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.
Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin-Luther University Halle-Wittenberg , Wolfgang-Langenbeck-Strasse 4, D-06120 Halle (Saale), Germany.
Jens
J
Pettelkau
Thomas
T
Schröder
Christian H
CH
Ihling
Björn E S
BE
Olausson
Knut
K
Kölbel
Christian
C
Lange
Andrea
A
Sinz
D000588:Amines / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D020134:Catalytic Domain; D002417:Cattle; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000378:metabolism*; D051604:Guanylate Cyclase-Activating Proteins / Q000737:chemistry* / Q000378:metabolism*; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019814:Myristic Acid / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism*; D012160:Retina / Q000201:enzymology*
D000588:Amines; D003432:Cross-Linking Reagents; D051604:Guanylate Cyclase-Activating Proteins; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D011956:Receptors, Cell Surface; C110715:guanylate cyclase 1; D019814:Myristic Acid; D006162:Guanylate Cyclase; D002118:Calcium