8UR6

Cryo-EM reconstruction of Staphylococcus aureus oleate hydratase (OhyA) dimer with a disordered C-terminal membrane-association domain

Summary for 8UR6

• Entry DOI: 10.2210/pdb8ur6/pdb
• EMDB information: 42480 42484 42487
• Descriptor: Oleate hydratase (1 entity in total)
• Functional Keywords: oleate hydratase (ohya), phospholipids, membrane binding domain, amphipathic helices, interfacial enzyme, peripheral membrane protein, hydrolase
• Biological source: Staphylococcus aureus
• Total number of polymer chains: 2
• Total formula weight: 139785.44

Authors

Oldham, M.L.,Qayyum, M.Z. (deposition date: 2023-10-25, release date: 2024-01-10, Last modification date: 2024-02-14)

Primary citation

Radka, C.D.,Grace, C.R.,Hasdemir, H.S.,Li, Y.,Rodriguez, C.C.,Rodrigues, P.,Oldham, M.L.,Qayyum, M.Z.,Pitre, A.,MacCain, W.J.,Kalathur, R.C.,Tajkhorshid, E.,Rock, C.O.
The carboxy terminus causes interfacial assembly of oleate hydratase on a membrane bilayer.
J.Biol.Chem., 300:105627-105627, 2024

PubMed 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 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.

PubMed: 38211817
DOI: 10.1016/j.jbc.2024.105627

Experimental method

ELECTRON MICROSCOPY (3.03 Å)