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

Abstract

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.

Pathways and disease-causing alterations in visual chromophore production for vertebrate vision

“…The protein is synthesized by photoreceptors and is secreted into the interphotoreceptor matrix in a highly glycosylated form. IRBP binds all- trans and 11- cis -retinoids in addition to unsaturated fatty acids within its four homologous modules ( 25 , 26 , 27 ). Using Irbp -/- mice it was shown that this protein is not essential for visual cycle operation ( 28 , 29 ).…”

“…Crystal structures of two modules were reported ( 32 , 33 ), revealing a conserved two-domain architecture consisting of an N-terminal ββα-spiral fold linked to a C-terminal αβα sandwich. The structure of full-length bovine IRBP was solved at a global resolution of 8.1 Å using cryoelectron microscopy ( 27 ), revealing a bent filament or “π”-shaped structure 14.1 nm in diameter, 39.1 nm in length, and 2.2 to 4 nm in width. This structure would enable IRBP to wrap around an unknown anchor to be retained in the extracellular space between the photoreceptors and RPE to prevent rapid clearance by the RPE.…”

Bioprocess Development and Characterization of a 13C-Labeled Hybrid Bispecific Antibody Produced in Escherichia coli

“…The different approaches used for mAb structural elucidation include spectroscopic methods [ 6 ], calorimetry [ 7 ], nuclear magnetic resonance [ 8 ], crystallography [ 9 ], electron microscopy [ 10 ], and mass spectrometry (MS)-based techniques [ 11 ]. Regardless of the technology used for structural characterizations, all of the methods face the inherent challenge of the homodimeric symmetry of antibodies.…”