A calbindin D9k mutant with reduced calcium affinity and enhanced cooperativity. Metal ion binding, stability, and structural studies.

Abstract:

:In the native calcium-binding protein calbindin D9k (M(r) 8.700; 75aa; 2 EF-hands), the backbone carbonyl oxygen of Glu60 coordinates the Ca2+ ion in the C-terminal site (site II). The carboxylate group of the same residue forms a hydrogen bond to a water molecule that constitutes a Ca2+ ligand in the N-terminal site (site I). The mutant E60D, with the charge-conserving substitution Glu60-->Asp, has been prepared to study the role of Glu60 in subjoining the two Ca(2+)-binding sites and its role in the cooperative Ca2+ binding. Ca(2+)-binding studies of the mutant show that the overall affinity for calcium has decreased by a factor of 38 in comparison with wild-type calbindin D9k. The largest reduction is seen in the first macroscopic binding step. The Ca2+ affinities for both sites in the protein are reduced to a similar extent. In contrast, the mutation leads to a large increase in the cooperativity of calcium binding. Differential scanning calorimetry has been used to determine the thermal stability which is almost as high as in the wild-type protein. Cadmium binding has been assessed with 1H and 113Cd NMR. X-ray crystallographic studies of the E60D mutant in its calcium-bound form show very small structural changes relative to the wild-type 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 found in the asymmetric unit in contrast to the single molecule in the wild-type crystal.(ABSTRACT TRUNCATED AT 250 WORDS)

journal_name

Biochemistry

journal_title

Biochemistry

authors

Linse S,Bylsma NR,Drakenberg T,Sellers P,Forsén S,Thulin E,Svensson LA,Zajtzeva I,Zajtsev V,Marek J

doi

10.1021/bi00207a015

subject

Has Abstract

pub_date

1994-10-18 00:00:00

pages

12478-86

issue

41

eissn

0006-2960

issn

1520-4995

journal_volume

33

pub_type

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