Structural differences in the two calcium binding sites of the porcine intestinal calcium binding protein: a multinuclear NMR study.
Cadmium-113 and calcium-43 NMR spectra of Cd2+ and Ca2+ bound to the porcine intestinal calcium binding protein (ICaBP; Mr 9000) contain two resonances. The first resonance is characterized by NMR parameters resembling those found for these cations bound to proteins containing the typical helix-loop-helix calcium binding domains of parvalbumin, calmodulin, and troponin C, which are defined as EF-hands by Kretsinger [Kretsinger, R. H. (1976) Annu. Rev. Biochem. 45, 239]. The second 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 two 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.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002413:Cations, Divalent; D004386:Duodenum / Q000378:metabolism*; D007082:Ileum / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D011485:Protein Binding; D011487:Protein Conformation; D013552:Swine
D002135:Calcium-Binding Proteins; D002413:Cations, Divalent; D002118:Calcium