Photoreceptor outer segment disk rim curvature relies on a tetraspanin interaction web

Abstract Rod and cone photoreceptors are sensory neurons required for the first steps of image-forming vision. Mutations in PRPH2 damage photoreceptor viability and produce a broad range of inherited retinal degenerations (IRDs) for which no treatments are available. The gene product, PRPH2 (peripherin-2), is an integral membrane tetraspanin that is required for the biogenesis and normal structure of photoreceptor outer segment (OS) disks. Peripherin-2 and its homologous partner Rom1 (rom1) self-assemble into non-covalent dimers that organize into three parallel belts that encircle each disk, forming a supramolecular scaffold that imposes extreme curvature on the disk rim. The mechanism by which curvature is generated and maintained is not yet understood. Here, we report the development of blue native (BN)-PAGE and molecular dynamics (MD) simulation approaches that suggest these proteins form an interaction web characterized by multiple interaction modes. BN-PAGE reveals that disk rim belts consist of a mixture of free peripherin-2 dimers and variously-sized chains of disulfide-linked dimers. MD simulations find that although free dimers can generate spontaneous curvature in a model membrane, dimers that are disulfide-linked into linear chains generate highly anisotropic curvature - a key hallmark of normal disk rims. The MD findings further suggest that although individual belts likely provide sufficient thermodynamic driving force to generate extreme curvature, non-covalent inter-belt interactions may also constrain rim diameter. The new findings advance understanding of peripherin-2 and rom1 structure and function in health and disease, and simultaneously provide promising new strategies for molecular phenotyping these clinically important proteins. Competing Interest Statement The authors have declared no competing interest. Footnotes Abbreviations - DTT - dithiothreitol - DDM - docecylmaltoside - EC2 - extracellular 2 - LOESS - locally estimated scatterplot smoothing - OS - outer segment - ROS - rod outer segment