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An inducible amphipathic α-helix mediates subcellular targeting and membrane binding of RPE65

Sheetal Uppal1, Tingting Liu1, Emily Galvan1

  • 1Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD, USA.

Life Science Alliance
|October 20, 2022
PubMed

Insights

Retinal pigment epithelium protein 65 (RPE65) binds membranes via a newly identified amphipathic alpha-helix (AH). This AH, formed by residues 107-125, is crucial for RPE65 function in the visual cycle.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Vision Science

Background:

  • Retinal pigment epithelium protein 65 (RPE65) is essential for the visual cycle, facilitating retinol isomerization.
  • Membrane association is critical for RPE65 function, but the underlying mechanism remains unclear.
  • Specific residues (107-125) and palmitoylation at C112 are implicated in membrane interaction.

Purpose of the Study:

  • To elucidate the mechanism of membrane recognition and binding by RPE65.
  • To investigate the role of residues 107-125 and palmitoylation in RPE65 membrane association.
  • To establish a working model for RPE65-membrane interaction.

Main Methods:

  • Synthesis and analysis of peptides corresponding to RPE65 residues 107-125.
  • Circular dichroism spectroscopy to study peptide structure transitions.
  • Assays to determine palmitoylation levels, membrane association, and isomerization activity.
  • Molecular dynamic simulations of peptide-membrane interactions.

Main Results:

  • The RPE65 peptide (aa107-125) transitions from an unstructured loop to an amphipathic alpha-helix (AH) upon binding to membrane-mimicking surfaces.
  • This AH transition is spontaneous in the C112-palmitoylated peptide.
  • The AH structure significantly influences RPE65 palmitoylation, membrane binding, and enzymatic activity.
  • Molecular dynamics simulations confirmed AH insertion into the membrane.

Conclusions:

  • Residues 107-125 act as a membrane sensor, forming an AH that serves as a membrane-targeting motif for RPE65.
  • Cysteine palmitoylation at C112 plays a novel role in modulating RPE65's membrane interaction and function.
  • A new model for RPE65-membrane binding is proposed, highlighting the importance of the AH and palmitoylation.

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