The linker of calmodulin — to helix or not to helix

doi:10.1016/0143-4160(92)90050-3

Cell Calcium

Volume 13, Issues 6–7, June–July 1992, Pages 363-376

https://doi.org/10.1016/0143-4160(92)90050-3 Get rights and content

Abstract

The linker regions of the central helices of calmodulin and of troponin C are observed to be α-helices in crystal and in solution. However, these linkers are predicted to be non-helical by standard algorithms. Further, there is strong evidence that when calmodulin interacts with some of its targets this linker helix bends. The linker appears to be delicately balanced between helical and non-helical conformations. A review of this subject suggests that one can anticipate more unpredicted conformations for the central helices of the score of other proteins that have four EF-hand domains.

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When cytosolic Ca2+ levels increase during signalling processes, the four Ca2+ sites of CaM are filled, and CaM undergoes conformational changes to expose hydrophobic patches (Chin and Means, 2000; Klee et al., 1986). In the presence of Ca2+, each lobe exposes a hydrophobic cleft with a significant number of phenylalanine and methionine residues (Babu et al., 1985, 1988; Kretsinger, 1992). As a consequence, the affinity for protein targets, including a range of kinases, is enhanced (Berridge et al., 2000; Tidow and Nissen, 2013; Hoeflich and Ikura, 2002; O'Connell et al., 2013).
Calmodulin (CaM) is a ubiquitous Ca²⁺ sensing protein that binds to and modulates numerous target proteins and enzymes during cellular signaling processes. A large number of CaM-target complexes have been identified and structurally characterized, revealing a wide diversity of CaM-binding modes. A newly identified target is creatine kinase (CK), a central enzyme in cellular energy homeostasis. This study reports two high-resolution X-ray structures, determined to 1.24 Å and 1.43 Å resolution, of calmodulin in complex with peptides from human brain and muscle CK, respectively. Both complexes adopt a rare extended binding mode with an observed stoichiometry of 1:2 CaM:peptide, confirmed by isothermal titration calorimetry, suggesting that each CaM domain independently binds one CK peptide in a Ca²⁺-depended manner. While the overall binding mode is similar between the structures with muscle or brain-type CK peptides, the most significant difference is the opposite binding orientation of the peptides in the N-terminal domain. This may extrapolate into distinct binding modes and regulation of the full-length CK isoforms. The structural insights gained in this study strengthen the link between cellular energy homeostasis and Ca²⁺-mediated cell signaling and may shed light on ways by which cells can ‘fine tune’ their energy levels to match the spatial and temporal demands.
Backbone dynamic properties of the central linker region of calcium-calmodulin in 35% trifluoroethanol
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The backbone dynamic properties of uniformly ¹⁵N-labeled calcium-saturated calmodulin (Ca²⁺-CaM) in 35% 2,2,2-trifluoroethanol (TFE) have been examined by ¹⁵N NMR relaxation methods. This particular solvent was chosen in order to mimic the conditions in which CaM was crystallized, which included the presence of alcohols. Special attention was paid to the central linker region of Ca²⁺-CaM, which is a long, solvent-exposed α-helix in the crystal structure but is known to be partially unwound and flexible in solution. ¹⁵N T₁, T₂, and ¹⁵N–{¹H} NOE values were determined for both Ca²⁺-CaM in H₂O and Ca²⁺-CaM in 35% TFE, and the results indicated that the presence of 35% TFE did indeed induce a more ordered conformation in the central linker, with order parameters for Asp78–Glu80 of 0.29, 0.17, and 0.27 in H₂O and 0.82, 0.66, and 0.64 in 35% TFE. However, ¹⁵N–{¹H} NOE values showed that these residues were still slightly more flexible than the rest of the molecule in 35% TFE (Asp78–Glu80 ¹⁵N–{¹H} NOE=0.46, 0.46, and 0.51). Furthermore, there is still independent motion of the two lobes of Ca²⁺-CaM in 35% TFE, with motional correlation times of ∼10 and ∼9 ns for the N- and C-lobes, respectively, indicating that 35% TFE was not sufficient to force Ca²⁺-CaM into a rigid dumbbell-shaped molecule as seen in the crystal structure. Additional factors that could further stabilize the structure of CaM in the crystal include pH, temperature, and crystal packing.
Calcium-dependent stabilization of the central sequence between Met<sup>76</sup> and Ser<sup>81</sup> in vertebrate calmodulin
2001, Biophysical Journal
Citation Excerpt :
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Spin-label electron paramagnetic resonance (EPR) provides optimal resolution of dynamic and conformational heterogeneity on the nanosecond time-scale and was used to assess the structure of the sequence between Met⁷⁶ and Ser⁸¹ in vertebrate calmodulin (CaM). Previous fluorescence resonance energy transfer and anisotropy measurements indicate that the opposing domains of CaM are structurally coupled and the interconnecting central sequence adopts conformationally distinct structures in the apo-form and following calcium activation. In contrast, NMR data suggest that the opposing domains of CaM undergo independent rotational dynamics and that the sequence between Met⁷⁶ and Ser⁸¹ in the central sequence functions as a flexible linker that connects two structurally independent domains. However, these latter measurements also resolve weak internuclear interactions that suggest the formation of transient helical structures that are stable on the nanosecond time-scale within the sequence between Met⁷⁶ and Asp⁸⁰ in apo-CaM (H. Kuboniwa, N. Tjandra, S. Grzekiek, H. Ren, C. B. Klee, and A. Bax, 1995, Nat. Struct. Biol. 2:768–776). This reported conformational heterogeneity was resolved using site-directed mutagenesis and spin-label EPR, which detects two component spectra for 1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)-methanethiosulfonate spin labels (MTSSL) bound to CaM mutants T79C and S81C that include a motionally restricted component. In comparison to MTSSL bound within stable helical regions, the fractional contribution of the immobilized component at these positions is enhanced upon the addition of small amounts of the helicogenic solvent trifluoroethanol (TFE). These results suggest that the immobilized component reflects the formation of stable secondary structures. Similar spectral changes are observed upon calcium activation, suggesting a calcium-dependent stabilization of the secondary structure. No corresponding changes are observed in either the solvent accessibility to molecular oxygen or the maximal hyperfine splitting. In contrast, more complex spectral changes in the line-shape and maximal hyperfine splitting are observed for spin labels bound to sites that undergo tertiary contact interactions. These results suggest that spin labels at solvent-exposed positions within the central sequence are primarily sensitive to backbone fluctuations and that either TFE or calcium binding stabilizes the secondary structure of the sequence between Met⁷⁶ and Ser⁸¹ and modulates the structural coupling between the opposing domains of CaM.
The calmodulin multigene family as a unique case of genetic redundancy: Multiple levels of regulation to provide spatial and temporal control of calmodulin pools?
2000, Cell Calcium
Calmodulin (CaM) is a ubiquitous, highly conserved calcium sensor protein involved in the regulation of a wide variety of cellular events. In vertebrates, an identical CaM protein is encoded by a family of non-allelic genes, raising questions concerning the evolutionary pressure responsible for the maintenance of this apparently redundant family. Here we review the evidence that the control of the spatial and temporal availability of CaM may require multiple regulatory levels to ensure the proper localization, maintenance and size of intracellular CaM pools. Differential transcription of the CaM genes provides one level of regulation to meet tissue-specific, developmental and cell-specific needs for altered CaM levels. Post-transcriptional regulation occurs at the level of mRNA stability, perhaps dependent on alternative polyadenylation and differences in the untranslated sequences of the multiple gene transcripts. Recent evidence indicates that trafficking of specific CaM mRNAs may occur to specialized cellular locales such as the dendrites of neurons. This could allow local CaM synthesis and thereby help generate local pools of CaM. Local CaM activity may be further regulated by post-translational mechanisms such as phosphorylation or storage of CaM in a ‘masked’ form. The spatial resolution of CaM activity is enhanced by the limited free diffusion of CaM combined with differential affinity for and availability of target proteins. Preserving multiple CaM genes with divergent noncoding sequences may be necessary in complex organisms to ensure that the many CaM-dependent processes occur with the requisite spatial and temporal resolution. Transgenic mouse models and studies on mice carrying single and double gene ‘knockouts’ promise to shed further light on the role of specificity versus redundancy in the evolutionary maintenance of the vertebrate CaM multigene family.
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Calmodulin (CaM) is an intracellular cooperative calcium-binding protein essential for activating many diverse target proteins. Biophysical studies of the calcium-induced conformational changes of CaM disagree on the structure of the linker between domains and possible orientations of the domains. Molecular dynamics studies have predicted that Ca4(2+)CaM is in equilibrium between an extended and compact conformation and that Arg74 and Arg90 are critical to the compaction process. In this study gel permeation chromatography was used to resolve calcium-induced changes in the hydrated shape of CaM at pH 7.4 and 5.6. Results showed that mutation of Arg 74 to Ala increases the R(s) as predicted; however, the average separation of domains in Ca4(2+)-CaM was larger than predicted by molecular dynamics. Mutation of Arg90 to Ala or Gly affected the dimensions of apo-CaM more than those of Ca4(2+)-CaM. Calcium binding to CaM and mutants (R74A-CaM, R90A-CaM, and R90G-CaM) lowered the Stokes radius (R(s)). Differences between R(s) values reported here and Rg values determined by small-angle x-ray scattering studies illustrate the importance of using multiple techniques to explore the solution properties of a flexible protein such as CaM.
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The linker of calmodulin — to helix or not to helix

