Site-site communication in the EF-hand Ca2+-binding protein calbindin D-9k

The cooperative binding of Ca2+ ions is an essential functional property of the EF-hand family of Ca2+-binding proteins. To understand how these prote ins function, it is essential to characterize intermediate binding states i n addition to the apo- and hole-proteins. The three-dimensional solution st ructure and fast time scale internal motional dynamics of the backbone have been determined for the half-saturated state of the N56A mutant of calbind in D-9k with Ca2+ bound only ii? the N-terminal site. The extent of conform ational reorganization and a loss of flexibility in the C-terminal EF-hand upon binding of an ion in the N-terminal EF-hand provide clear evidence of the importance of site-site interactions in this family of proteins, and de monstrates the strength of long-range effects in the cooperative EF-hand Ca 2+-binding domain.