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- Evenäs, Johan, et al.
(författare)
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Ca2+ binding and conformational changes in a calmodulin domain
- 1998
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 37:39, s. 13744-13754
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Tidskriftsartikel (refereegranskat)abstract
- 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 glutmate Ca2+ ligand in the binding loops is emphasized by the striking effects resulting from a mutation of this glutantic 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 [Evenas 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 μs 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.
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| 2. |
- Evenäs, Johan, et al.
(författare)
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NMR studies of the E140Q mutant of the carboxy-terminal domain of calmodulin reveal global conformational exchange in the Ca2+-saturated state
- 1997
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 36:12, s. 3448-3457
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Tidskriftsartikel (refereegranskat)abstract
- In the present investigation, the Ca2+ activation of the C-terminal domain of bovine calmodulin and the effects of replacing the bidentate Ca2+-coordinating glutamic acid residue in the 12th and last position of loop IV with a glutamine are studied by NMR spectroscopy. The mutation E140Q results in sequential Ca2+ binding in this domain and has far-reaching effects on the structure of (Ca2+)2 TR2C, thereby providing further evidence for the critical role of this glutamic acid residue for the Ca2+- induced conformational change of regulatory EF-hand proteins. Analyses of the NOESY spectra of the mutant under Ca2+-saturated conditions, such that 97% of the protein is in the (Ca2+)2 form, revealed two sets of mutually exclusive NOEs. One set of NOEs is found to be consistent with the closed structure observed in the apo state of the C-terminal domain of the wild- type protein, while the other set supports the open structure observed in the Ca2+-saturated state. In addition, several residues in the hydrophobic core exhibit broadened resonances. We conclude that the (Ca2+)2 form of the mutant experiences a global conformational exchange between states similar to the closed and open conformations of the C-terminal domain of wild-type calmodulin. A population of 65 ± 15% of the open conformation and an exchange rate of (1-7) x 104 s-1 were estimated from the NMR data and the chemical shifts of the wild-type protein. From a Ca2- titration of the 15N-labeled mutant, the macroscopic binding constants (log(K1) = 4.9 ± 0.3 and log(K2) = 3.15 ± 0.10] and the inherent chemical shifts of the intermediate (Ca2+)1 form of the mutant were determined using NMR. Valuable information was also provided on the mechanism of the Ca2+ activation and the roles of the structural elements in the two Ca2+- binding events. Comparison with the wild-type protein indicates that the (Ca2+)1 conformation of the mutant is essentially closed but that some rearrangement of the empty loop IV toward the Ca2+-bound form has occurred.
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