An inducible amphipathic α-helix mediates subcellular targeting and membrane binding of RPE65.
An inducible amphipathic α-helix mediates subcellular targeting and membrane binding of RPE65.
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DOI：
10.26508/lsa.202201546
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发表时间：
2023-01
影响因子：
4.4
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中科院分区：
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RPE 65的氨基酸残基107-125与C112处的棕榈酰基团一起形成膜感测两亲性螺旋，其将RPE 65靶向至平滑内质网以获得其底物。RPE 65视黄醇异构酶是脊椎动物视网膜和RPE之间的视觉循环中不可或缺的参与者。尽管膜缔合对于RPE 65的功能至关重要，但其机制尚不清楚。残基107-125被认为与膜相互作用，但在所有RPE 65晶体结构中均未解析，而C112处的棕榈酰化也起作用。我们报告了RPE 65的膜识别和结合机制。aa 107 -125合成肽与膜模拟胶束表面的结合诱导从非结构化环到两亲性α-螺旋（AH）结构的转变，但这种转变在C112-棕榈酰化肽中是自动的。我们证明，AH显着影响棕榈酰化水平，膜协会，和异构化活性的RPE 65。此外，aa 107 -125作为膜传感器和AH作为膜靶向基序。分子动力学模拟清楚地表明AH-膜插入，支持我们的实验结果。总的来说，这些研究使我们能够提出RPE 65膜结合的工作模型，并提供半胱氨酸棕榈酰化的新作用。
Amino acid residues 107–125 of RPE65, in concert with a palmitoyl group at C112, form a membrane-sensing amphipathic helix that targets RPE65 to smooth endoplasmic reticulum to acquire its substrate. 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.