Interaction of smooth muscle caldesmon with calmodulin mutants

Abstract

The interaction of avian smooth muscle caldesmon with calmodulin (CAM) was investigated by studying the ability of selected mutant calmodulins to induce fluorescence changes in caldesmon. Different types of CaM mutants were used including point charge mutants, cluster mutations, and mutations which alter the calcium binding of CaM. The caldesmon binding properties were only slightly affected by E84K-CaM or by the double mutation E84Q/E120Q-CaM. Affinity of calmodulin to caldesmon was decreased 2-4 times by point mutation G33V-CaM, double mutation E841(dEI20K-CaM, deletion of residues 82-84, and by cluster mutations DEEll8-120-->KKK or EEE82-84--~KKK. Mutations of the first (E31A-CaM) and the second (E67A-CaM) calcium binding sites reduced the affinity of calmodulin to caldesmon by at least 5-fold; in addition these calmodulin mutants exhibited smaller changes in the fluorescence spectra of caldesmon. Simultaneous mutation of the two negatively charged clusters of calmodulin EEE82-84--~ KKK and DEEll8-120 --> KKK resulted in a more than 15-fold decrease in the affinity of calmodulin for caldesmon. The data indicate that charged and uncharged amino acids in both halves of CaM play an important role in the binding of calmodulin to caldesmon, and that Ca 2+ binding must be maintained in the amino-terminal sites for maximal interaction with caldesmon.

Mapping of contact sites in the caldesmon–calmodulin complex

“…Caldesmon-binding sites of calmodulin are not so well determined. It is supposed that calmodulin interacts with caldesmon in an extended conformation [9] and that both N-and C-terminal globular domains as well as the central α-helix of calmodulin are involved in caldesmon binding [10]. The data obtained on proteolytic fragments of calmodulin [11] and calmodulin mutants [12] agree with this suggestion and indicate that multiple sites of calmodulin interact with caldesmon.…”

“…Knowing the total concentration of calmodulin and the stoichiometries of the complexes formed, we determined the total concentration of caldesmonbinding sites. This value was used to estimate apparent dissociation constants of caldesmon fragments from intact calmodulin by using a non-linear binding equation [10,11]. A similar approach was used when tryptic peptides of calmodulin were titrated with caldesmon fragments.…”

Multiple-sited interaction of caldesmon with Ca2+-calmodulin

“…Different positioning of methionine side chains in the mutant may be the cause of this result, as three methionines, 71, 72 and 76, are close to the disulphide bond of C41\75. Medvedeva et al [35] also observed altered calmodulininduced changes in the fluorescence spectra of caldesmon for two N-terminal calmodulin mutants with alteration of the first or of the second Ca# + -binding loop of calmodulin.…”

“…Similarly we have observed that the C-terminal fragments, H2 and H7 with Trp-716 and -749 (659 and 692 in the chicken sequence) and 658C and H9 with Trp-749 and -779 (692 and 722), show a fluorescence intensity increase of the spectrum maximum of 33 % to 49 % and a shift of the maximum of 8 nm to 18 nm on interaction with calmodulin. Previous fluorescence intensity measurements for the interaction of caldesmon with calmodulin were performed at a fixed wavelength (320 nm) [7,9,14,35]. However, the intensities measured at 320 nm are the function of the fluorescence intensity change and the blue shift.…”

Crystallographic snapshots of initial steps in the collapse of the calmodulin central helix

“…Several studies have been carried out on various mutant variants of the CaM central helix, including mutation or deletion of the acidic residues (Craig et al, 1987;Persechini et al, 1989Persechini et al, , 1991Gulati et al, 1990;VanBerkum et al, 1990;Raghunathan et al, 1993;Medvedeva et al, 1995Medvedeva et al, , 1999Tabernero et al, 1997). In general, the outcome has been CaM that has been functional but with a lower affinity towards target proteins.…”