Thermodynamic Properties and Nmr Data Indicate An Inverse Calcium-Myristoyl Switch of Gcap2

Abstract

Guanylyl cyclase-activating protein-2 (GCAP-2) is a neuronal calcium sensor protein (NCS) present in vertebrate photoreceptor cells. Depending on the Ca2+ concentration, GCAP-2, and its homologue GCAP-1, inhibit or activate their target protein, the rod outer segment guanylate cyclase (ROS-GC). This plays an important role in shaping the photoreceptor light response. Like all members of the NCS, GCAP-2 is myristoylated at the N-terminus. This fatty acid modification is not essential for the basic function of GCAP-2, but required for full activation of the ROS-GC. Up to now, the biological role of this modification has not been fully understood. In order to gain insight into the Ca2+-dependent conformational changes of GCAP-2, we measured the thermodynamic stability of the protein in dependence of Ca2+ binding and myristoylation by monitoring thermally and chemically induced folding / unfolding transitions of myristoylated and non-myristoylated GCAP-2. Stabilities observed for myristoylated and non-myristoylated GCAP-2 in absence of Ca2+ were indistinguishable. Addition of Ca2+ exerted a strong stabilising effect. This effect was more pronounced for the myristoylated GCAP-2 than for the non-myristoylated, indicating a structural role of the myristoyl moiety in Ca2+-bound but not in the Ca2+-free state. Furthermore, from deuterium solid state experiments we have evidence that the myristoyl moiety is highly flexible in the Ca2+-free state when bound to liposomes. In contrast to the Ca2+-myristoyl switch for the prototype NCS Recoverin, which exposes its myristoyl moiety in the Ca2+-bound state, but buries it when Ca2+ is missing, the myristoyl moiety ofGCAP-2 appears to be fully solvent-exposed in the Ca2+-free state. As we could show, myristoylation does not significantly enhance membrane binding of GCAP-2. These results are in agreement with a possible direct interaction of the myristoyl moiety with the target protein, the ROS-GC.