Stabilization of Meta‐I Rhodopsin Conformation by a Nanobody

• DOI: 10.1096/fasebj.2022.36.s1.r2572
• Publication Date: 2022-05-03T08:48:22Z
• Authors: David Salom; Arum Wu; Christopher L. Sa...; Els Pardon; Jan Steyaert; Philip D. Kiser; Krzysztof Palczewski
• Keywords: Opsin; Retinaldehyde

Binding of an extracellular signaling molecule to a G protein-coupled receptor (GPCR) results in protein activation, which turn triggers the production second messengers. Although many advances have been achieved understanding GPCR early steps this process are still poorly understood. In case rhodopsin, paradigmatic member class A GPCRs, endogenous ligand is retinal covalently bound K296 through Schiff base. The activation rhodopsin triggered by absorption photon and isomerization 11-cis-retinal all-trans-retinal. When ground state (λmax = 500 nm) photoactivated, it progresses short-lived intermediates leading Meta-I ~478 nm), then establishes equilibrium with Meta-II 380 G-protein binding state. We developed llama antibody (nanobody Nb2) that binds bovine stabilizes its intermediate Meta-I. Here we present crystal structures Nb2 complex ground-state as well apo-rhodopsin (opsin). site includes rhodopsin's N-terminus loops ECL2 ECL3. Whereas structure rhodopsin/Nb2 virtually identical alone, induces dramatic structural change opsin, trapping conformation similar rhodopsin. way, when solution deprotonated (Meta-II) shifts towards protonated base corresponding conformation. Finally, co-expression P23H mutant HEK293 cell lines able rescue phenotype misfolding degradation vitro, suggesting therapeutic potential nanobody. summary, nanobody traps photoactivated elusive state; addition, misfolding-prone might also be useful tool facilitate crystallization wild type mutants different conformational states ligands. Current efforts include solving rhodopsin/nanobody complex.