Calmodulin Binds to Caldesmon in an Antiparallel Manner

Wang, Enzhong; Zhuang, Shaobin; Kordowska, Jolanta; Grabarek, Zenon; Wang, Chih‐Lueh Albert

doi:10.1021/bi963075h

Cited by 22 publications

(12 citation statements)

References 49 publications

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“…We have observed that the CaD-B sequence binds relatively poorly to CaM, which is in agreement with other work (24,58). Intact caldesmon appears to bind CaM in an antiparallel fashion such that the CaD-B sequence interacts with the N-lobe of CaM (72). Therefore, it seems likely that the CaD-A sequence binds first to the C-terminal lobe of CaM, which raises the local concentration of the CaD-B sequence, enabling it to bind to the N-terminal lobe of CaM.…”

Section: Thermodynamics Of Calmodulin-peptide Recognitionsupporting

confidence: 93%

Energetics of Target Peptide Binding by Calmodulin Reveals Different Modes of Binding

Brokx

López

Vogel

et al. 2001

Journal of Biological Chemistry

112

155

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Understanding detailed molecular mechanisms that govern macromolecular interactions represents one of the major goals of structural biology. One of the important prerequisites for success in this line of research depends on the choice of an appropriate biological system. Calcium-dependent target recognition by calmodulin might be an ideal model system, because of its well characterized nature and its central role in cellular metabolism. Calmodulin (CaM) 1 is a small, acidic, eukaryotic Ca 2ϩ

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Section: Thermodynamics Of Calmodulin-peptide Recognitionsupporting

confidence: 93%

Energetics of Target Peptide Binding by Calmodulin Reveals Different Modes of Binding

Brokx

López

Vogel

et al. 2001

Journal of Biological Chemistry

112

155

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“…It has been suggested that CaD may contain two distinct CaM-binding domains, and that these may bind simultaneously to Ca 2ϩ -CaM (Marston et al, 1994;Mezgueldi et al, 1994;Wang et al, 1997;Zhou et al, 1997). This prediction was investigated using DLS.…”

Section: Dls Of Caldesmon-calmodulin Interactionsmentioning

confidence: 99%

Dynamic Light Scattering Study of Calmodulin–Target Peptide Complexes

Papish

Tari²,

Vogel

2002

Biophysical Journal

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Dynamic light scattering (DLS) has been used to assess the influence of eleven different synthetic peptides, comprising the calmodulin (CaM)-binding domains of various CaM-binding proteins, on the structure of apo-CaM (calcium-free) and Ca(2+)-CaM. Peptides that bind CaM in a 1:1 and 2:1 peptide-to-protein ratio were studied, as were solutions of CaM bound simultaneously to two different peptides. DLS was also used to investigate the effect of Ca(2+) on the N- and C-terminal CaM fragments TR1C and TR2C, and to determine whether the two lobes of CaM interact in solution. The results obtained in this study were comparable to similar solution studies performed for some of these peptides using small-angle x-ray scattering. The addition of Ca(2+) to apo-CaM increased the hydrodynamic radius from 2.5 to 3.0 nm. The peptides studied induced a collapse of the elongated Ca(2+)-CaM structure to a more globular form, decreasing its hydrodynamic radius by an average of 25%. None of the peptides had an effect on the conformation of apo-CaM, indicating that either most of the peptides did not interact with apo-CaM, or if bound, they did not cause a large conformational change. The hydrodynamic radii of TR1C and TR2C CaM fragments were not significantly affected by the addition of Ca(2+). The addition of a target peptide and Ca(2+) to the two fragments of CaM, suggest that a globular complex is forming, as has been seen in nuclear magnetic resonance solution studies. This work demonstrates that dynamic light scattering is an inexpensive and efficient technique for assessing large-scale conformational changes that take place in calmodulin and related proteins upon binding of Ca(2+) ions and peptides, and provides a qualitative picture of how this occurs. This work also illustrates that DLS provides a rapid screening method for identifying new CaM targets.

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“…Measurement of conformational change as a function of [Ca# + ] by means of trinitrobenzene sulphonate fluorescence, which has been used with some calpain fragments [34], has not been successful with the whole enzyme. Terbium chloride is frequently used to study Ca# +binding sites, because of the characteristic enhancement of Tb$ + fluorescence when bound to protein, and in favourable cases binding at specific sites can be monitored by tyrosine-or tryptophan-sensitized Tb$ + luminescence [35,36]. It has been shown previously that Tb$ + can activate both µand m-calpain, but it also causes calpain aggregation [24,25].…”

Section: Ca 2 + Titrations In the Whole Enzyme And Comparisons Of K Dmentioning

confidence: 99%

Roles of individual EF-hands in the activation of m-calpain by calcium

DUTT¹,

Arthur²,

GROCHULSKI³

et al. 2000

Biochem. J.

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m-Calpain is a heterodimeric, cytosolic, thiol protease, which is activated by Ca(2+)-binding to EF-hands in the C-terminal domains of both subunits. There are four potential Ca(2+)-binding EF-hands in each subunit, but their relative affinities for Ca(2+) are not known. In the present study mutations were made in both subunits to reduce the Ca(2+)-binding affinity at one or more EF-hands in one or both subunits. X-ray crystallography of some of the mutated small subunits showed that Ca(2+) did not bind to the mutated EF-hands, but that its binding at other sites was not affected. The structures of the mutant small subunits in the presence of Ca(2+) were otherwise identical to that of the Ca(2+)-bound wild-type small subunit. In the whole enzyme the wild-type macroscopic Ca(2+) requirement (K(d)) was approx. 350 microM. The mutations did not affect the maximum specific activity of the enzyme, but caused increases in K(d), which were characteristic of each site. All the EF-hands could be mutated in various combinations without loss of activity, but preservation of at least one wild-type EF-hand 3 sequence was required to maintain K(d) values lower than 1 mM. The results suggest that all the EF-hands can contribute co-operatively to calpain activation, but that EF-hand 3, in both subunits, has the highest intrinsic affinity for Ca(2+) and provides the major driving force for conformational change.

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Calmodulin Binds to Caldesmon in an Antiparallel Manner

Cited by 22 publications

References 49 publications

Energetics of Target Peptide Binding by Calmodulin Reveals Different Modes of Binding

Energetics of Target Peptide Binding by Calmodulin Reveals Different Modes of Binding

Dynamic Light Scattering Study of Calmodulin–Target Peptide Complexes

Roles of individual EF-hands in the activation of m-calpain by calcium

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