Structural differences in the two calcium binding sites of the porcine intestinal calcium binding protein: a multinuclear NMR study

Publisher Website
Google Scholar
Abstract
Cd 113 and Ca-43 NMR spectra of Cd2+ and Ca2+ bound to the porcine intestinal Ca binding protein (ICaBP; MW 9000) contain 2 resonances. The 1st resonance is characterized by NMR parameters resembling those found for these cations bound to proteins containing the typical helix-loop-helix Ca binding domains of parvalbumin, calmodulin, and troponin C, which are defined as EF-hands by Kretsinger. The 2nd resonance in both spectra has a unique chemical shift and is consequently assigned to the metal ion bound in the N-terminal site of ICaBP. This site is characterized by an insertion of a proline in the loop of the helix-loop-helix domain and will be called the pseudo-EF-hand site. The binding of Cd2+ to the apo form of ICaBP is sequential. The EF-hand site is filled first. Both binding sites have similar, but not identical, affinities for Ca2+: at a Ca2+ to protein ratio of 1:1, 65% of the ion is bound in the EF-hand site and 35% in the pseudo-EF-hand site. The 2 sites do not appear to act independently; thus, replacement of Ca2+ or Cd2+ by La3+ in the EF-hand site causes changes in the environment of the ions in the pseudo-EF-hand site. In addition, the chemical shift of Cd2+ bound to the EF-hand site is dependent on the presence or absence of Ca2+ or Cd2+ in the pseudo-EF-hand site. These data show the existence of interactions between the 2 Ca binding sites that may be of a cooperative nature. 1H NMR studies demonstrate that the changes in the tertiary structure of ICaBP induced by saturating levels of Ca2+ and Cd2+, respectively, are virtually indistinguishable. 1H NMR studies also confirm the different modes of binding of the 2 cations.