The functional significance of pathogenic variants in RPE65^107-125 amphipathic helix in RPE65-membrane recognition and binding

Abstract

Purpose : RPE65 retinoid isomerase plays an indispensable role in the regeneration of 11-cis retinal visual chromophore essential for vision. Despite extensive knowledge of the physiological role of RPE65, we know less about how RPE65 interacts with membranes. While association with the smooth endoplasmic reticulum (sER) membrane is crucial for catalytic function, RPE65 lacks a defined membrane insertion domain. We recently showed that aa107-125 of RPE65 (RPE65^107-125), which is unresolved crystallographically, mediates association with the ER. We wish to elucidate how pathogenic variants in RPE65^107-125 affect RPE65-membrane binding.

Methods : Human pathogenic mutations of RPE65^107-125 were identified from LOVD. Circular dichroism (CD) spectroscopy and gel filtration chromatography were performed on synthetic peptides representing wild-type (WT) and pathogenic variants of RPE65^107-125. Also, we generated WT and pathogenic variant GFP-RPE65^107-125 fusion protein constructs for immunofluorescence studies to assess RPE65-membrane targeting. We evaluated the expression and isomerase activity of RPE65 WT and mutant expression constructs.

Results : Binding of the synthetic aa107-125 peptide to membrane-mimicking surfaces induces transition from a random loop structure to an amphipathic α-helix (AH), suggesting that it functions as a membrane sensor and as a membrane-targeting motif. This AH is capable of redirecting GFP, typically localizing to the nucleus, to the ER. Notably, many of the pathogenic mutations in AH^107-125 impair the localization of GFP-AH fusions to the ER. Furthermore, the pathogenic mutations in AH^107-125 significantly affect the expression level, membrane association, and isomerase activity of RPE65, highlighting the essential role of AH^107-125 in RPE65 function.

Conclusions : We have proposed a working model for RPE65-ER membrane binding: in the presence of membranes, aa107-125 undergoes a disorder-to-order transition, forming an AH that facilitates ER binding. Palmitoylation of C112 accentuates this transition, “locking” the AH in place, a phenomenon not seen in other proteins to date. However, we show here that many of the pathogenic mutations in RPE65^107-125 disrupt this transition, thereby impeding RPE65's membrane binding and its ability to access its substrate, providing a rationale for their deleterious action.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.