Calcium-binding sites geometry

As shown in Table 3, the relocated natural calcium binding sites have small U(p) values (<25). They all have the smallest U(p) values among all the constructed sites for each protein. Therefore, the U(p) number can be used to rank the constructed potential sites according to their deviations from the target site. Our parameters used to describe the geometry of calcium binding sites allow us to accurately identify natural calcium binding sites. 

Figure 5. The deviation of the designed sites fiom the ideal pentagonal bipyramidal geometry (pseudoenergy U(p)) as a function the designed calcium binding sites. The natural calcium binding sites I-IV of calmodulin are shown as black dots.

The calcium sites in troponin C have been studied by X-ray absorption near edge structure (XANES).244 In all four cases, Ca2+ appears to be coordinated to carboxylate and carbonyl groups, and no structural differences could be found between the two classes of sites. Binding of Mg2+ causes a distortion of the geometry of the calcium site. Thus, the reduced affinity for Ca2+ of the Ca2+-Mg2+ sites in the presence of Mg2+ may not simply be due to competition with Mg2+, but due to some conformational change induced at these sites by Mg2+. The similarity of all four Ca2+ sites means that local bonding effects do not explain the inability of Mg2+ to bind to the calcium-specific sites I and II. The XANES of parvalbumin differs from that of troponin C. 

Asp-49/35 is essential to optimal calcium binding and catalysis. Replacement of Asp-49 with other amino acids or chemical modification of the side-chain carboxylate reduces Acat to less than 5% of native rates (Fleer etai, 1981 Van den Berghe/a/., 1989). The close spatial coupling of Asp-49/35 with its respective catalytic histidine (His-48/34) ensures a fixed active site geometry. Stability of this bihelical substructure is critical because His-48/34 is supported by a side chain from an adjacent segment of the same helix (Tyr-52) and must hydrogen bond with a residue from the opposed helix (Asp-99/64) for function. 

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