Obtaining and characterization of EF‐hand mutants of recoverin

Алексеев, А. М.; Shulga-Morskoy, S. V.; Zinchenko, D. V.; Shulga-Morskaya, Svetlana; Suchkov, Dmitry V.; Vaganova, Svetlana; Senin, Ivan I.; Zargarov, A.A.; Липкин, В. М.; Akhtar, Muhammad; Philippov, Pavel P.

doi:10.1016/s0014-5793(98)01426-4

Cited by 19 publications

(23 citation statements)

References 27 publications

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“…In the first, a myristoylated calcium-binding mutant still associated weakly with reconstituted ROS membranes and even exhibited limited inhibition of RK (34). In the second, Rv was found in lipid rafts even in the presence of EGTA (35).…”

Section: Discussionmentioning

confidence: 98%

“…Our results have direct implications for the mechanism of Rv action. The studies that led to the calcium-myristoyl switch model of Rv regulation utilized recombinant myristoylated Rv for structural analysis (4,8,26) or for in vitro association with reconstituted ROS membranes or membrane vesicles (27,34). The only pertinent study employing Rv in its natural environment showed the protein to remain associated with ROS lipid rafts even upon calcium chelation (35).…”

Section: Discussionmentioning

confidence: 99%

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A Flagellum-specific Calcium Sensor

Buchanan

Ames

Asfaw

et al. 2005

Journal of Biological Chemistry

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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...

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Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

A Flagellum-specific Calcium Sensor

Buchanan

Ames

Asfaw

et al. 2005

Journal of Biological Chemistry

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“…EF-hand 2 seems to be critical for the control of rhodopsin kinase activity (31). Alekseev et al (27) found no inhibition of rhodopsin kinase by myristoylated Rc E85Q in the range of 1 to 100 M Ca 2ϩ . However, a small fraction of Rc E85Q (25%) can bind to membranes.…”

Section: Ca 2ϩ -Myristoyl Switch Of Recoverin 50370mentioning

confidence: 97%

“…To understand the mechanism of the Ca 2ϩ -myristoyl switch in greater molecular detail, we used recoverin mutants with impaired EF-hand Ca 2ϩ -binding sites (27). For this purpose, we introduced the substitutions E85Q (mutant Rc E85Q ) and E121Q (mutant Rc E121Q ) in EF-hand 2 and EF-hand 3 of recoverin, respectively, because we know that substitutions in the Z position of the loop of the EF-hand motif decrease the affinity of the Ca 2ϩ -binding site for Ca 2ϩ by about 1000-fold (28,29).…”

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

Senin¹,

Fischer²,

Komolov³

et al. 2002

Journal of Biological Chemistry

128

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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

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Structure and Membrane-Targeting Mechanism of Retinal Ca2+-Binding Proteins, Recoverin and GCAP-2

Ames

Ikura

2002

Advances in Experimental Medicine and Biology

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Obtaining and characterization of EF‐hand mutants of recoverin

Cited by 19 publications

References 27 publications

A Flagellum-specific Calcium Sensor

A Flagellum-specific Calcium Sensor

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

Structure and Membrane-Targeting Mechanism of Retinal Ca2+-Binding Proteins, Recoverin and GCAP-2

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