CALCIUM BINDING PROTEINS

Calcium has been chosen by evolution to become the most important and versatile messenger inside cells. This selection process was based on the ability of constituents of the cell to bind calcium.

Nowadays many cellular calcium binding proteins with specific functions are known and for many more the function has yet to be discovered.

Table 1.2. calcium binding proteins in cells.

calcium binding protein Kd calcium (microM) localisation function

calmodulin ~1 (tablet.4.) throughout cell buffer, confers calcium sensitivity onto other proteins

calbindin D28K 0.3-0.5 nucleus/cytosol (3) buffer?

parvalbumin cytosol buffer?

annexing I and II >1 to >100 (2) not clear

troponin-C 1.8 (N-term)(l) calcium sensor in

muscle contraction

S-IOOP 1.6 site 1

800 site 2

?

calgizzarin (SIOOC) 520 (EC50) ?

synaptotagmin 4-6 membrane/vesicles calcium sensor in

exocytosis

calretinin 0.3-0.5 buffer?

calpain ~1 calpain I

-1000 calpain II

protease

1. (Davis, J.P. e t a l , 1999), 2. (Gerke, V. and Moss, S.E., 1997), 3.(German, D C . e t a l , 1997).

Calcium binding proteins can be classified into three different categories, based on a common theme in their calcium binding domains.

The first group consists of proteins that contain a calcium binding domain characterised by a ~30 amino acid helix-loop-helix repeat, called an EF-hand. EF-hand regions show a high affinity for calcium and consist of a 1 2 amino acid pocket with a helices on either side (England, P.J., 1986). EF-hand domains bind calcium complexed with seven oxygenated ligands in a structure which can be described as pentagonal bipyramid. Six of the oxygenated groups are contributed by the amino acids of calmodulin and one comes from a contributing water molecule (Fig 1.3). Calmodulin, parvalbumin, troponin C, myosin light chains, S-100 proteins and the P subunit of calcineurin are calcium binding proteins of this type. A second group of calcium binding proteins is characterised by conserved repeat units that bind calcium and interact with phospholipids in a calcium

dependent manner. These are the annexing. The last group is formed by proteins that bind calcium in a similar manner to protein kinase C, using the C2 domain. Proteins like this include protein kinase C and synaptotagmin.

1.8.1. EF-hand calcium binding domains

I will now describe EF-hand domains in more detail. The requirements for a protein to be able to bind calcium become clear if we look at what some known EF-hands have in common (table 1.3). First of all they contain a high number of aspartate and glutamate residues. These amino acids are negatively charged and can therefore help to complex the positively charged calcium ion at the binding site. Having a binding site with a strong negative charge, however, will also attract other positive ions like sodium, potassium and magnesium, all of which are present in millimolar concentrations, and hydrogen, which is present at similar concentrations to calcium in a resting cell.

Table 1.3. sequence of calcium binding sites adapted (Levine, B.A., Williams, R.J.P., 1988).

Calcium binding protein amino acids involved in calcium binding

(for each site the amino acids and their position are shown) Asp-5 Ï. Asp-5 3. Ser-5 5. Phe-5 7. GÏu-5 ^

Asp-90. Asp-92. Asp-94. Lys-96. Glu-101. H2O

Ala-15.Glu-17.Asp-19.Gln-22.Ser-24.Glu-27

Asp-54. Asn-56. Asp-5 8. Glu-60. Ser-62. Glu-65 Asp-27. Asp-29. Gly-31. Asp-3 3. Ser-3 5. Giu-3 8 Asp-63. Asp-65.Ser-67.Thr-69. Asp-71.Glu-74 Asp-103.Asn-105.Asp-107.Tyr-109.Asp-lll.Glu-114 Asp-139. Asn-141. Asp-143. Arg-145.Asp-147.Glu-150 Asp-20. Asp-22. Asn-24. Thr-26. Thr-2 8. Glu-31

Asp-56. Asp-58.Asn-60.Thr-60.Thr-62. Asp-64.Glu-67 Asp-93. Asp-95. Asn-97.Tyr-99. Ser-101. Glu-104 Asn-109. Asp-131. Asp-133.Glu-135.Asn-137.Glu-140 parvalbumin

intestinal calcium binding protein

troponin C

The intracellular calcium concentration on the other hand, is less than a microM. So how do calcium binding sites obtain selectivity for calcium over the other cations? The ionic radius of the calcium ion is 0.099 nm and that for the magnesium ion 0.066 nm, hydrogen, potassium and sodium are even smaller (Vogel, H.J., 1988). It is found that other polar amino acids such as Ser, Thr, Tyr, Asn, Gin, Arg, Lys, contribute to the calcium binding site, helping to shape the binding site to give an ideal fit for the calcium ion. Other cations, like magnesium, are therefore not able to interact as strongly as calcium with the charges in the binding site and hence show a much lower affinity (Malmendal, A. et a l, 1998). This Tif for calcium is illustrated in figure 1.3. In addition, the binding of magnesium is about 1 0^ times slower than calcium binding as measured by the rate of H2O displacement on oxygenated ligands (Levine, B.A., Williams, R.J.P.,

1988). T28 T26 D24 E31 D22 D20

Figure 1.3 EF-hand calcium binding domain.

A m ino acids that participate in calcium binding are indicated w ith their single letter am ino acid code and num ber in the calm odulin sequence. A m ino acid side chains are show n as green sticks, the m ain protein backbone as a grey ribbon and hydrogen bonds as dashed lines. C alcium is represented by a red sphere (Figure adapted from N elson-M R and C hazin-W J (1998) in C alm odulin and signal transduction (Van E ldik-L J and W aterson-D M eds) A cadem ic Press, N Y, pp 17-64; Fig 1).

EF-hand calcium binding domains usually occur in facing pairs, where the presence of one domain stabilises the other and increases the relative affinity of the binding site pair for calcium compared with single EF-hands (Nelson, M.R., Chazin, W.J., 1998).