Abstract

The soluble flavoprotein oleate hydratase (OhyA) hydrates the 9-cis double bond of unsaturated fatty acids. OhyA sub- strates 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 phosphatidylgly- cerol (PG) bilayer. Super-resolution microscopy of Staphylo- coccus 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 2 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 sur- face to obtain bilayer-embedded unsaturated fatty acids.

Document Type

Article

Publication Date

2024

Notes/Citation Information

© 2024 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Digital Object Identifier (DOI)

https://doi.org/10.1016/j.jbc.2024.105627

Funding Information

This work was supported by National Institutes of Health Grants R01-GM034496 (C. O. R.), R00-AI166116 (C. D. R.), R01-GM123455, P41-GM104601, and R24-GM145965 (E. T.), Cancer Center Support Grant CA21765, and the American Lebanese Syrian Associated Charities. The sim- ulations reported in this study are supported by XSEDE allocation (grant MCA06N060 to E. T.), Microsoft Azure, and Blue Waters at the National Center for Supercomputing Application (NCSA) at the University of Illinois at Urbana-Champaign. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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