Structural Insights into retinal guanylylcyclase-GCAP-2 interaction determined by cross-linking and mass spectrometry

The retinal guanylylcyclases ROS-GC 1 and 2 are regulated via the intracellular site by guanylylcyclase-activating proteins (GCAPs). The mechanisms of how GCAPs activate their target proteins remain elusive as exclusively structures of nonactivating calcium-bound GCAP-1 and -2 are available. In this work, we apply a combination of chemical cross-linking with amine-reactive cross-linkers and photoaffinity labeling followed by a mass spectrometric analysis of the created cross-linked products to study the interaction between N-terminally myristoylated GCAP-2 and a peptide derived from the catalytic domain of full-length ROS-GC 1. In our studies, only a few cross-linked products were obtained for calcium-bound GCAP-2, pointing to a well-defined structure of the GCAP-2-GC peptide complex. A much larger number of cross-links were detected in the absence of calcium, indicating a high flexibility of calcium-free GCAP-2 in the complex with the GC peptide. On the basis of the distance constraints imposed by the cross-links, we were able to create a structural model of the calcium-loaded complex between myristoylated GCAP-2 and the GC peptide.