Calcium‐Binding Proteins


Calcium ions act as critical second messengers in all eukaryotes and plants and are of fundamental importance to the normal development and functioning of all higher animal species. In mammals, calcium influences almost all aspects of cellular physiology and is particularly relevant for normal cardiac and neuronal function. Calcium signals are generated by the complex interplay of two opposing systems the first of which mediates calcium entry into the cell cytoplasm and a second clearance system that removes it. Exactly how these two systems integrate in space and time determines the precise nature of a given calcium signal. When the cytosolic calcium is elevated, it can be detected by a large number of diverse binding proteins, many of which decode specific signals into a biological output. According to well‐conserved structural elements, these proteins can be grouped into different families including annexins, C2 domain proteins and EF‐hand proteins.

Key Concepts

  • Calcium is an evolutionarily ancient and fundamental intracellular second messenger used by plants, animals and bacteria.
  • Complex spatiotemporal calcium signals generated within cells are coupled to changes in biological activity. This is largely achieved through the presence of proteins that are able to rapidly bind Ca2+ with high affinity
  • These calcium‐binding proteins, or CaBPs, contain unique calcium chelating motifs formed by multiple amino acids in the protein primary sequence.
  • Calcium binding to a CaBP can induce significant conformational rearrangements in the protein tertiary structure, which can expose binding sites for specific target interaction partners, thereby coupling the calcium signal to a unique cell signalling pathway.
  • Calcium signalling events mediated by various CaBPs are becoming increasingly important as potential biomarkers and therapeutic targets for a range of human diseases.