Abstract

J. Mol. Biol.

J. Biol. Chem.

J. Mol. Biol.

J. Inorg. Biochem.

Biochim. Biophys. Acta

J. Biol. Chem.

FEBS. Lett

Biochem. Biophys. Res. Commun.

J. Mol. Biol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Mol. Biol.

Biochimie

Structure of the calcium regulatory muscle protein troponin-C at 2.8 Å resolution

Nature

Molecular structure of troponin C from chicken skeletal muscle at 3-angstrom resolution

Science

Three-dimensional structure of calmodulin

Nature

Kinetics of Ca2+ release show interactions between the two classes of sites of troponin C

J. Biol. Chem.

A study of the interactions between residues in the C-terminal half of calmodulin by one and two-dimensional NMR methods and computer modelling

Eur. J. Biochem.

1H-NMR studies of calmodulin. The nature of the Ca2+-dependent conformational change

Eur. J. Biochem.

Metal ion and drug binding to proteolytic fragments of calmodulin: proteolytic, cadmium-113, and proton nuclear magnetic resonance studies

Biochemistry

Kinetics of calcium dissociation from calmodulin and its tryptic fragments. A stopped-flow fluorescence study using Quin 2 reveals a two-domain structure

Eur. J. Biochem.

Calcium release from intact calmodulin and calmodulin fragment 78–148 measured by stopped-flow fluorescence with 2-p-toluidinylnaphthalene sulfonate. Effect of calmodulin fragments on cardiac sarcoplasmic reticulum

Eur. J. Biochem.

Kinetics of Ca²⁺ release show interactions between the two classes of sites of troponin C

¹H-NMR studies of calmodulin. The nature of the Ca²⁺-dependent conformational change