Abstract
Purpose :
Interaction with the membrane is a critical requirement for RPE65's catalytic activity, even though it lacks any hydrophobic transmembrane domain. Despite detailed understanding of its physiological function, the structural characterization of RPE65-membrane interaction is still quite limited. A loop region consisting of residues 107-125, is apparently disordered and unresolved in all crystal structures of RPE65. This missing loop region is thought to interact with the membrane. We have confirmed recently that palmitoylation at C112 residue is important for the RPE65-membrane association. To gain more insight into the mechanism of the RPE65 membrane association, we used in silico and in vitro biochemical approaches to determine the functional significance of this missing loop.
Methods :
Secondary structure prediction and sequence composition analysis were performed using web-based servers. Extensive mutational analysis was performed and various biochemical methods, such as palmitoylation detection assay, subcellular fractionation, and isomerase activity in non-RPE minimal visual cycle system, were used to study the role of the aa107-125 residues on RPE65-membrane association and catalytic function.
Results :
Our in silico results reveal that this segment can form a strongly amphipathic alpha-helical structure. The palmitoylated C112 residue is found in the hydrophobic face surrounded by hydrophobic residues. In vitro mutational analysis revealed that most of the residues in the amphipathic helix significantly affect the palmitoylation level and membrane association of RPE65. Moreover, mutation of almost all residues in the hydrophobic face and of some charged or polar residues in the hydrophilic face have severe (>50% reduction) or total loss of RPE65 isomerase activity. In addition to C112 mutation, our results show that R118 mutation also significantly affects the membrane association and isomerase activity of RPE65.
Conclusions :
We conclude that the amphipathic helix might play a role in the regulation of RPE65 palmitoylation and membrane interaction and, thus, the function of RPE65. Taking all the findings into consideration, we hypothesize that the amphipathic helix (residues 107-125) may act as an intrinsic membrane-binding motif and that palmitoylation of C112 stabilizes the affinity of RPE65 with the membrane. This needs further investigation in detail, currently ongoing in the laboratory.
This is a 2020 ARVO Annual Meeting abstract.