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- PDB-8aq2: In meso structure of the membrane integral lipoprotein N-acyltran... -

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Basic information

Entry
Database: PDB / ID: 8aq2
TitleIn meso structure of the membrane integral lipoprotein N-acyltransferase Lnt from P. aeruginosa covalently linked with TITC
ComponentsApolipoprotein N-acyltransferase
KeywordsTRANSFERASE / Lnt / apolipoprotein N-acyltransferase / Bacterial lipoproteins.
Function / homology
Function and homology information


apolipoprotein N-acyltransferase / N-acyltransferase activity / lipoprotein biosynthetic process / membrane => GO:0016020 / plasma membrane
Similarity search - Function
Apolipoprotein N-acyltransferase / Carbon-nitrogen hydrolase superfamily / Carbon-nitrogen hydrolase / Carbon-nitrogen hydrolase domain profile. / Carbon-nitrogen hydrolase
Similarity search - Domain/homology
CITRATE ANION / (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate / ~{N}-tridecylmethanethioamide / Apolipoprotein N-acyltransferase
Similarity search - Component
Biological speciesPseudomonas aeruginosa PAO1 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.6 Å
AuthorsHuang, C.-Y. / Weichert, D. / Boland, C. / Smithers, L. / Olieric, V. / Wang, M. / Caffrey, M.
Funding support Ireland, 2items
OrganizationGrant numberCountry
Science Foundation Ireland16/IA/4435 Ireland
Irish Research CouncilGOIPD/2021/40 Ireland
CitationJournal: Sci Adv / Year: 2023
Title: Structure snapshots reveal the mechanism of a bacterial membrane lipoprotein -acyltransferase.
Authors: Luke Smithers / Oksana Degtjarik / Dietmar Weichert / Chia-Ying Huang / Coilín Boland / Katherine Bowen / Abraham Oluwole / Corinne Lutomski / Carol V Robinson / Eoin M Scanlan / Meitian ...Authors: Luke Smithers / Oksana Degtjarik / Dietmar Weichert / Chia-Ying Huang / Coilín Boland / Katherine Bowen / Abraham Oluwole / Corinne Lutomski / Carol V Robinson / Eoin M Scanlan / Meitian Wang / Vincent Olieric / Moran Shalev-Benami / Martin Caffrey /
Abstract: Bacterial lipoproteins (BLPs) decorate the surface of membranes in the cell envelope. They function in membrane assembly and stability, as enzymes, and in transport. The final enzyme in the BLP ...Bacterial lipoproteins (BLPs) decorate the surface of membranes in the cell envelope. They function in membrane assembly and stability, as enzymes, and in transport. The final enzyme in the BLP synthesis pathway is the apolipoprotein -acyltransferase, Lnt, which is proposed to act by a ping-pong mechanism. Here, we use x-ray crystallography and cryo-electron microscopy to chart the structural changes undergone during the progress of the enzyme through the reaction. We identify a single active site that has evolved to bind, individually and sequentially, substrates that satisfy structural and chemical criteria to position reactive parts next to the catalytic triad for reaction. This study validates the ping-pong mechanism, explains the molecular bases for Lnt's substrate promiscuity, and should facilitate the design of antibiotics with minimal off-target effects.
History
DepositionAug 11, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 12, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 7, 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

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Assembly

Deposited unit
A: Apolipoprotein N-acyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)63,11721
Polymers58,2301
Non-polymers4,88620
Water54030
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1640 Å2
ΔGint-6 kcal/mol
Surface area21940 Å2
MethodPISA
Unit cell
Length a, b, c (Å)78.060, 116.230, 73.220
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number18
Space group name H-MP21212

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Apolipoprotein N-acyltransferase / ALP N-acyltransferase / Copper homeostasis protein CutE homolog


Mass: 58230.461 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa PAO1 (bacteria)
Strain: ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1
Gene: lnt, cutE, PA3984 / Production host: Escherichia coli (E. coli)
References: UniProt: Q9ZI86, Transferases; Acyltransferases; Transferring groups other than aminoacyl groups

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Non-polymers , 5 types, 50 molecules

#2: Chemical
ChemComp-OLC / (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate / 1-Oleoyl-R-glycerol


Mass: 356.540 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C21H40O4
#3: Chemical ChemComp-QGT / ~{N}-tridecylmethanethioamide / Tetradecyl-1-isothiocyanate (reacted form) / TITC (reacted form)


Mass: 243.452 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C14H29NS / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-FLC / CITRATE ANION


Mass: 189.100 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H5O7
#5: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C3H8O3
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 30 / Source method: isolated from a natural source / Formula: H2O

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Details

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: 2.85 Å3/Da / Density % sol: 56.87 %
Crystal growTemperature: 293 K / Method: lipidic cubic phase
Details: 30 %(v/v) PEG-500 DME, 0.1 M sodium citrate pH 5.0 and 0.1 M sodium acetate

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: Y
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06SA / Wavelength: 1 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Mar 27, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.6→48.526 Å / Num. obs: 21051 / % possible obs: 99.7 % / Redundancy: 14.63 % / CC1/2: 0.99 / Rrim(I) all: 0.35 / Net I/σ(I): 6.93
Reflection shellResolution: 2.6→2.7 Å / Redundancy: 14.59 % / Mean I/σ(I) obs: 0.85 / Num. unique obs: 2200 / CC1/2: 0.35 / Rrim(I) all: 6.29 / % possible all: 99.7
Serial crystallography sample deliveryMethod: fixed target
Serial crystallography sample delivery fixed targetDescription: IMISX method / Sample dehydration prevention: COC film sandwitch / Sample holding: IMISX method/COC film / Support base: standard goniometer base

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Processing

Software
NameVersionClassification
XSCALEdata scaling
PHENIXdev_3494refinement
PDB_EXTRACT3.25data extraction
XDSdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5N6M

5n6m


Resolution: 2.6→48.526 Å / SU ML: 0.42 / Cross valid method: THROUGHOUT / σ(F): 1.33 / Phase error: 30.08 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2571 1054 5.01 %
Rwork0.2278 19979 -
obs0.2292 21033 99.6 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 191.66 Å2 / Biso mean: 73.4173 Å2 / Biso min: 30.74 Å2
Refinement stepCycle: final / Resolution: 2.6→48.526 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4009 0 294 30 4333
Biso mean--92 69.05 -
Num. residues----520
LS refinement shell

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

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.6-2.71820.37021360.3434242099
2.7182-2.86150.35771260.31732466100
2.8615-3.04080.30851280.29652464100
3.0408-3.27550.31841320.26732462100
3.2755-3.6050.28581240.2366249499
3.605-4.12640.2541380.20522493100
4.1264-5.19790.22111290.19152532100
5.1979-48.5260.21811410.212648100
Refinement TLS params.Method: refined / Origin x: -10.1329 Å / Origin y: 19.4728 Å / Origin z: -16.6794 Å
111213212223313233
T0.3645 Å20.021 Å2-0.0129 Å2-0.3386 Å2-0.0157 Å2--0.4078 Å2
L1.7644 °20.4885 °2-0.5021 °2-1.0389 °2-0.4354 °2--2.2988 °2
S-0.1705 Å °0.3129 Å °0.0677 Å °-0.1908 Å °0.113 Å °-0.0117 Å °0.1138 Å °-0.2235 Å °0.0589 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allA-10 - 509
2X-RAY DIFFRACTION1allB2 - 22
3X-RAY DIFFRACTION1allS1 - 44

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