Effects of mutations in the calcium-binding sites of recoverin on its calcium affinity: evidence for successive filling of the calcium binding sites

The flagellar calcium-binding protein (FCaBP) of the flagellated protozoan Trypanosoma cruzi associates with the flagellar membrane via its N-terminal myristate and palmitate moieties in a calcium-modulated, conformation-dependent manner. This mechanism of localization is similar to that described for neuronal calcium sensors, which undergo calcium-dependent changes in conformation, which modulate the availability of the acyl groups for membrane interaction and partner association. To test whether FCaBP undergoes a calcium-dependent conformational change and to explore the role of such a change in flagellar targeting, we first introduced point mutations into each of the two EF-hand calcium-binding sites of FCaBP to define their affinities. Analysis of recombinant EF-3 mutant (E151Q), EF-4 mutant (E188Q), and double mutant proteins showed EF-3 to be the high affinity site (K d ϳ9 M) and EF-4 the low affinity site (K d ϳ120 M). These assignments also correlated with partial (E188Q), nearly complete (E151Q), and complete (E151Q,E188Q) disruption of calcium-induced conformational changes determined by NMR spectrometry. We next expressed the FCaBP E151Q mutant and the double mutant in T. cruzi epimastigotes. These transproteins localized to the flagellum, suggesting the existence of a calcium-dependent interaction of FCaBP that is independent of its intrinsic calcium binding capacity. Several proteins were identified by FCaBP affinity chromatography that interact with FCaBP in a calcium-dependent manner, but with differential dependence on calcium-binding by FCaBP. These findings may have broader implications for the calcium acyl switch mechanism of protein regulation. Flagellar calcium-binding protein (FCaBP)2 is a 24-kDa highly immunogenic protein found in the flagellum of the protozoan parasite Trypanosoma cruzi (1). FCaBP contains four EF-hand calcium-binding domains, the third and fourth (EF-3 and EF-4) of which bind calcium (2). The protein is modified at the N terminus by the addition of myristate and palmitate, both of which are required for association with the inner leaflet of the flagellar membrane (3). Calcium is required for stable flagellar localization as well, since FCaBP can be washed out of detergent-permeabilized trypanosomes if calcium chelators are included in the wash solutions. Because of the size, acylation, calcium binding, membrane association, and calcium-dependent localization of FCaBP, we concluded that the protein is a calcium acyl switch protein. These proteins undergo calcium-dependent membrane association by virtue of calcium-regulated extrusion or sequestration of a myristate moiety that mediates membrane binding (4).The best studied calcium acyl switch protein is recoverin (Rv), a myristoylated calcium-binding protein of the mammalian retina (5) that inhibits rhodopsin kinase (RK) (6) and regulates recovery of the photoreceptor cell after photoexcitation (7). Rv regulates RK via the calcium myristoyl switch mechanism (4). In the resting state of the cell, Rv binds two calcium ions a...

Section: Discussionmentioning

confidence: 93%

A Flagellum-specific Calcium Sensor

Buchanan

Ames

Asfaw

et al. 2005

“…1C). A change of the wavelength maximum of fluorescence emission ( max ) at increasing temperature indicates thermal unfolding of the protein (13). Fluorescence emission of the mutant was slightly blue-shifted by 2 nm at temperatures below the transition point, which indicates small changes in the microenvironment of tryptophan residues caused by the removal of twelve C-terminal amino acids.…”

Section: Resultsmentioning

confidence: 99%

“…Fluorescence Measurements-Thermal stability of recoverin variants was studied by recording the wavelength maximum ( max ) of tryptophan fluorescence emission at different temperatures essentially as described before (13). Measurements were performed on a Cary Eclipse spectrofluorimeter (Varian Inc.), equipped with a Peltier-controlled cell holder.…”

