Cog-Wheel Octameric Structure of RS1, the Discoidin Domain Containing Retinal Protein Associated with X-Linked Retinoschisis

Abstract

RS1, also known as retinoschisin, is a disulphide-linked, discoidin domain containing homo-oligomeric protein that plays a crucial role in maintaining the cellular and synaptic organization of the retina. This is highlighted by the finding that over 130 mutations in RS1 cause X-linked retinoschisis, a retinal degenerative disease characterized by the splitting of the retinal cell layers, disruption of the photoreceptor-bipolar synapses, degeneration of photoreceptors, and severe loss in central vision. In this study, we investigated the arrangement of the RS1 subunits within the oligomer complex using single particle electron microscopy. RS1 was seen as two stacked rings with each ring displaying a symmetrical cog wheel-like structure with eight teeth or projections corresponding to the RS1 subunits. Three dimensional reconstruction and molecular modelling indicated that the discoidin domain, the principal functional unit of RS1, projects outward, and the Rs1 domain and C-terminal segment containing intermolecular disulphide bonds are present in the inner ring to form the core octameric structure. These studies provide a basis for further understanding the role of the novel core RS1 octameric complex in retinal cell biology and X-linked retinoschisis.

Fig 1. Linear diagram of two RS1 subunits showing the various domains and key disulphide bonds.

The signal peptide (1–23 amino acids) is cleaved during biosynthesis. In the mature protein there are two intramolecular disulphide bonds (C63 –C219; C110 –C142) and one intermolecular disulphide bond (C59 –C223) responsible for homo-oligomeric assembly of subunits.

Fig 2. Analysis of RS1 by SDS gel electrophoresis and size exclusion chromatography (SEC).

RS1 was purified from the culture fluid of Sf21 insect cells by ion exchange chromatography followed by galactose affinity chromatography and SEC. A. The sample was analysed on SDS gels under disulphide reducing (left) and nonreducing (right) conditions. B. Nondenatured RS1 sample was subjected to size exclusion chromatography on a calibrated column. Standards were 1: Thyroglobulin 669KDa, 2: Ferritin 440kDa, 3: Aldolase, 158kDa, 4: Conalbumin 75kDa, 5: Ovalbumin 44kDa. Inset: Plot of the Log MW vs. elution volume. RS1 (black trace) eluted with an apparent molecular mass of 180 kDa.

Fig 3. Single particle EM images and 3-D reconstruction of RS1.

A. Electron micrograph of purified RS1. B. Represented refined images of RS1 in 2-dimensions. On-face images show a cog-wheel-like structure. Each box width = 30 nm. Also present are side views of double cog-wheel-like structures. C. 3-D on-face reconstruction from 10,664 EM images. D. Tilted 3-D image of a double cog-wheel showing interaction between two cog-wheels at the outer edge.

Fig 4. Molecular model of the mature RS1 subunit.

A. The RS1 subunit model shows the cysteine residues involved in intramolecular disulphide bonds (C110-C142 and C63-C219) in blue and two cysteine residues involved in intermolecular disulphide bonds (C59 and C223) that generate the octamer complex in magenta. B. Ramachandran plot for the RS1 model.

Fig 5. Modelling RS1 into EM map.

Eight RS1 monomers fit within the EM framework. The discoidin domains project outward and the intermolecular disulphide bonds in the inner ring are essential for maintaining the octameric structure.