Single particle cryo‐EM of the complex between interphotoreceptor retinoid‐binding protein and a monoclonal antibody

• Published in: The FASEB journal 2020-10, Vol.34 (10), p.13918-13934
• Main Authors: Sears, Avery E., Albiez, Stefan, Gulati, Sahil, Wang, Benlian, Kiser, Philip, Kovacik, Lubomir, Engel, Andreas, Stahlberg, Henning, Palczewski, Krzysztof
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - immunology; Antigen-Antibody Complex - chemistry; Cattle; Cryoelectron Microscopy; cryo‐EM; Eye Proteins - chemistry; Eye Proteins - immunology; Female; interphotoreceptor retinoid‐binding protein; Mice; Mice, Inbred C57BL; monoclonal antibody; Retinol-Binding Proteins - chemistry; Retinol-Binding Proteins - immunology; Single Molecule Imaging; single particle analysis; visual cycle
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fj.202000796RR

Interphotoreceptor retinoid‐binding protein (IRBP) is a highly expressed protein secreted by rod and cone photoreceptors that has major roles in photoreceptor homeostasis as well as retinoid and polyunsaturated fatty acid transport between the neural retina and retinal pigment epithelium. Despite two crystal structures reported on fragments of IRBP and decades of research, the overall structure of IRBP and function within the visual cycle remain unsolved. Here, we studied the structure of native bovine IRBP in complex with a monoclonal antibody (mAb5) by cryo‐electron microscopy, revealing the tertiary and quaternary structure at sufficient resolution to clearly identify the complex components. Complementary mass spectrometry experiments revealed the structure and locations of N‐linked carbohydrate post‐translational modifications. This work provides insight into the structure of IRBP, displaying an elongated, flexible three‐dimensional architecture not seen among other retinoid‐binding proteins. This work is the first step in elucidation of the function of this enigmatic protein.