Changes in Biological Activity and Folding of Guanylate Cyclase-Activating Protein 1 as a Function of Calcium

• Published in: Biochemistry (Easton) Vol. 37; no. 1; pp. 248 - 257
• Main Authors: Rudnicka-Nawrot, Maria, Surgucheva, Irina, Hulmes, Jeffrey D, Haeseleer, Françoise, Sokal, Izabela, Crabb, John W, Baehr, Wolfgang, Palczewski, Krzysztof
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 06.01.1998
• Subjects: Amino Acid Sequence; Amino Acid Substitution - genetics; Animals; Binding, Competitive; Calcium - physiology; Calcium-Binding Proteins - genetics; Calcium-Binding Proteins - metabolism; Calcium-Binding Proteins - physiology; Cattle; Cell Line; Enzyme Activation - drug effects; Guanylate Cyclase - antagonists & inhibitors; Guanylate Cyclase - metabolism; Guanylate Cyclase-Activating Proteins; Hydrolysis; Insecta; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Photoreceptor Cells - enzymology; Protein Folding; S100 Proteins - pharmacology; Trypsin
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi972306x

Guanylate cyclase-activating protein 1 (GCAP1), a photoreceptor-specific Ca2+-binding protein, activates retinal guanylate cyclase 1 (GC1) during the recovery phase of phototransduction. In contrast to other Ca2+-binding proteins from the calmodulin superfamily, the Ca2+-free form of GCAP1 stimulates the effector enzyme. In this study, we analyzed the Ca2+-dependent changes in GCAP1 structure by limited proteolysis and mutagenesis in order to understand the mechanism of Ca2+-sensitive modulation of GC1 activity. The change from a Ca2+-bound to a Ca2+-free form of GCAP1 increased susceptibility of Ca2+-free GCAP1 to proteolysis by trypsin. Sequencing data revealed that in the Ca2+-bound form, only the N-terminus (myristoylated Gly2−Lys9) and C-terminus (171−205 fragment) of GCAP1 are removed by trypsin, while in the Ca2+-free form, GCAP1 is readily degraded to small fragments. Successive inactivation of each of the functional EF loops by site-directed mutagenesis showed that only EF3 and EF4 contribute to a Ca2+-dependent inactivation of GCAP1. GCAP1(E75D,E111D,E155D) mutant did not bind Ca2+ and stimulated GC1 in a [Ca2+]-independent manner. GCAP1 and GCAP2, but not S-100β, a high [Ca2+]free activator of GC1, competed with the triple mutant at high [Ca2+]free, inhibiting GC1 with similar IC50's. These competition results are consistent with comparable affinities between GC1 and GCAPs. Our data suggest that GCAP1 undergoes major conformational changes during Ca2+ binding and that EF3 and EF4 motifs are responsible for changes in the GCAP1 structure that converts this protein from the activator to the inhibitor of GC1.