Calcium-induced Shape Change of Calmodulin with Mastoparan Studied by Solution X-ray Scattering

Yoshino, Hidenori; Minari, Osamu; Matsushima, Norio; Ueki, Tatzuo; Miyake, Yasuhiro; Matsuo, T; Izumi, Yoshinobu

doi:10.1016/s0021-9258(19)47170-6

Cited by 42 publications

(14 citation statements)

References 25 publications

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“…Each lobe also contains a hydrophobic patch believed to serve as a binding site for calmodulin targets (LaPorte et al, 1980; Tanaka & Hidaka, 1980;Vogel et al, 1983;Newton et al, 1984). The size and shape of the globular domains determined by small-angle X-ray scattering studies of calmodulin in solution are consistent with those in the crystal, but the distance between them is significantly shorter (Seaton et al, 1985; Heidorn & Trewhella, 1988;Yoshino et al, 1989). The structure of Ca2+-saturated calmodulin in solution, currently being determined by three-dimensional nuclear magnetic resonance (NMR),1 may help to explain this discrepancy.2 Very little is known about the structure of Ca2+-free calmodulin except that it is different from that of Ca2+-saturated calmodulin [for reviews, see Forsen et al (1986), Evans et al (1988].…”

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confidence: 73%

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Calcium-induced sensitization of the central helix of calmodulin to proteolysis

Mackall¹,

Klee²

1991

Biochemistry

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The rate of proteolysis of trypsin-sensitive bonds was used to examine the nature of the structural changes accompanying Ca2+ and Mg2+ binding to calmodulin. In the Ca(2+)-free form, the rates of proteolysis at Arg-106 and Arg-37 are rapid (greater than 300 and 28 nmol min-1 mL-1, respectively), the bonds at Arg-74, Lys-75, and Lys-77, in the central helix, are cleaved more slowly (10 nmol min-1 mL-1), and a lag in the cleavage at the remaining bonds (Lys-13, Lys-30, Arg-86, Arg-90, and Arg-126) suggests that they are not cleaved in the native protein. High concentrations of Ca2+, but not Mg2+, almost completely abolish proteolysis at Arg-106 and drastically reduce the rate of cleavage at Arg-37. Both Ca2+ and Mg2+ exert a moderate protective effect on the proteolysis of the central helix. These results suggest that the F-helix of domains III and, to a lesser extent, the F-helix of domain I are somewhat flexible in the Ca(2+)-free form and are stabilized by Ca2+. Whereas full occupancy of the four Ca(2+)-binding sites produces little change in the susceptibility of the central helix to proteolytic attack, binding of two Ca2+ produces a 10-fold enhancement of the rate of proteolysis in this part of the molecule. We propose that at intermediate Ca2+ levels the flexibility of the central helix of calmodulin is greatly increased, resulting in the transient formation of intermediates which have not been detected by spectroscopic techniques but are trapped by the irreversible action of trypsin.

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confidence: 73%

“…the NMR spectra and small-angle X-ray scattering measurements of calmodulin complexed with mastoporan or the calmodulin-binding domain of myosin light chain kinase (Heidorn et al, 1989;Kataoka et al, 1989;Yoshino et al, 1989;Ikura et al, 1989). Flexibility in the central helix could therefore be essential for the mechanism of action of calmo-…”

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confidence: 99%

Calcium-induced sensitization of the central helix of calmodulin to proteolysis

Mackall¹,

Klee²

1991

Biochemistry

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“…The calmodulin-C20W complex appears to be a dumbbell shape, while the calmodulin-C24W complex is globular. In forming a globular complex, C24W appears to fall in to the same group as the calmodulin-binding peptides melittin (Kataoka et al, 1989), mastoparan (Matsushima et al, 1989;Yoshino et al, 1989), and the Ml3 peptide derived from the calmodulin-binding domain of myosin light chain kinase (Heidorn et al, 1989).…”

Section: Discussionmentioning

confidence: 99%

Small-angle x-ray scattering study of calmodulin bound to two peptides corresponding to parts of the calmodulin-binding domain of the plasma membrane calcium pump

