PT  - JOURNAL ARTICLE
AU  - Sheetal Uppal
AU  - Tingting Liu
AU  - Emily Galvan
AU  - Fatima Gomez
AU  - Tishina Tittley
AU  - Eugenia Poliakov
AU  - Susan Gentleman
AU  - T Michael Redmond
TI  - An inducible amphipathic α-helix mediates subcellular targeting and membrane binding of RPE65
AID  - 10.26508/lsa.202201546
DP  - 2023 Jan 01
TA  - Life Science Alliance
PG  - e202201546
VI  - 6
IP  - 1
4099  - https://www.life-science-alliance.org/content/6/1/e202201546.short
4100  - https://www.life-science-alliance.org/content/6/1/e202201546.full
SO  - Life Sci. Alliance2023 Jan 01; 6
AB  - RPE65 retinol isomerase is an indispensable player in the visual cycle between the vertebrate retina and RPE. Although membrane association is critical for RPE65 function, its mechanism is not clear. Residues 107–125 are believed to interact with membranes but are unresolved in all RPE65 crystal structures, whereas palmitoylation at C112 also plays a role. We report the mechanism of membrane recognition and binding by RPE65. Binding of aa107–125 synthetic peptide with membrane-mimicking micellar surfaces induces transition from unstructured loop to amphipathic α-helical (AH) structure but this transition is automatic in the C112-palmitoylated peptide. We demonstrate that the AH significantly affects palmitoylation level, membrane association, and isomerization activity of RPE65. Furthermore, aa107–125 functions as a membrane sensor and the AH as a membrane-targeting motif. Molecular dynamic simulations clearly show AH-membrane insertion, supporting our experimental findings. Collectively, these studies allow us to propose a working model for RPE65-membrane binding, and to provide a novel role for cysteine palmitoylation.