515
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 423 through 446 )A423 - 446
2X-RAY DIFFRACTION2chain 'A' and (resid 447 through 563 )A447 - 563
3X-RAY DIFFRACTION3chain 'A' and (resid 564 through 675 )A564 - 675
4X-RAY DIFFRACTION4chain 'A' and (resid 676 through 810 )A676 - 810

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