A CALBINDIN D-9K MUTANT WITH REDUCED CALCIUM AFFINITY AND ENHANCED COOPERATIVITY - METAL-ION BINDING, STABILITY, AND STRUCTURAL STUDIES

In the native calcium-binding protein calbindin D-9k (M(r) 8.700; 75aa ; 2 EF-hands), the backbone carbonyl oxygen of Glu60 coordinates the C a2+ ion in the C-terminal site (site II). The carboxylate group of the same residue forms a hydrogen bond to a water molecule that constitut es a Ca2+ ligand in the N-terminal site (site I). The mutant E60D, wit h the charge-conserving substitution Glu60-->Asp, has been prepared to study the role of Glu60 in subjoining the two Ca2+-binding sites and its role in the cooperative Ca2+ binding. Ca2+-binding studies of the mutant show that the overall affinity for calcium has decreased by a f actor of 38 in comparison with wild-type calbindin D-9k. The largest r eduction is seen in the first macroscopic binding step. The Ca2+ affin ities for both sites in the protein are reduced to a similar extent. I n contrast, the mutation leads to a large increase in the cooperativit y of calcium binding. Differential scanning calorimetry has been used to determine the thermal stability which is almost as high as in the w ild-type protein. Cadmium binding has been assessed with H-1 and Cd-11 3 NMR. X-ray crystallographic studies of the E60D mutant in its calciu m-bound form show very small structural changes relative to the wild-t ype protein. Almost all differences are within the error limits of the method. The largest crystallographic effects are seen in the crystal packing. Two E60D molecules with slightly different structure are foun d in the asymmetric unit in contrast to the single molecule in the wil d-type crystal. The distance between the carboxylate group of residue 60 and the Ca2+-coordinating water in site I has increased by 0.2-0.3 Angstrom. The distance between the side chain oxygen of Gln22 and the Ca2+ coordinating water in site II has increased by 0.7 Angstrom in on e of the E60D molecules but is unchanged in the other. Shortening of t he side chain in position 60 is therefore suggested to lead to more la bile water ligands, which in part could explain the lower calcium affi nity. H-1 NMR chemical shift analyses in the apo and calcium-loaded fo rms show that also in solution the structural consequences of the muta tion are minor. The NMR results for the calcium form are in perfect ag reement with the crystallographic structure determination; all chemica l shift changes beyond the error limits occur for residues for which a small but significant rearrangement is observed in the crystal.