The mechanism of Ca(2+)-coordination in the EF-hand of TnC, by cassette mutagenesis.

Genetic engineering of TnC and skinned fiber physiology on rabbit psoas muscle are combined to study the mechanisms of Ca2+-binding in the EF-hand in TnC. Of the six coordinating positions (X,Y,Z,-Y,-X & -Z) for Ca2+-binding in the loop, the X position is invariably occupied by an aspartate, and the -Z position by a glutamate. X-ray analysis has indicated that both oxygen atoms of the β-carboxylate in aspartate (in X) are extensively hydrogen bonded to other residues in the loop. When this aspartate in site II was replaced by a glutamate (γ-carboxylate), Ca2+-binding was annihilated, and the mutant was unable to regulate force development in the fiber. Similarly, glutamate for aspartate exchange in the -Z position of site I also inactivated the site as well as its function in skinned fiber. Mutations in the Y position indicated that a glutamate was unacceptable in place of aspartate but that an asparagine was acceptable. The Ca2+-sensitivity with asparagine was also similar to that of the wild type. The study indicates a powerful approach for defining the physicochemical principles governing Ca-coordination and sensitivity in Ca-binding proteins. Furthermore, by comparison with findings on chemically synthesized peptides, the results show that behavior of the EF-hand in TnC is modified by quaternary structure of the molecule.