There is communication between all four Ca(2+)-bindings sites of calcineurin B.
We have used site-directed mutagenesis, flow dialysis, and Fourier transform infrared (FTIR) spectroscopy to study Ca(2+)-binding to the regulatory component of calcineurin. Single Glu-Gln(E --> Q) mutations were used to inactivate each of the four Ca(2+)-binding sites of CnB in turn, generating mutants Q1, Q2, Q3, and Q4, with the number indicating which Ca(2+) site is inactivated. The binding data derived from flow dialysis reveal two pairs of sites in the wild-type protein, one pair with very high affinity and the other with lower affinity Ca(2+)-binding sites. Also, only three sites are titratable in the wild-type protein because one site cannot be decalcified. Mutation of site 2 leaves the protein with only two titratable sites, while mutation of sites 1, 3, or 4 leave three titratable sites that are mostly filled with 3 Ca(2+) equiv added. The binding data further show that each of the single-site mutations Q2, Q3, and Q4 affects the affinities of at least one of the remaining sites. Mutation in either of sites 3 or 4 results in a protein with no high-affinity sites, indicating communication between the two high-affinity sites, most likely sites 3 and 4. Mutation in site 2 decreases the affinity of all three remaining sites, though still leaving two relatively high-affinity sites. The FTIR data support the conclusions from the binding data with respect to the number of titratable sites as well as the impact of each mutation on the affinities of the remaining sites. We conclude therefore that there is communication between all four Ca(2+)-binding sites. In addition, the Ca(2+) induced changes in the FTIR spectra for the wild-type and Q4 mutant are most similar, suggesting that the same three Ca(2+)-binding sites are being titrated, i.e., site 4 is the very high-affinity site under the conditions of the FTIR experiments.
Gallagher
S C
SC
Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA.
Gao
Z H
ZH
Li
S
S
Dyer
R B
RB
Trewhella
J
J
Klee
C B
CB
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D019703:Calcineurin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002264:Carboxylic Acids / Q000737:chemistry; D017931:DNA Primers; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared
D002264:Carboxylic Acids; D017931:DNA Primers; D019703:Calcineurin; D002118:Calcium