Cryo-EM structures of peripherin-2 and ROM1 suggest multiple roles in photoreceptor membrane morphogenesis

Abstract

Mammalian peripherin-2 (PRPH2) and rod outer segment membrane protein 1 (ROM1) are retina-specific tetraspanins that partake in the constant renewal of stacked membrane discs of photoreceptor cells that enable vision. Here, we present single-particle cryo-electron microscopy structures of solubilized PRPH2-ROM1 heterodimers and higher-order oligomers. High-risk PRPH2 and ROM1 mutations causing blindness map to the protein-dimer interface. Cysteine bridges connect dimers forming positive-curved oligomers, whereas negative-curved oligomers were observed occasionally. Hexamers and octamers exhibit a secondary micelle that envelopes four carboxyl-terminal helices, supporting a potential role in membrane remodeling. Together, the data indicate multiple structures for PRPH2-ROM1 in creating and maintaining compartmentalization of photoreceptor cells.

Fig. 1.. Cryo-EM structure of PRPH2-ROM1 heterodimer.

(A) Overall cryo-EM map with ROM1 and PRPH2 is colored red and orange, respectively. Detergent micelle density is transparently visible (left). Nanobody19 is shown in gray. (B) Ribbon representation of PRPH2-ROM1 dimer in the same orientation and colors as the map in (A). (C) Topology diagram of PRPH2-ROM1 showing secondary structure elements, same colors as in (A). Free cysteines in the AB loops and N-acetylglucosamine shown as green squares and sticks, respectively.

Fig. 2.. PRPH2-ROM1 heterodimer interface and structural interpretation of degenerative retinal diseases mutations.

(A) Open-book surface representation of the PRPH2-ROM1 interface is colored orange and red, respectively. High-risk human mutations are mapped onto the structure in black. Cysteine residues that permit oligomerization via intermolecular disulfide bond formation, PRPH2 C150 and ROM1 C153, are shown in green. (B to D) Zoomed-in presentations of PRPH2-ROM1 interactions.

Fig. 3.. Cryo-EM structures of PRPH2-ROM1 oligomers.

(A) Cryo-EM map of PRPH2-ROM1 tetramer (green), hexamer (violet), and octamer (blue). Side view and top view are shown. High-resolution dimers fitted into corresponding density are transparently visible. (B) Cryo-EM model of PRPH2-ROM1 octamer with each dimer colored separately. (C) Ribbon representation of PRPH2-ROM1 octamer in the same orientation and colors as the map in (B). Intermolecular cysteines connecting dimers are shown in yellow.

Fig. 4.. Disc morphogenesis model.

(A) Twisted ribbon model of 50-nucleotide oligomer PRPH2-ROM1. Particles present in micrographs highlighted using a white 460-Å circle. (B) Schematic representation of steps in disc morphogenesis based on Steinberg et al. (5) and a model depicting how PRPH2 and ROM1 play a role in this process: (1) membrane evagination of the ciliary plasma membrane (PM), (2) bilateral membrane expansion and enclosure, and (3) mature disc with PRPH2-ROM1 scaffold at disc rims according to Pöge et al. (19). PRPH2-ROM1 dimers are schematically indicated in red, and membrane is indicated in dark gray. (C) 2D classes of PRPH2-ROM1 hexamers showing negative curvature (right) and positive curvature (left). Negative curvature reflects a scaffold of three noncovalent dimers as observed in mature disc rim (19). Positive curvature complexes reveal dimers covalently linked through disulfide bonds exhibiting a secondary micelle that we hypothesize surrenders the C-terminal tails and may support the first steps of disc morphogenesis.