Structural characterization of the second intra‐discal loop of the photoreceptor tetraspanin RDS

• Published in: The FEBS journal 2013-01, Vol.280 (1), p.127-138
• Main Authors: Chakraborty, Dibyendu, Rodgers, Karla K., Conley, Shannon M., Naash, Muna I.
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Animals; CD spectroscopy; Circular Dichroism; Escherichia coli; Eye diseases; Eye Proteins - chemistry; Gene Expression; Intermediate Filament Proteins - biosynthesis; Intermediate Filament Proteins - chemistry; Intermediate Filament Proteins - genetics; macular dystrophy; Maltose-Binding Proteins - biosynthesis; Maltose-Binding Proteins - chemistry; Maltose-Binding Proteins - genetics; Membrane Glycoproteins - biosynthesis; Membrane Glycoproteins - chemistry; Membrane Glycoproteins - genetics; Membrane Proteins - chemistry; Mice; Models, Molecular; Mutagenesis; Mutation, Missense; Nerve Tissue Proteins - biosynthesis; Nerve Tissue Proteins - chemistry; Nerve Tissue Proteins - genetics; Peripherins; Protein Binding; Protein Stability; Protein Structure, Tertiary; RDS; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Retina; retinitis pigmentosa; tetraspanin; Tetraspanins
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.12055

Vertebrate photoreceptors contain a unique tetraspanin protein known as ‘retinal degeneration slow’ (RDS). Mutations in the RDS gene have been identified in a variety of human retinal degenerative diseases, and more than 70% of these mutations are located in the second intra‐discal (D2) loop, highlighting the importance of this region. Here we examined the conformational and thermal stability properties of the D2 loop of RDS, as well as interactions with ROM–1, a non‐glycosylated homolog of RDS. The RDS D2 loop was expressed in Escherichia coli as a fusion protein with maltose binding protein (MBP). The fusion protein, referred to as MBP–D2, was purified to homogeneity. Circular dichroism spectroscopy showed that the wild‐type (WT) D2 loop consists of approximately 21% α–helix, approximately 20% β–sheet and approximately 59% random coil. D2 loop fusion proteins carrying disease‐causing mutations in RDS (e.g. R172W, C214S, N244H/K) were also examined, and conformational changes were observed (compared to wild‐type D2). In particular, the C150S, C214S and N244H proteins showed significant reductions in α–helicity. However, the thermal stability of the mutants was unchanged compared to wild‐type, and all the mutants were capable of interacting with ROM–1, indicating that this functional aspect of the isolated D2 loop remained intact in the mutants despite the observed conformational changes. An I–TASSER model of the RDS D2 loop predicted a structure consistent with the circular dichroism experiments and the structure of the conserved region of the D2 loop of other tetraspanin family members. These results provide significant insight into the mechanism of RDS complex formation and the disease process underlying RDS‐associated retinal degeneration. The second intradiscal loop of the photoreceptor tetraspanin RDS contains mutations which cause widely varying blinding phenotypes. Here we conduct circular dichroism studies of this protein region to understand the effects disease‐causing mutations have on the secondary protein structure. The majority of examined mutations cause decreases in the α‐helical content of this loop but no change in thermal stability