Kataoka

Head²,

Vorherr³

et al. 1991

Biochemistry

107

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The interaction between calmodulin (CaM) and two synthetic peptides, C20W and C24W, corresponding to parts of the calmodulin-binding domain of the Ca2+ pump of human erythrocytes, has been studied by using small-angle X-ray scattering (SAXS). The total length of the CaM-binding domain of the enzyme is estimated to be 28 amino acids. C20W contains the 20 N-terminal amino acids of this domain, C24W the 24 C-terminal amino acids. The experiments have shown that the binding of either peptide results in a complex with a radius of gyration (Rg) smaller than that of CaM. The complex between CaM and C20W revealed an interatomic length distribution function, P(r), similar to that of calmodulin alone, indicating that the complex retains an extended, dumbbell-shaped structure. By contrast, the binding of C24W resulted in the formation of a globular structure similar to those observed with many other CaM-binding peptides.

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“…Therefore, while the crystal structure of calcium-saturated CaM indicates that CaM contains two homologous globular domains which include an extended eight-turn R-helix often referred to as the central helix (9)(10)(11), high-field NMR measurements suggest that in solution the central helix is disrupted between Asp 78 and Ser 81 , resulting in the independent rotational motion of the opposing globular domains of CaM (7,(12)(13). However, the observed structural uncoupling between the opposing globular domains observed using NMR spectroscopy is inconsistent with a range of other biochemical and biophysical measurements, including small-angle X-ray scattering and time-resolved fluorescence spectroscopy, which suggest that there are important structural transitions associated with each of the opposing globular domains upon calcium activation that result in concerted conformational changes involving both amino-and carboxyl-terminal domains (6,(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33). The apparent discrepancy between results obtained using NMR spectroscopy and these later results may be related to the fact that NMR spectroscopy detects only conformational states that are both highly populated and long-lived (34).…”

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confidence: 97%

Calcium-Dependent Structural Coupling between Opposing Globular Domains of Calmodulin Involves the Central Helix

1999

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We have used fluorescence spectroscopy to investigate the average structure and extent of conformational heterogeneity associated with the central helix in calmodulin (CaM), a sequence that contributes to calcium binding sites 2 and 3 and connects the amino- and carboxyl-terminal globular domains. Using site-directed mutagenesis, a double mutant was constructed involving conservative substitution of Tyr(99) --> Trp(99) and Leu(69) --> Cys(69) with no significant effect on the secondary structure of CaM. These mutation sites are at opposite ends of the central helix. Trp(99) acts as a fluorescence resonance energy transfer (FRET) donor in distance measurements of the conformation of the central helix. Cys(69) provides a reactive group for the covalent attachment of the FRET acceptor 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS). AEDANS-modified CaM fully activates the plasma membrane (PM) Ca-ATPase, indicating that the native structure is retained following site-directed mutagenesis and chemical modification. We find that the average spatial separation between Trp(99) and AEDANS covalently bound to Cys(69) decreases by approximately 7 +/- 2 A upon calcium binding. However, irrespective of calcium binding, there is little change in the conformational heterogeneity associated with the central helix under physiologically relevant conditions (i.e., pH 7.5, 0.1 M KCl). These results indicate that calcium activation alters the spatial arrangement of the opposing globular domains between two defined conformations. In contrast, under conditions of low ionic strength or pH the structure of CaM is altered and the conformational heterogeneity of the central helix is decreased upon calcium activation. These results suggest the presence of important ionizable groups that affect the structure of the central helix, which may play an important role in mediating the ability of CaM to rapidly bind and activate target proteins.

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Calcium-induced Shape Change of Calmodulin with Mastoparan Studied by Solution X-ray Scattering

Cited by 42 publications

References 25 publications

Calcium-induced sensitization of the central helix of calmodulin to proteolysis

Calcium-induced sensitization of the central helix of calmodulin to proteolysis

Small-angle x-ray scattering study of calmodulin bound to two peptides corresponding to parts of the calmodulin-binding domain of the plasma membrane calcium pump

Calcium-Dependent Structural Coupling between Opposing Globular Domains of Calmodulin Involves the Central Helix

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