Abstract
Purpose :
Rhodopsin is the class A archetypical G-protein-coupled receptor (GPCR). Despite the progress in biophysical characterization of rhodopsin at different points of the photo-activation process, the first stages of this process are still poorly understood at the structural level. We have developed several llama antibodies (nanobodies, Nbs) as tools to facilitate the structural characterization of rhodopsin, especially in its early activation state, Meta-I.
Methods :
Several nanobodies that bind rhodopsin were obtained upon immunization of llamas with bovine rod outer segments. UV-visible spectroscopy was used to characterize the conformational/spectral changes of rhodopsin upon binding of Nbs. Finally, we performed trials for the co-crystallization of several nanobodies with ground state rhodopsin, activated rhodopsin and apo-rhodopsin (opsin).
Results :
Here we present the crystal structures of a nanobody (Nb2) in complex with ground-state bovine rhodopsin, as well as Nb2 in complex with opsin. The binding site of Nb2 includes rhodopsin’s N-terminus and extracellular loops, ECL2 and ECL3. Whereas rhodopsin’s structure in the ground-state rhodopsin/Nb2 complex is virtually identical to that of rhodopsin by itself, Nb2 binding induces a dramatic structural change in opsin, trapping it in a conformation similar to that of ground-state rhodopsin. The crystal lattice is maintained by Nb2/bRho, bRho/bRho and Nb2/Nb2 protein contacts that allow the small conformational changes required by rhodopsin to transition from the ground state to Meta-I. In solution, several of these nanobodies bind to photoactivated, deprotonated rhodopsin (Meta-II), shifting the equilibrium towards a protonated Schiff base state corresponding to the Meta-I conformation.
Conclusions :
We have developed several nanobodies that shift the equilibrium of photoactivated rhodopsin towards the elusive Meta-I state. These nanobodies might be a useful tool to facilitate the crystallization of wild type rhodopsin in different conformational states, and/or bound to different ligands. Current efforts include solving the crystal structure of the Meta-I rhodopsin/Nb2 complex.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.