-transparent.png){kind=logo}
Calcium activation of the C-terminal domain of calmodulin was studied using 1H and 15N NMR spectroscopy. The important role played by the conserved bidentate glutamate Ca2+ ligand in the binding loops is emphasized by the striking effects resulting from a mutation of this glutamic acid to a glutamine, i.e. E104Q in loop III and E140Q in loop IV. The study involves determination of Ca2+ binding constants, assignments, and structural characterizations of the apo, (Ca2+)1, and (Ca2+)2 states of the E104Q mutant and comparisons to the wild-type protein and the E140Q mutant [Evenäs et al. (1997) Biochemistry 36, 3448-3457]. NMR titration data show sequential Ca2+ binding in the E104Q mutant. The first Ca2+ binds to loop IV and the second to loop III, which is the order reverse to that observed for the E140Q mutant. In both mutants, the major structural changes occur upon Ca2+ binding to loop IV, which implies a different response to Ca2+ binding in the N- and C-terminal EF-hands. Spectral characteristics show that the (Ca2+)1 and (Ca2+)2 states of the E104Q mutant undergo global exchange on a 10-100 micros time scale between conformations seemingly similar to the closed and open structures of this domain in wild-type calmodulin, paralleling earlier observations for the (Ca2+)2 state of the E140Q mutant, indicating that both glutamic acid residues, E104 and E140, are required for stabilization of the open conformation in the (Ca2+)2 state. To verify that the NOE constraints cannot be fulfilled in a single structure, solution structures of the (Ca2+)2 state of the E104Q mutant are calculated. Within the ensemble of structures the precision is good. However, the clearly dynamic nature of the state, a large number of violated distance restraints, ill-defined secondary structural elements, and comparisons to the structures of calmodulin indicate that the ensemble does not provide a good picture of the (Ca2+)2 state of the E104Q mutant but rather represents the distance-averaged structure of at least two distinct different conformations.
J Evenäs, and
A Malmendal, and
E Thulin, and
G Carlström, and
S Forsén
January 2008,
Proceedings of the National Academy of Sciences of the United States of America,
J Evenäs, and
A Malmendal, and
E Thulin, and
G Carlström, and
S Forsén
July 1986,
Biochemical and biophysical research communications,
J Evenäs, and
A Malmendal, and
E Thulin, and
G Carlström, and
S Forsén
September 1988,
Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme,
J Evenäs, and
A Malmendal, and
E Thulin, and
G Carlström, and
S Forsén
October 1982,
The Journal of biological chemistry,
J Evenäs, and
A Malmendal, and
E Thulin, and
G Carlström, and
S Forsén
January 1990,
Biophysical chemistry,
J Evenäs, and
A Malmendal, and
E Thulin, and
G Carlström, and
S Forsén
April 1991,
The Journal of biological chemistry,
J Evenäs, and
A Malmendal, and
E Thulin, and
G Carlström, and
S Forsén
June 2017,
Biochimica et biophysica acta. Proteins and proteomics,
J Evenäs, and
A Malmendal, and
E Thulin, and
G Carlström, and
S Forsén
July 2006,
Biochemistry,
J Evenäs, and
A Malmendal, and
E Thulin, and
G Carlström, and
S Forsén
November 1982,
Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme,
J Evenäs, and
A Malmendal, and
E Thulin, and
G Carlström, and
S Forsén
August 2008,
American journal of physiology. Cell physiology,
