Impact of N-terminal Myristoylation on the Ca2+-dependent Conformational Transition in Recoverin

Abstract

Recoverin is a Ca2؉ -regulated signal transduction modulator found in vertebrate retina that has been shown to undergo dramatic conformational changes upon Ca 2؉ binding to its two functional EF-hand motifs. To elucidate the differential impact of the N-terminal myristoylation as well as occupation of the two Ca 2؉ binding sites on recoverin structure and function, we have investigated a non-myristoylated E85Q mutant exhibiting virtually no Ca 2؉ binding to EF-2. Crystal structures of the mutant protein as well as the non-myristoylated wild-type have been determined. Although the non-myristoylated E85Q mutant does not display any functional activity, its three-dimensional structure in the presence of Ca 2؉ resembles the myristoylated wildtype with two Ca 2؉ but is quite dissimilar from the myristoylated E85Q mutant. We conclude that the N-terminal myristoyl modification significantly stabilizes the conformation of the Ca 2؉ -free protein (i.e. the T conformation) during the stepwise transition toward the fully Ca 2؉ -occupied state. On the basis of these observations, a refined model for the role of the myristoyl group as an intrinsic allosteric modulator is proposed.Recoverin belongs to an ancient family of calcium-binding proteins termed the neuronal calcium sensor family (1, 2) and is mainly expressed in vertebrate photoreceptor cells (3, 4). The 23-kDa protein is composed of two domains, each of them harboring one non-functional and one functional EF-hand helix-loop-helix motif (5). Vertebrate photoreceptor cells respond to illumination by a decrease of the intracellular transmitters of excitation and adaptation, cGMP and Ca 2ϩ , respectively. In the dark, at high Ca 2ϩ concentration, the two functional EFhands of recoverin (EF-2 and -3) are occupied by Ca 2ϩ (6 -8). In this state, recoverin is able to inhibit the G-protein-coupled receptor kinase GRK1 1 (rhodopsin kinase), thereby prolonging the lifetime of photoexcited rhodopsin (9 -12). Upon illumination, decrease of cytoplasmic Ca 2ϩ causes this inhibition to be relieved. This regulatory circuit is thought to be one out of several Ca 2ϩ -dependent mechanisms that control adaptation of phototransduction to changing background light intensities (13,14).At its N terminus recoverin is heterogeneously acylated, the prevailing modification being a myristoyl chain (15). The observation of a Ca 2ϩ -dependent partitioning of recoverin to membranes led to the proposal that it underwent a Ca 2ϩ -myristoyl switch (16). The mechanics of this switch were unraveled by determining the solution structures of Ca 2ϩ -free and Ca 2ϩ -bound myristoylated recoverin via NMR spectroscopy. In the Ca 2ϩ -free state of recoverin (T state) the myristoyl moiety is buried within a hydrophobic pocket, whereas in the Ca 2ϩ -bound form (R state), the acyl group is extruded and thus available for interaction with other proteins or insertion into a lipid bilayer (17-19). Moreover, the myristoyl chain has been proposed to act as an intrinsic allosteric effector modifying the conformational...

Tuning of a Neuronal Calcium Sensor

“…While the electron density indicated the presence of a Ca 2ϩ ion in EF-hand 3, the lower affinity EF-hand 2 turned out to be unoccupied. Therefore, like the crystal structure of wildtype recoverin (22), our structure of Rc represents an intermediate state resulting from Ca 2ϩ binding to the high affinity EF-hand 3. The N-terminal 8 amino acids could not be located in the electron density and are thus likely to be disordered.…”

“…Rhodopsin Kinase Assay-The kinase assay was performed as described before (22). Briefly, the assay mixture in a final volume of 50 l contained 10 M rhodopsin (urea-washed ROS), 20 mM Tris-HCl (pH 7.5), 2 mM MgCl 2 , 1 mM [␥ 32 P]ATP (30 -100 dpm/pmol), 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 0.3-0.5 units of rhodopsin kinase.…”

“…The search model was created from the crystal structure of wild-type bovine recoverin (PDB code 1OMR, Ref. 22) by deleting residues 191-202. Crystals were found to contain four molecules per asymmetric unit, related by a proper 4-fold non-crystallographic symmetry.…”

“…To this end, we have determined the crystal structure of a truncated mutant lacking the C-terminal cluster of charged residues and compared it with available structures of wild-type recoverin (12,15,16,21,22). Furthermore, we have investigated key biochemical properties of the mutant, such as Ca 2ϩ binding, association with rod outer segment (ROS) membranes and inhibition of rhodopsin kinase.…”

The Diversity of Calcium Sensor Proteins in the Regulation of Neuronal Function

“…The structure of recoverin has been extensively studied by X-ray crystallography and NMR studies to interrogate its structure in its Ca 2þ -bound and -free forms (Flaherty et al 1993;Ames et al 1995;Tanaka et al 1995;Ames et al 1997;Ames et al 2002;Weiergraber et al 2003). Recoverin is composed of two distinct domains connected through a bent linker and forms a compact structure in the absence of Ca 2þ .…”