Abstract
Purpose :
RPE65 association with RPE smooth endoplasmic reticulum (sER) is required to regenerate 11-cis retinal. How RPE65 binds to sER is unclear as it lacks transmembrane motifs, but a crystallographically unresolved “missing loop” (ML) at residues 107-125 is proposed to be involved. This contains a highly conserved PDPCK motif. Our previous studies reveal that C112 in the missing loop region is palmitoylated and partly important for RPE65-membrane binding and visual cycle function. To elucidate the mechanism of RPE65-membrane binding, we targeted the functional significance of the crystallographically unresolved region in RPE65 membrane binding.
Methods :
Secondary structure analyses of the ML were studied using web-based servers (I-TASSER and HELIQUEST). Circular dichroism (CD) spectroscopy and gel filtration chromatography (GFC) was performed using synthethic peptides corresponding to the ML of RPE65. Extensive mutational analyses, palmitoylation detection, subcellular fractionation, and isomerase assays were done. We made a GFP-RPE65107-125 construct for immunofluorescence (IF) studies. We predicted the orientation of RPE65 containing the modelled ML and employed molecular dynamic (MD) simulations to study binding.
Results :
In silico analysis of the ML predicts this region to form an amphipathic alpha (α)-helical (AH) structure. CD and GFC analyses of unpalmitoylated synthetic peptide confirmed the capacity of the disordered loop to fold into α-helices upon association with lipid-like molecules, while the palmitoylated synthetic peptide was always α-helical. Mutagenesis revealed that most of the residues forming the AH significantly affect the palmitoylation level of RPE65 and membrane association of RPE65. Moreover, we found that mutation of almost all residues in the hydrophobic face and some charged/polar residues in the hydrophilic face have severe (>50% reduction) to total loss of RPE65 isomerase activity. IF studies reveal that this sequence functions as a membrane sensor and a membrane targeting motif. MD simulations clearly show AH-membrane insertion, strongly validating our experimental findings.
Conclusions :
We conclude that the AH plays a major role in the regulation of RPE65 palmitoylation and sER membrane interaction and, thus, the catalytic function of RPE65. Taking all these findings into consideration, we propose a hypothetical working model for RPE65-membrane binding.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.