Structure and Calcium-Binding Studies of a Recoverin Mutant (E85Q) in an Allosteric Intermediate State

Abstract

Recoverin, a member of the EF-hand superfamily, serves as a calcium sensor in retinal rod cells. A myristoyl or related fatty acyl group covalently attached to the N-terminus of recoverin facilitates the binding of recoverin to retinal disk membranes by a mechanism known as the Ca2+-myristoyl switch. Previous structural studies revealed that the myristoyl group of recoverin is sequestered inside the protein core in the absence of calcium. The cooperative binding of two calcium ions to the second and third EF-hands (EF-2 and EF-3) of recoverin leads to the extrusion of the fatty acid. Here we present nuclear magnetic resonance (NMR), fluorescence, and calcium-binding studies of a myristoylated recoverin mutant (myr-E85Q) designed to abolish high-affinity calcium binding to EF-2 and thereby trap the myristoylated protein with calcium bound solely to EF-3. Equilibrium calcium-binding studies confirm that only one Ca2+ binds to myr-E85Q under the conditions of this study with a dissociation constant of 100 microM. Fluorescence and NMR spectra of the Ca2+-free myr-E85Q are identical to those of Ca2+-free wild type, indicating that the E85Q mutation does not alter the stability and structure of the Ca2+-free protein. In contrast, the fluorescence and NMR spectra of half-saturated myr-E85Q (one bound Ca2+) look different from those of Ca2+-saturated wild type (two bound Ca2+), suggesting that half-saturated myr-E85Q may represent a structural intermediate. We report here the three-dimensional structure of Ca2+-bound myr-E85Q as determined by NMR spectroscopy. The N-terminal myristoyl group of Ca2+-bound myr-E85Q is sequestered within a hydrophobic cavity lined by many aromatic residues (F23, W31, Y53, F56, F83, and Y86) resembling that of Ca2+-free recoverin. The structure of Ca2+-bound myr-E85Q in the N-terminal region (residues 2-90) is similar to that of Ca2+-free recoverin, whereas the C-terminal region (residues 100-202) is more similar to that of Ca2+-bound wild type. Hence, the structure of Ca2+-bound myr-E85Q represents a hybrid between the structures of recoverin with zero and two Ca2+ bound. The binding of Ca2+ to EF-3 leads to local structural changes within the EF-hand that alter the domain interface and cause a 45 degrees swiveling of the N- and C-terminal domains, resulting in a partial unclamping of the myristoyl group. We propose that Ca2+-bound myr-E85Q may represent a stable intermediate state in the kinetic mechanism of the calcium-myristoyl switch.

Genetic polymorphism and protein conformational plasticity in the calmodulin superfamily: Two ways to promote multifunctionality

“…2 A). The exposed hydrophobic residues in this region are conserved and correspond to residues of recoverin that interact with the myristoyl group in the Ca 2ϩ -free state (69,95,96). Upon Ca 2ϩ binding, these exposed residues in the hydrophobic patch have been implicated in target recognition from mutagenesis studies (97-100), and these residues form intermolecular contacts with target proteins as observed in the recent crystal structure of KChIP1 (101).…”

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 .…”

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

“…Consequently, the myristoyl group is still sequestered within a hydrophobic cavity and only partially unclamped. Biochemical studies on the myristoylated E85Q mutant confirmed that it can only bind one Ca 2ϩ at saturating concentration (21,22). Intriguingly, a fraction of myristoylated E85Q recoverin binds to membranes already at low free Ca 2ϩ (2-9 M), indicating that the presence of lipid bilayers may shift the conformational equilibrium toward a form of the E85Q mutant in which the myristoyl chain is exposed (22).…”

“…An intermediate state with Ca 2ϩ bound solely to EF-3 can be prepared using myristoylated recoverin harboring the mutation E85Q, which virtually abolishes Ca 2ϩ binding to EF-hand 2. Recently the three-dimensional structure of the myristoylated E85Q mutant was determined by NMR spectroscopy (21). It was shown to exhibit a hybrid fold with the N-and C-terminal domains resembling the corresponding portions of Ca 2ϩ -free and Ca 2ϩ -bound myristoylated wild-type recoverin, respectively.…”

“…In the present study we address the impact of the myristoyl group on the Ca 2ϩ -dependent conformational transition in recoverin. To this end, we have determined the x-ray structure of the non-myristoylated E85Q mutant and compared it to the non-myristoylated wild-type as well as to the myristoylated mutant published recently (21). Furthermore, to gain more insight into the biochemical properties of the non-acylated E85Q variant, we investigated key functional parameters such as Ca 2ϩ binding, association with hydrophobic matrices, and inhibition of rhodopsin kinase.…”