[Show abstract][Hide abstract] ABSTRACT: The topology of the GCAP-2 homodimer was investigated by chemical cross-linking and high resolution mass spectrometry. Complementary conducted size-exclusion chromatography and analytical ultracentrifugation studies indicated that GCAP-2 forms a homodimer both in the absence and in the presence of Ca(2+). In-depth MS and MS/MS analysis of the cross-linked products was aided by (15) N-labeled GCAP-2. The use of isotope-labeled protein delivered reliable structural information on the GCAP-2 homodimer, enabling an unambiguous discrimination between cross-links within one monomer (intramolecular) or between two subunits (intermolecular). The limited number of cross-links obtained in the Ca(2+)-bound state allowed us to deduce a defined homodimeric GCAP-2 structure by a docking and molecular dynamics approach. In the Ca(2+)-free state, GCAP-2 is more flexible as indicated by the higher number of cross-links. We consider stable isotope-labeling to be indispensable for deriving reliable structural information from chemical cross-linking data of multi-subunit protein assemblies.
Full-text · Article · Sep 2013 · Journal of the American Society for Mass Spectrometry
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Guanylate cyclase activating protein-2 (GCAP-2) is a Ca²⁺-binding protein of the neuronal calcium sensor (NCS) family. Ca²⁺-free GCAP-2 activates the retinal rod outer segment guanylate cyclases ROS-GC1 and 2. Native GCAP-2 is N-terminally myristoylated. Detailed structural information on the Ca²⁺-dependent conformational switch of GCAP-2 is missing so far, as no atomic resolution structures of the Ca²⁺-free state have been determined. The role of the myristoyl moiety remains poorly understood. Available functional data is incompatible with a Ca²⁺-myristoyl switch as observed in the prototype NCS protein, recoverin. For the homologous GCAP-1, a Ca²⁺-independent sequestration of the myristoyl moiety inside the proteins structure has been proposed. In this article, we compare the thermodynamic stabilities of myristoylated and non-myristoylated GCAP-2 in their Ca²⁺-bound and Ca²⁺-free forms, respectively, to gain information on the nature of the Ca²⁺-dependent conformational switch of the protein and shed some light on the role of its myristoyl group. In the absence of Ca²⁺, the stability of the myristoylated and non-myristoylated forms was indistinguishable. Ca²⁺ exerted a stabilizing effect on both forms of the protein, which was significantly stronger for myr GCAP-2. The stability data were corroborated by dye binding experiments performed to probe the solvent-accessible hydrophobic surface of the protein. Our results strongly suggest that the myristoyl moiety is permanently solvent-exposed in Ca²⁺-free GCAP-2, whereas it interacts with a hydrophobic part of the protein's structure in the Ca²⁺-bound state.
[Show abstract][Hide abstract] ABSTRACT: Guanylate cyclase-activating proteins (GCAPs) are neuronal Ca(2+) sensors that play a central role in shaping the photoreceptor light response and in light adaptation through the Ca(2+)-dependent regulation of the transmembrane retinal guanylate cyclase. GCAPs are N-terminally myristoylated, and the role of the myristoyl moiety is not yet fully understood. While protein lipid chains typically represent membrane anchors, the crystal structure of GCAP-1 showed that the myristoyl chain of the protein is completely buried within a hydrophobic pocket of the protein, which stabilizes the protein structure. Therefore, we address the question of the localization of the myristoyl group of GCAP-2 in the absence and in the presence of lipid membranes as well as DPC detergents (as a membrane substitute amenable to solution state NMR). We investigate membrane binding of both myristoylated and nonmyristoylated GCAP-2 and study the structure and dynamics of the myristoyl moiety of GCAP-2 in the presence of POPC membranes. Further, we address structural alterations within the myristoylated N-terminus of GCAP-2 in the presence of membrane mimetics. Our results suggest that upon membrane binding the myristoyl group is released from the protein interior and inserts into the lipid bilayer.
No preview · Article · Feb 2011 · Biophysics of Structure and Mechanism
[Show abstract][Hide abstract] ABSTRACT: Guanylate cyclase-activating protein-2 (GCAP-2) is a retinal Ca2+ sensor protein. It is responsible for the regulation of both isoforms of the transmembrane photoreceptor guanylate cyclase, a key enzyme of vertebrate phototransduction. GCAP-2 is N-terminally myristoylated and full activation of its target proteins requires the presence of this lipid modification. The structural role of the myristoyl moiety in the interaction of GCAP-2 with the guanylate cyclases and the lipid membrane is currently not well understood. In the present work, we studied the binding of Ca2+-free myristoylated and non-myristoylated GCAP-2 to phospholipid vesicles consisting of dimyristoylphosphatidylcholine or of a lipid mixture resembling the physiological membrane composition by a biochemical binding assay and 2H solid-state NMR. The NMR results clearly demonstrate the full-length insertion of the aliphatic chain of the myristoyl group into the membrane. Very similar geometrical parameters were determined from the 2H NMR spectra of the myristoyl group of GCAP-2 and the acyl chains of the host membranes, respectively. The myristoyl chain shows a moderate mobility within the lipid environment, comparable to the acyl chains of the host membrane lipids. This is in marked contrast to the behavior of other lipid-modified model proteins. Strikingly, the contribution of the myristoyl group to the free energy of membrane binding of GCAP-2 is only on the order of -0.5 kJ/mol, and the electrostatic contribution is slightly unfavorable, which implies that the main driving forces for membrane localization arises through other, mainly hydrophobic, protein side chain-lipid interactions. These results suggest a role of the myristoyl group in the direct interaction of GCAP-2 with its target proteins, the retinal guanylate cyclases.
Full-text · Article · Jan 2008 · Biochimica et Biophysica Acta