Molecular Details of Retinal Guanylyl Cyclase 1/GCAP-2 Interaction

Publication year: 2018
Authors: Rehkamp A. 1, Tänzler D. 1, Iacobucci C. 1, Golbik R.P. 2, Ihling C.H. 1, Sinz A. 1

1 - Department of Pharmaceutical Chemistry and Bioanalytics, Charles Tanford Protein Center, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle, Germany
2 - Department of Microbial Biotechnology, Charles Tanford Protein Center, Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Halle, Germany

Published in: Frontiers in Molecular Neuroscience, 2018, Vol. 11
doi: 10.3389/fnmol.2018.00330

The rod outer segment guanylyl cyclase 1 (ROS-GC1) is an essential component of photo-transduction in the retina. In the light-induced signal cascade, membrane-bound ROS-GC1 restores cGMP levels in the dark in a calcium-dependent manner. With decreasing calcium concentration in the intracellular compartment, ROS-GC1 is activated via the intracellular site by guanylyl cyclase-activating proteins (GCAP-1/-2). Presently, the exact activation mechanism is elusive. To obtain structural insights into the ROS-GC1 regulation by GCAP-2, chemical cross-linking/mass spectrometry studies using GCAP-2 and three ROS-GC1 peptides were performed in the presence and absence of calcium. The majority of cross-links were identified with the C-terminal lobe of GCAP-2 and a peptide comprising parts of ROS-GC1's catalytic domain and C-terminal extension. Consistently with the cross-linking results, surface plasmon resonance and fluorescence measurements confirmed specific binding of this ROS-GC peptide to GCAP-2 with a dissociation constant in the low micromolar range. These results imply that a region of the catalytic domain of ROS-GC1 can participate in the interaction with GCAP-2. Additional binding surfaces upstream of the catalytic domain, in particular the juxtamembrane domain, can currently not be excluded.

MP-SPR keywords: binding affinity, CMD sensor slides, neuronal calcium sensor proteins in retina, protein immobilization using amine coupling chemistry, protein-enzyme interaction, protein-protein