Mutation of Lys-75 affects calmodulin conformation

AbstractSome properties of synthetic calmodulin and its five mutants with replacement of Lys-75 were analyzed by means of electrophoresis, limited proteolysis and MALDI mass-spectrometry. A double mutant of calmodulin containing insert KGK between residues 80 and 81 and replacement of Lys-75 by Pro has a highly flexible central helix which is susceptible to trypsinolysis in the presence of Ca2+. Two mutants, K75P and K75E, having a distorted central helix demonstrate high resistance to trypsinolysis in the absence of Ca2+. Arg-90 and Arg-106 being the primary site of trypsinolysis of synthetic calmodulin are partially-protected in K75P and K75E mutants. The central helix of K75A and K75V mutants is stabilized by hydrophobic interactions between residues located in positions 71, 72 and 75. In the presence of Ca2+, the central helix of K75V is resistant to trypsinolysis. Mutations K75A and K75V decrease the rate of trypsinolysis of the central helix with a simultaneous increase of the rate of trypsinolysis in the C-terminal domain of calmodulin. It is concluded that the point mutation in the central helix has a long distance effect on the structure of calmodulin

Interaction of smooth muscle caldesmon with calmodulin mutants

AbstractThe 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 E84K/E120K-CaM, deletion of residues 82–84, and by cluster mutations DEE118-120 → KKK or EEE8284 → 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 DEE118-120 → 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 Ca2+ binding must be maintained in the amino-terminal sites for maximal interaction with caldesmon

Unique sequence of a high molecular weight myosin light chain kinase is involved in interaction with actin cytoskeleton

AbstractMyosin light chain kinase (MLCK) is the key regulator of cell motility and smooth muscle contraction in higher vertebrates. We searched for the features of the high molecular weight MLCK (MLCK-210) associated with its unique N-terminal sequence not found in a more ubiquitous lower molecular weight MLCK (MLCK-108). MLCK-210 demonstrates stronger association with the Triton-insoluble cytoskeletons than MLCK-108, suggesting the role for this sequence in subcellular targeting. Indeed, the expressed unique domain of MLCK-210 binds and bundles F-actin in vitro and colocalises with the microfilaments in transfected cells reproducing endogenous MLCK-210 distribution. Thus, MLCK-210 features an extensive actin binding interface and, perhaps, acts as an actin cytoskeleton stabiliser