Ca2+ Binding and Conformational Changes in a Calmodulin Domain
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- 作者：Johan Evenä ; s ; Anders Malmendal ; Eva Thulin ; Gö ; ran Carlströ ; m ; and Sture Forsé ; n
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：September 29, 1998
- 年：1998
- 卷：37
- 期：39
- 页码：13744 - 13754
- 全文大小：160K
- 年卷期：v.37,no.39(September 29, 1998)
- ISSN：1520-4995
文摘
Calcium activation of the C-terminal domain of calmodulin was studied using 1H and 15NNMR spectroscopy. The important role played by the conserved bidentate glutamate Ca2+ ligand in thebinding loops is emphasized by the striking effects resulting from a mutation of this glutamic acid to aglutamine, 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 ofthe 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 forthe 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. Spectralcharacteristics show that the (Ca2+)1 and (Ca2+)2 states of the E104Q mutant undergo global exchange ona 10-100 
s time scale between conformations seemingly similar to the closed and open structures ofthis domain in wild-type calmodulin, paralleling earlier observations for the (Ca2+)2 state of the E140Qmutant, indicating that both glutamic acid residues, E104 and E140, are required for stabilization of theopen conformation in the (Ca2+)2 state. To verify that the NOE constraints cannot be fulfilled in a singlestructure, solution structures of the (Ca2+)2 state of the E104Q mutant are calculated. Within the ensembleof structures the precision is good. However, the clearly dynamic nature of the state, a large number ofviolated distance restraints, ill-defined secondary structural elements, and comparisons to the structuresof calmodulin indicate that the ensemble does not provide a good picture of the (Ca2+)2 state of theE104Q mutant but rather represents the distance-averaged structure of at least two distinct differentconformations.
s time scale between conformations seemingly similar to the closed and open structures ofthis domain in wild-type calmodulin, paralleling earlier observations for the (Ca2+)2 state of the E140Qmutant, indicating that both glutamic acid residues, E104 and E140, are required for stabilization of theopen conformation in the (Ca2+)2 state. To verify that the NOE constraints cannot be fulfilled in a singlestructure, solution structures of the (Ca2+)2 state of the E104Q mutant are calculated. Within the ensembleof structures the precision is good. However, the clearly dynamic nature of the state, a large number ofviolated distance restraints, ill-defined secondary structural elements, and comparisons to the structuresof calmodulin indicate that the ensemble does not provide a good picture of the (Ca2+)2 state of theE104Q mutant but rather represents the distance-averaged structure of at least two distinct differentconformations.