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- PDB-8um1: Structure of the Carboxy terminus of Oleate Hydratase -

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Basic information

Entry
Database: PDB / ID: 8um1
TitleStructure of the Carboxy terminus of Oleate Hydratase
ComponentsMyosin-cross-reactive antigen
KeywordsLIPID TRANSPORT / Oleate Hydratase
Function / homologyoleate hydratase activity / Oleate hydratase / MCRA family / FAD binding / fatty acid metabolic process / FAD/NAD(P)-binding domain superfamily / Myosin-cross-reactive antigen
Function and homology information
Biological speciesStaphylococcus aureus (bacteria)
MethodSOLUTION NMR / simulated annealing
AuthorsGrace, C.R. / Radka, C.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)GM034496, AI166116,GM123455 United States
CitationJournal: J Biol Chem / Year: 2024
Title: The carboxy terminus causes interfacial assembly of oleate hydratase on a membrane bilayer.
Authors: Christopher D Radka / Christy R Grace / Hale S Hasdemir / Yupeng Li / Carlos C Rodriguez / Patrick Rodrigues / Michael L Oldham / M Zuhaib Qayyum / Aaron Pitre / William J MacCain / Ravi C ...Authors: Christopher D Radka / Christy R Grace / Hale S Hasdemir / Yupeng Li / Carlos C Rodriguez / Patrick Rodrigues / Michael L Oldham / M Zuhaib Qayyum / Aaron Pitre / William J MacCain / Ravi C Kalathur / Emad Tajkhorshid / Charles O Rock /
Abstract: The soluble flavoprotein oleate hydratase (OhyA) hydrates the 9-cis double bond of unsaturated fatty acids. OhyA substrates are embedded in membrane bilayers; OhyA must remove the fatty acid from the ...The soluble flavoprotein oleate hydratase (OhyA) hydrates the 9-cis double bond of unsaturated fatty acids. OhyA substrates are embedded in membrane bilayers; OhyA must remove the fatty acid from the bilayer and enclose it in the active site. Here, we show that the positively charged helix-turn-helix motif in the carboxy terminus (CTD) is responsible for interacting with the negatively charged phosphatidylglycerol (PG) bilayer. Super-resolution microscopy of Staphylococcus aureus cells expressing green fluorescent protein fused to OhyA or the CTD sequence shows subcellular localization along the cellular boundary, indicating OhyA is membrane-associated and the CTD sequence is sufficient for membrane recruitment. Using cryo-electron microscopy, we solved the OhyA dimer structure and conducted 3D variability analysis of the reconstructions to assess CTD flexibility. Our surface plasmon resonance experiments corroborated that OhyA binds the PG bilayer with nanomolar affinity and we found the CTD sequence has intrinsic PG binding properties. We determined that the nuclear magnetic resonance structure of a peptide containing the CTD sequence resembles the OhyA crystal structure. We observed intermolecular NOE from PG liposome protons next to the phosphate group to the CTD peptide. The addition of paramagnetic MnCl indicated the CTD peptide binds the PG surface but does not insert into the bilayer. Molecular dynamics simulations, supported by site-directed mutagenesis experiments, identify key residues in the helix-turn-helix that drive membrane association. The data show that the OhyA CTD binds the phosphate layer of the PG surface to obtain bilayer-embedded unsaturated fatty acids.
History
DepositionOct 17, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 31, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 21, 2024Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.2May 15, 2024Group: Database references / Category: database_2 / Item: _database_2.pdbx_DOI

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Myosin-cross-reactive antigen


Theoretical massNumber of molelcules
Total (without water)4,9071
Polymers4,9071
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: NMR Distance Restraints, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 100target function
RepresentativeModel #1lowest energy

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Components

#1: Protein/peptide Myosin-cross-reactive antigen


Mass: 4906.807 Da / Num. of mol.: 1 / Fragment: resdidues 500-591 / Source method: obtained synthetically / Source: (synth.) Staphylococcus aureus (bacteria) / References: UniProt: A0A7H9C8C7

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic12D 1H-1H COSY
121isotropic12D 1H-1H NOESY
131isotropic12D 1H-1H TOCSY
141isotropic12D 1H-13C HSQC
151isotropic12D 1H-15N HSQC

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Sample preparation

DetailsType: solution
Contents: 1 mM C-terminus of Oleate Hydratase, 20 mM TRIS, 150 mM sodium chloride, 90% H2O/10% D2O
Label: Unlabeled_sample / Solvent system: 90% H2O/10% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
1 mMC-terminus of Oleate Hydratasenatural abundance1
20 mMTRISnatural abundance1
150 mMsodium chloridenatural abundance1
Sample conditionsIonic strength: 150 mM / Label: conditions_1 / pH: 6.0 / Pressure: 1 atm / Temperature: 303 K

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NMR measurement

NMR spectrometerType: Bruker AVANCE NEO / Manufacturer: Bruker / Model: AVANCE NEO / Field strength: 600 MHz

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Processing

NMR software
NameDeveloperClassification
NMRFAM-SPARKYWoonghee Leechemical shift assignment
CNSBrunger, Adams, Clore, Gros, Nilges and Readstructure calculation
CNSBrunger, Adams, Clore, Gros, Nilges and Readrefinement
NMRFAM-SPARKYWoonghee Leepeak picking
RefinementMethod: simulated annealing / Software ordinal: 3
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: target function / Conformers calculated total number: 100 / Conformers submitted total number: 20

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