The S100 proteins represent the largest subgroup within the EF-hand Ca2+ protein family.
Whereas calmodulin exists as a monomer with four very similar EF-hands the S100 proteins contains two different types of EF-hands. S100 proteins form dimers and bind besides Ca2+ (Kd = 20-500 µM), also Zn2+ (Kd = 0.1-2000 µM) and Cu2+ (Kd = 0.4-5 µM) with high affinities. A total of 21 members in human have currently been assigned to the S100 family with sequence identities ranging from 22% to 57% (Figure 1.5.) (Fritz and Heizmann, 2004; Heizmann et al., 2003; Marenholz et al., 2006).
Figure 1.5: Figure legend see next page.
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Figure 1.5: Multiple sequence alignment of human S100 proteins. The Ca2+-coordinating residues are highlighted in dark red (side chain coordination), and in light red (backbone oxygen coordination). The Zn2+-coordinating residues in S100A7 and the conserved binding site in S100A12 are highlighted in magenta. Hydrophobic residues that are essential for dimerization are highlighted in green. Residues that are putative Zn2+ ligands in other S100 proteins are highlighted in yellow (cysteine) and blue (histidine), respectively. Adapted & modified from Fritz and Heizmann, 2004; Marenholz et al., 2004.
The first S100 protein was discovered in 1965 by Moore. He investigated proteins specific for the nervous system in higher animals and isolated a protein from bovine brain that turned out to be completely soluble in 100% ammonium sulfate (Moore, 1965). Therefore, he called the isolated molecule S100 protein. Later research has shown that Moore's original preparation primarily contained two polypeptides, which are now called S100A1 and S100B (Hilt and Kligman, 1991). S100 proteins are small acidic proteins with a size of 10-12 kDa and except calbindin D9k (S100G, only monomeric) they form non-covalent homo- and heterodimers. An example for a typical structure of a Ca2+-S100 protein is shown in Figure 1.6. (Otterbein et al., 2002).
Figure 1.6: Schematic representation of human Ca -S100A6 (PDB code 1K96) showing the homodimer and bound Ca ions (yellow). Adapted from Fritz and Heizmann, 2004.
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Each S100 subunit consists of two EF-hands, an N-terminal EF-hand and a C-terminal EF-hand, which is followed by a C-terminal extension. The two EF-hands are connected by an intermediate region, referred as the hinge region. The S100 members differ from one another mostly in the length and sequence of the hinge region and the C-terminal extension, which are thus suggested to determine the biological activity of individual proteins (Donato, 2003). The plane for dimerization (~2500 Å2) of S100 proteins is composed by hydrophobic residues which are absent in the case for calbindin. Generally, S100 proteins are characterized by the presence of two Ca2+ binding sites of the EF-hand type (i.e., helix-loop-helix), whereby the N-terminal EF-motif is composed of 14 residues which is specific for S100 proteins and a classical EF-hand motif with 12 residues at the C-terminus (Figure 1.5.) (Fritz and Heizmann, 2004). Interestingly, the N-terminal EF-hand of S100 proteins exhibits only small conformational changes upon Ca2+ binding. Helix HII undergoes a small rearrangement in direction of the Ca2+ binding site with virtually no change in the interhelical angle of helix HI and HII (Figure 1.7. A).
Whereas the C-terminal canonical EF-hand that represents the target interaction site of S100 proteins undergoes a dramatic change in its conformation. In the Ca2+ free state, the helices HIII and HIV flanking the EF-hand loop adopt an antiparallel conformation similar to the EF-hands in Ca2+ free calmodulin (Ishida et al., 2002). Upon Ca2+ binding to S100 proteins, there is a large change in the orientation of helix HIII (Figure 1.7. B) whereas helix HIV that is engaged in the dimer interface does not move (Fritz and Heizmann, 2004). One consequence of the different length in the different EF-hands is that Ca2+
binding to the individual EF hands occur with different affinities. With a lower affinity in the case of the N-terminal site and a ~100-times higher affinity in the case of the C-terminal site.
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Figure 1.7: Conformational change in EF-hands of S100 proteins. The S100 specific EF-hand is depicted in (A), the canonical EF-hand in (B). The Ca free (apo) protein is shown in blue, the Ca loaded form in red. Ca ions are shown as yellow spheres. The coordinates were taken from the crystal structures of human S100A6 (Otterbein et al., 2002). Adapted from Fritz and Heizmann, 2004.
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So far S100 proteins have been isolated only from vertebrates were they serve intracellularly as Ca2+ sensors, except S100G, which serves as a Ca2+ buffer. Other EF-hand proteins like troponin C and calmodulin are also found in invertebrates, which imply that the S100 family is an evolutionary rather young group within the EF-hand superfamily. Interestingly most of the S100 proteins (16 out of 21) are found on one gene cluster that is highly conserved in the human chromosome region 1q21, which points towards multiple gene duplication (Marenholz et al., 2006; Fritz and Heizmann, 2004;
Ridinger et al., 1998).
The S100 family has received increasing interest in recent years due to their cell- and tissue-specific expression and their involvement in widely different processes such as contraction, cell cycle regulation, cell growth, cell differentiation, mobility, transcription, differentiation and secretion. Some S100 proteins are known to be associated with human diseases like cardiomyopathy and cancer. S100A4 has been implicated in cancer and metastasis, where it shows an increased expression. S100A8/A9 which forms a heterodimer is associated with rheumatoid arthritis. Moreover, certain diseases are associated with altered expression levels of S100 proteins and can be largely classified 12
into four categories: diseases of the heart, diseases of the central nervous system, inflammatory disorders, and cancer (Marenholz et al., 2004). The variation of these physiological functions of the S100 proteins is at least in part controlled by their cell-type specific expression. Besides their versatile intracellular functions, several S100 proteins such as S100A4, S100A8/A9, S100A12, S100A16 and S100B can be secreted and act extracellularly in a cytokine like manner (Heizmann et al., 2003, Marenholz et al., 2004; Sturcheler et al., 2006). For example, the S100A8/A9 heterodimer acts as a chemotactic molecule in inflammation (Newton and Hogg, 1998), whereas S100B exhibits neurotrophic activity (Davey et al., 2001). A further diversity within the S100 proteins results from the ability to bind other metal ions in addition to Ca2+. Many S100 proteins can bind Zn2+ and Cu2+ at sites different from the EF-hand site (Ambartsumian et al., 2001).