A structure-function relationship for the calcium affinities of regulatory proteins containing 'EF-hand' pairs

Using a series of homologous calcium-binding proteins, a quantitative structure-activity relationship (QSAR), log(1/Kd) = -18.986 - 1.6278(X1) + 0.7981(X2) + 0.2312(X3), has been established, which relates the calcium-binding affinities (1/Kd) of the regulatory proteins with (i) the net ligand charge (X1) of the two calcium binding loops, (ii) the hydrophobicity (X2) of the beta-sheet segment of the loops and (iii) the hydrophobicity (X3) of the four 'EF-hand' helices. It is found that the binding affinities are influenced by the 'EF-hand' pair rather than the individual 'EF-hands'. The QSAR, in addition to explaining satisfactorily the large variation in the observed calcium affinities, can predict the affinities of the 'EF-hand' pairs in other proteins from the amino acid sequence and can also account for the changes in the affinities caused by substitution in the hydrophobic and/or metal-coordinating residues. Thus, this relationship can be employed in protein design and engineering. The method is potentially useful in the development of similar relationships for the binding of other proteins to substrates, inhibitors, drugs and co-factors.