Keywords: calcium; EF‐hand; annexins; C2 domain; gamma glutamate

Figure 1. Structure of the annexins. (a) Annexins consist of an N‐terminal domain of variable length and a protein core composed of four annexin folds which contain the binding sites for Ca2+ and phospholipids. Boxes (a–e) represent α helices as observed in the crystal structure of annexin V. Annexin VI is made up of eight annexin folds. Shown here for annexin II, in the N‐terminal region, phosphorylation sites and binding sites for targets (S100A10) are present. (b) Crystal structure of annexin A1, which binds to S100A11. (i) The calcium‐free structure of annexin A1 (Rosengarth et al., ); RCSB Protein Data bank ( PDB entry code:1HM6. (ii) The calcium‐bound structure (orange spheres) showing the form of the protein that interacts with negatively charged phospholipids (Rosengarth and Luecke, ); PDB entry code: 1MCX. Of note in these structures, the yellow helix in (i) represents the N‐terminal amphipathic helix of Annexin A1 which, in calcium‐free conditions, is buried within the core of the protein. Upon calcium binding, the N‐terminus is ejected from the core and is disordered. This domain could not be resolved in the calcium‐bound structure (ii) but may contribute to membrane binding. Reproduced with permission from Rosengarth and Luecke (2003) © Elsevier.
Figure 2. Typical structure of a C2‐domain protein. (a) Ribbon diagram showing the eight strands of an S‐family or type I topology (top) and P‐family of type II C2 domain (bottom). Potential Ca2+‐binding regions (CBRs) exist in the loop domains located at one end of the folded domain. Illustrative crystal structures of the PKCα S‐type C2 domain (green) and PLCδ P‐family C2 domain (purple). The overall architecture of both classes of C2 domain is very similar (shown in more detail in (b)). Nt, N‐terminus; Ct, C‐terminus; Yellow spheres, bound Ca2+ ions. (b) A representative Type I C2 domain from protein kinase‐Cβ (green) superimposed with a representative Type II C2 domain from phospholipase‐Cδ. Both topologies have highly similar folds and are related by circular permutation. The calcium‐binding loop regions of both domains are highlighted by the red sphere, which represents a Ca2+ ion. (c) Representative domain architecture of C2 domain containing proteins synaptotagmin (Syt, Type I), protein kinase C alpha (PKC, Type I) and phospholipase C beta (Type II). Other domains present in these proteins include: TM, trans‐membrane; C1, diacylglycerol binding domain; sTKc and sTKx, kinase domains; EF, EF‐hand calcium binding motif; PLCXC and PLCYC, phospholipase C catalytic domains. Reproduced with permission from Corbalan‐Garcia and Gomez‐Fernandez (2014) © Elevier and Jimenez et al. (2003) © Elsevier.
Figure 3. EF‐hand motif. (a) The three‐dimensional arrangement of the EF‐hand motif can be simulated by the right hand, with the index finger representing the E‐helix (residues 1–10), the bent middle finger symbolising the Ca2+‐binding loop (10–21) and the thumb depicting the F‐helix (19–29). The geometrical arrangement of the seven oxygen ligands coordinating the Ca2+ ion can be best described as a pentagonal bipyramid. Modified from Celio et al. (). (b) Crystal structure from EF‐domain of PV. Modified from Kretsinger and Nockolds (). (c) Coordination of the Ca2+ ion in CaM with the seven oxygen ligands (five from side chains, one from a carbonyl group of the backbone and one from a water molecule). (d) Consensus sequence for the canonical EF‐hand domain. The symbol n denotes nonpolar side chains and the positions X, Y, Z, –Y, –X and –Z provide the oxygen ligand for the Ca2+ binding. At position –Y, a carbonyl oxygen bonds to the Ca2+ ion. The –X ligand (usually glutamate) binds Ca2+ with both oxygen atoms from the carboxylate group (Kawasaki et al., ). * Any amino acid; I, isoleucine. (e) Solution NMR structure of the high‐affinity Ca2+‐binding EF‐hand 1 domain from the CaM‐related protein CaBP7 (McCue et al., ), illustrating the classical three‐dimensional helix–loop–helix arrangement described in (a). PDB entry code: 2LV7.
Figure 4. The mechanism of SOCE. This diagram is reproduced from Feske et al. () and outlines the pathway for SOCE specifically in lymphocytes; however, the basic STIM‐Orai machinery exists ubiquitously and it is only the specific cell signalling pathway that induces initial ER Ca2+ release that differs between cell types. Activation of T‐cell receptor ultimately couples to phospholipase C activation and liberation of IP3 from PM PIP2. IP3 induces ER Ca2+ release and store depletion that is sensed by STIM1 and accompanied by unfolding of the EF‐hand‐SAM domain on loss of Ca2+ binding. Oligomerisation of the unfolded luminal STIM1 domains leads to recruitment of STIM cytoplasmic domains to defined PM domains rich in negatively charged phospholipids. Orai is then recruited to these ER–PM junctions, where it is activated to permit influx of extracellular Ca2+. Reproduced with permission from Feske et al. (2012) © Nature Publishing Group.
Figure 5. Structure of CaM. (Apo) In the Ca2+‐free apo form, CaM consists of two pairs of EF‐hand domains (N‐terminal, green and C‐terminal, orange) that are connected by a flexible central linker that is partially unstructured (Babu et al., ). (Ca2+) On Ca2+ binding, CaM undergoes significant conformational rearrangements in the EF‐hand lobes, becoming more globular with the central linker domain adopting a helical structure that can wrap around target proteins as shown in Ca2+/peptide, where CaM is illustrated bound to its target peptide from CaM‐dependent kinase II (Wall et al., ). (PDB entry codes: Apo, 1CFD; Ca2+, 3CLN and Ca2+/peptide, 1CM1)
Figure 6. Vitamin K‐dependent proteins. (a) Structure of the modified amino acid γ‐glutamate (Gla). (b) Picture of prothrombin fragment 1. The peptide is in wire‐frame form except for Ala1, Phe5, Leu6 and Val9 (Brookhaven Data Base, PDB entry code: 2PF2.). Modified from Nelsestuen and Ostrowski (1999).


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Further Reading

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Haynes, Lee(Feb 2016) Calcium‐Binding Proteins. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0001347.pub3]