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Beata Jastrzebska, Tivadar Orban, Marcin Golczak, Andreas Engel, Krzysztof Palczewski; EM-structure and functional properties of the rhodopsin-transducin complex. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2462.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the molecular assembly, structure and functional properties of the visual G protein-coupled receptor (GPCR), rhodopsin (Rho), in the complex with its cognitive heterotrimeric G protein, transducin (Gt).
By photoactivation of Rho (Meta II or Rho*) and nucleotide depletion, we trapped and purified the nucleotide-free Rho*-Gt complex and calculated its molecular envelope from projections of negatively stained Rho*-Gt particles. Using sConA as a probe, we identified the quaternary organization of Rho molecules in this complex. Functional properties of Rho molecules bound to Gt within the stable Rho*-Gt complex was investigated by UV-visible spectroscopy and HPLC retinoid analysis.
The 3-D envelope determined for the Rho*-Gt complex accommodated two Rho molecules and one Gt heterotrimer (pentameric assembly). Within the complex, the photoactivated Rho dimer serves as a platform for binding the Gt heterotimer. We found that binding of Gt stabilizes one Rho in its active Meta II state, whereas the second progresses toward the opsin conformation. Therefore, each monomer contributes unevenly to the pentameric complex, indicating an asymmetry of the Rho dimer.
This study unequivocally demonstrates dimeric assembly of Rho* in the active complex with transducin and provides a clear functional distinction between each monomer, indicating that oligomeric assembly and activation of family A GPCRs are intimately associated.
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