Characterization of the N‐terminal half‐saturated state of calbindin D_9k: NMR studies of the N56A mutant

Abstract

Calbindin D9k is a small EF-hand prc )tein that binds two calcium ions with positive cooperativity. The molea llar basis of cooperativity for the binding pathway where the first ion binds in the N-terminal site (I) is investigated by NMR experiments on the half-saturated state of the N56A mutant, which exhibits sequential yet cooperative binding (Linse S, Chazin WJ, 1995, Protein Sci 4: 1038-1044). Analysis of calcium-induced changes in chemical shifts, amide proton exchange rates, and NOES indicates that ion binding to the N-terminal binding loop causes significant changes in conformation and/or dynamics throughout the protein. In particular, all three parameters indicate that the hydrophobic core undergoes a change in packing to a conformation very similar to the calciumloaded state. These results are similar to those observed for the (Cd2+), state of the wild-type protein, a model for the complementary half-saturated state with an ion bound in the C-terminal site (11). Thus, with respect to cooperativity in either of the binding pathways, binding of the first ion drives the conformation and dynamics of the protein far toward the (Ca2+)2 state, thereby facilitating binding of the second ion. Comparison with the half-saturated state of the analogous E65Q mutant confirms that mutation of this critical bidentate calcium ligand at position 12 of the consensus EF-hand binding loop causes very significant structural perturbations. This result has important implications regarding numerous studies that have utilized mutation of this critical residue for site deactivation.

Effects of Calcium Binding on the Side-chain Methyl Dynamics of Calbindin D9k: A 2H NMR Relaxation Study

“…We note that wellcharacterized model systems have been developed for both the half-saturated states of calbindin. [44][45][46][47] These model systems should provide an initial test of the generality of our current findings. …”

Quantitative measurements of the cooperativity in an EF‐hand protein with sequential calcium binding

“…For mutants designed to bind sequentially and enable study of the cooperative mechanism, it is therefore absolutely essential to establish that the system retains cooperativity. For N56A, it has been shown that the high degree of coupling between the binding sites is conserved, thereby justifying its use as a model for the site I (Ca2+)' state of wild-type calbindin D,, (Wimberly et al, 1995). In addition, the evidence showing that Cd2+ binding at one site enhances the metal ion affinity of the other site justifies the use of the wild-type calbindin Dgk with one cadmium ion occupying site I1 as a model for the site I1 (Ca"), state of wild-type calbindin D,, (Akke et a]., 1991(Akke et a]., , 1993(Akke et a]., , 1995.…”

“…In an effort to closely match the conditions under which the detailed NMR analysis was carried out (see accompanying paper, Wimberly et al, 1995), the concentration of protein in the binding measurements was in the range of 1-2 mM. To rule out any unusual effects associated with these relatively high protein concentrations, a second Ca2+ titration was performed at a protein concentration of 30 pM N56A.…”

Metals and Breast Cancer

“…In support of this hypothesis, cadmium replaces calcium in many biological systems and assays [33-35]. In the case of ER α , cadmium and the other bivalent cationic metalloestrogens activate it through the LBD and require the same amino acids (cys381, glu523, and asp538) as calcium [3, 5].…”