Sequence variations within the S100 protein family were used as a starting point in an effort to determine differences at the amino acid level that could have an important
role in stabilizing the open form of S100A10. Generally, if a residue is highly conserved in all S100 proteins it would be less likely to contribute to the structural differences in S100A10. Similarly, if there is widespread variation at a particular site, it is equally unlikely to be important for the stabilization of the active form of S100A10.
Figure 2.1 shows the multiple sequence alignment of S100A10 and other S100 proteins for which the “closed” three-dimensional structures in the absence of calcium have been reported. The sequence of S100A10 shows differences primarily in the composition of the calcium-binding loops. The pseudo canonical EF-hand has a three- residue deletion and lacks the conserved glutamate at position –z of the calcium coordination sphere. In addition, S100A10 is the only protein that contains a penultimate negatively charged residue at the end of loop I (E28) that corresponds to a polar, neutral (T/S) or basic (K) residue in the remaining S100 proteins, possibly affecting the helix dipole of helix II. In the second calcium-binding site S100A10 contains two critical substitutions (C61 and S70) at calcium coordinating positions y and –z. Replacements occur at the conserved aspartate at the N-terminus of the loop (Ca2+ coordinating position 3) and at the bidentate glutamate found at the end of the loop (position 12). The residue at position 12in either of the EF-hand loops provides two oxygens for calcium coordination and its replacement leads to the inability to bind calcium, as has been shown by site directed mutagenesis in S100B [18], calmodulin [19, 20] and calbindin D9K [21, 22].
Further, all EF-hand calcium-binding loops are rich in negatively charged residues (~4/site), which provide carboxylate ligands for ion coordination. Remarkably, site II in S100A10 contains only two acidic residues (D59, D63) and more neutral (Q60,
Figure 2.1 Multiple sequence alignment of S100 proteins .
Multiple sequence alignment of S100 proteins for which three-dimensional structures in the apo-form have been reported performed using Clustal W 2.01. The Ca2+-coordinating positions are highlighted in pink. Residues unique in the S100A10 sequence are highlighted in yellow and the secondary structures shown above the sequences. Major differences between the primary sequence of S100A10 and other S100 proteins occur in the calcium-binding sites and the N-terminal portions of the helices and the linker region.
R62 and K65) amino acids, which contribute to its net charge of 0 at a pH near 7.0. In other S100 proteins this canonical loop is more negatively charged and must overcome the destabilizing electrostatic interactions in their loop arrangements [23].
It is worth noting that distinctive substitutions in S100A10 do not occur randomly along the protein sequence but seem to be grouped at the N-terminal portion of each α-
helix and the linker region, while higher identity or conservative substitutions occur near the C-terminus of each section (Figure 2.1). For example, residues at the N-terminus of the linker in S100A10 (38FPGF41) are nearly identical to those in S100P (LPGF) but significantly different from those of S100A1 (LSSF), S100B (LSHF), S100A12 (LANT), S100A6 (LITG) and S100A11 (LAAF), whereas the C-terminus is identical to S100A11 (NQKD) and highly similar to S100A1 (VQKD), S100A12 (NIKD) and S100P (SGKD).
Three S100 proteins share high sequence identity and similarity with S100A10: S100A1 (48%, 72%), S100P (45%, 67%) and S100A11 (41%, 63%) and could be used as templates to test hypotheses as to which residues govern the permanently open form of S100A10. S100A11 is a particularly interesting candidate as its Ca2+-bound form presents a nearly identical fold to that of S100A10 (discussed later in this chapter). In addition, Ca2+-S100A11 can form complexes with proteins of the annexin family and potentially intervene in membrane trafficking events similarly to S100A10 [24, 25]. This resemblance in structure and biological function has not been observed for any other pair of S100 proteins.
2.3.1.1Sequence similarity between S100A10 and S100A11
S100A10 and S100A11 are highly related proteins (identity 41%, similarity 63%, conservation 93%), well conserved amongst species. They display strong sequence identity in the linker region, helix III and the N-terminus of helix IV (Figure 2.1 and 1.13). Interestingly, these are the regions that form the surface for biological target recognition, and could explain the capability of these two proteins to interact with similar biological partners, such as annexin A2.
The most evident primary sequence differences between S100A10 and S100A11, as with other S100 proteins, are a three-residue gap in the non-canonical EF-hand and substitutions in calcium coordinating residues in both EF-hand loops. Further, amino acid substitutions appear to be clustered at the N-terminus of the helices. A helical wheel representation (Figure 2.2) was plotted to appreciate the distribution of the amino acid substitutions in S100A10 compared to S100A11. The figure shows that all the helices in the proteins are amphipathic, with hydrophobic residues facing their inner portions. Moreover, the representation illustrates how differences in amino acid composition between S100A10 and S100A11 are enriched on the accessible faces of the helices, except for helix I where substitutions occur all throughout the helix. This suggests that the buried core of S100A10 is more similar to that in S100A11 than the exposed outer portions of the helices.
Overall, sequence alignment comparisons show that S100A10 presents unique amino acid composition in the calcium binding loops and the N-terminus of helices II, III, IV and the linker region, which correspond to the exposed face of the helices. There are
54 non-identical residues comparing human-S100A10 and rabbit-S100A11 that could potentially lead to differences in residues interactions allowing S100A10 to adopt a calcium-bound like conformation in the absence of calcium. Moreover, S100A10 contains 22 unique residues in its sequence, with half of them located in the loop regions (Figure 2.3 highlighted in yellow). Noteworthy are substitutions in S100A10 at positions F38 and R31, which correspond to conserved leucine and lysine (except S100A11 which corresponds to L36) residues in other S100 proteins respectively. To further analyze the relationship between sequence and structure of these two closely related members of the S100 family, in depth analyses of the three-dimensional structures of S100A11 and S100A10 were conducted.