Section: Methodsmentioning

confidence: 99%

Tuning of a Neuronal Calcium Sensor

Weiergräber

Senin

Zernii

et al. 2006

Self Cite

Recoverin is a Ca2؉ -regulated signal transduction modulator expressed in the vertebrate retina that has been implicated in visual adaptation. An intriguing feature of recoverin is a cluster of charged residues at its C terminus, the functional significance of which is largely unclear. To elucidate the impact of this segment on recoverin structure and function, we have investigated a mutant lacking the C-terminal 12 amino acids. Whereas in myristoylated recoverin the truncation causes an overall decrease in Ca 2؉ sensitivity, results for the non-myristoylated mutant indicate that the truncation primarily affects the high affinity EF-hand 3. The three-dimensional structure of the mutant has been determined by x-ray crystallography. In addition to significant changes in average coordinates compared with wild-type recoverin, the structure provides strong indication of increased conformational flexibility, particularly in the C-terminal domain. Based on these observations, we propose a novel role of the C-terminal segment of recoverin as an internal modulator of Ca 2؉ sensitivity.Many biological processes are triggered or regulated by transient intracellular Ca 2ϩ signals. Because these signals elicit specific cellular responses, the precise detection of changes in cytoplasmic Ca 2ϩ concentration is a crucial step in many signaling pathways and requires sensing of Ca 2ϩ within very different concentration ranges. Ca 2ϩ -binding proteins work as intracellular Ca 2ϩ sensors and regulate their targets with high specificity and high spatial and temporal resolution. To achieve these remarkable tasks, Ca 2ϩ is recognized by specific amino acid sequence motifs, for example the C 2 domain and the EF-hand motif (1, 2). These motifs can detect subtle changes in Ca 2ϩ concentration and allow a fine tuning of Ca 2ϩ signaling. However, it remains a challenging problem to understand at a structural level how minimal changes in cytoplasmic Ca 2ϩ are reliably detected.The EF-hand superfamily of Ca 2ϩ -binding proteins includes, among others, the family of neuronal calcium sensor (NCS) 3 proteins (3), which are named because of their predominant expression in neuronal tissue. NCS proteins are grouped into five subfamilies and show a rather heterogeneous localization and function in the nervous system (4). In the photoreceptor cells of the vertebrate retina, for instance, recoverin and several isoforms of guanylate cyclase activating protein (GCAP) detect changes in Ca 2ϩ concentration during or after illumination and regulate their target proteins in Ca 2ϩ -dependent feedback loops (5).Recoverin inhibits rhodopsin kinase at high cytoplasmic Ca 2ϩ concentration (6 -9), a process that is thought to contribute to light adaptation of photoreceptor cells (9, 10). Recoverin harbors a myristoyl group at its N terminus (11), which is buried in a hydrophobic cleft in the Ca 2ϩ -free state (12). Upon Ca 2ϩ binding to the two functional EF-hands (EF-hand 2 and EFhand 3) (13) the acyl chain is exposed to the solvent. This socalled Ca 2ϩ -myrist...

“…NMR studies of WT-recoverin and fluorescence studies of mutants Rc E85Q and Rc E121Q suggest that the binding of Ca 2ϩ to recoverin is a sequential process: Ca 2ϩ binds first to EF-hand 3 and then to EF-hand 2 (17,31,39 …”

Section: Discussionmentioning

confidence: 99%

Ca2+-Myristoyl Switch in the Neuronal Calcium Sensor Recoverin Requires Different Functions of Ca2+-binding Sites

Senin¹,

Fischer²,

Komolov³

et al. 2002

Self Cite

128

Recoverin is an EF-hand Ca2؉ -binding protein that is suggested to control the activity of the G-protein-coupled receptor kinase GRK-1 or rhodopsin kinase in a Ca 2؉ -dependent manner. It undergoes a Ca 2؉ -myristoyl switch when Ca 2؉ binds to EF-hand 2 and 3. We investigated the mechanism of this switch by the use of point mutations in EF-hand 2 (E85Q) and 3 (E121Q) that impair their Ca 2؉ binding. EF-hand 2 and 3 display different properties and serve different functions. Binding of Ca 2؉ to recoverin is a sequential process, wherein EFhand 3 is occupied first followed by the filling of EFhand 2. After EF-hand 3 bound Ca 2؉ , the subsequent filling of EF-hand 2 triggers the exposition of the myristoyl group and in turn binding of recoverin to membranes. In addition, EF-hand 2 controls the mean residence time of recoverin at membranes by decreasing the dissociation rate of recoverin from membranes by 10-fold. We discuss this mechanism as one critical step for inhibition of rhodopsin kinase by recoverin.G-protein-coupled receptor kinases provide desensitization of G-protein-coupled receptors by phosphorylation of serine and threonine residues at their cytoplasmic C terminus (1). A wellknown system represents the light absorbing pigment rhodopsin and the corresponding kinase, rhodopsin kinase, or GRK-1, which phosphorylates (and thus desensitizes) photobleached rhodopsin. Arrestin then binds to phosphorylated rhodopsin and thereby stops any further activation of the G-protein transducin (2, 3). Illumination causes the decrease in the concentration of the intracellular transmitters of excitation and adaption, cGMP and cytoplasmic [Ca 2ϩ ], respectively. The decrease in cytoplasmic [Ca 2ϩ ] is sensed by Ca 2ϩ sensor proteins such as recoverin (4 -6; for a recent review, see Ref. 7). Recoverin or the amphibian orthologue S-modulin (8, 9) inhibit rhodopsin kinase at high levels of free [Ca 2ϩ ], thereby relieving inhibition when the Ca 2ϩ level decreases. The Ca 2ϩ