Crystal Structure of Recoverin with Calcium Ions Bound to Both Functional EF Hands

Abstract

Recoverin (Rv), a small Ca2+-binding protein that inhibits rhodopsin kinase (RK), has four EF hands, two of which are functional (EF2 and EF3). Activation requires Ca2+ in both EF hands, but crystal structures have never been observed with Ca2+ ions in both sites; all previous structures have Ca2+ bound only to EF3. We suspected that this was due to an intermolecular crystal contact between T80 and a surface glutamate (E153) that precluded coordination of a Ca2+ ion in EF2. We constructed the E153A mutant, determined its X-ray crystal structure to 1.2 Å resolution, and show that 2 Ca2+ ions are bound, one in EF3 and one in EF2. Additionally, several other residues are shown to adopt conformations in the 2Ca2+-structure not seen previously and not seen in a second structure of the E153A mutant containing Na+ instead of Ca2+ in the EF2 site. The side-chain rearrangements in these residues form a 28 Å long allosteric cascade along the surface of the protein connecting the Ca2+-binding site of EF2 with the active-site pocket responsible for binding RK.

Binding of a Myristoylated Protein to the Lipid Membrane Influenced by Interactions with the Polar Head Group Region

“…Points represent values obtained from two independent experiments. A black line in the right panels shows the content of α-helices in Rv and n-Rv as determined in refs , , , , and . The experimental error bars were calculated for the lowest and highest contents of α-helices in recoverin (a) and n-recoverin (b) as reported in refs , , , , and .…”

“…2% of Rv. In the Ca 2+ -bound state of n-Rv, the content of α-helices varies between 55 and 62% and of β-sheets between 2 and 4%. ,,, IR transmission spectra of Rv and n-Rv in 50 mM NaNO 3 with 2 mM Ca­(NO 3 ) 2 in D 2 O are used to determine the secondary structure elements of these proteins (SI.6). The α-helical content in Rv is 52 ± 2% and in n-Rv 55 ± 2%, being in a very good agreement with literature. ,,,,,, …”

“…Acylation is an important post-translational modification of a large number of cytoplasmic proteins, facilitating their binding to cell membranes. − Some acylated proteins experience a dynamic change in their three-dimensional structure, which can be triggered by factors like Ca 2+ or guanosine-5′-triphosphate and involves the movement of a myristoyl (or palmitoyl) chain. , The neuronal calcium sensor protein recoverin (Rv) is the prototype of this kind of myristoyl-switch proteins. It has a covalently attached myristoyl (C14:0) group at the N-terminus .…”

Zebrafish Recoverin Isoforms Display Differences in Calcium Switch Mechanisms

“…The Gly in zRec2b decreases the degree of hydrophobicity thereby lowering binding of ANS, whereas Ala in zRec2a has a side chain of lower hydrophobicity than Ile, but probably sufficient for ANS binding ( Supplementary Tables S1 , S2 ). In non-myristoylated bRec, the large hydrophobic patch is solvent accessible in both forms of bRec, with one or two Ca 2+ bound ( Supplementary Figure S4 Weiergräber et al, 2003 ; Kumar et al, 2015 ). The hydrophobic canyon-like cleft seems to be exposed increasing the general hydrophobicity, although ANS binding to bRec occurred to nearly the same extent independent on the presence of the myristoyl group.…”

Bringing the Ca 2+ sensitivity of myristoylated recoverin into the physiological range

“…NMR spectroscopy [ 9 – 11 ] and X-ray crystallography [ 12 – 15 ] have greatly contributed to our understanding of the structure-function characteristics of Rec highlighting the existence of a ‘tense' (T), compact conformation and a ‘relaxed' (R), more elongated conformation, in which the four EF-hands motifs in Rec acquire different relative orientations ( figure 1 ). Since EF1 and EF4 are non-functional EF-hands [ 13 ] Rec binds only two Ca 2+ ions in the EF2 (low-affinity site) and EF3 (high-affinity site) motifs located, respectively, in the N and in the C terminal domains [ 16 , 17 ].…”