The central helix of calmodulin functions as a flexible tether.
Using site-directed mutagenesis we have created an altered calmodulin in which Gln-3 and Thr-146 have both been replaced by cysteines. We have reacted this protein with the bifunctional reagent, bismaleimidohexane, forming an intramolecular cross-link between the two cysteines. In the crystal structure of native calmodulin alpha-carbons at positions 3 and 146 are 37 A apart. In the bismaleimidohexane cross-linked protein these atoms can be no more than 19 A apart, and model building studies indicate that there is probably a bend in the central helix of calmodulin. A second modified calmodulin was generated by cleaving the central helix of the cross-linked protein at Lys-77 with trypsin. In this molecule, the two lobes of calmodulin are joined solely by the bismaleimidohexane cross-link, which bridges Cys-3 and Cys-146. Vm and Kact values for activation of myosin light chain kinase activity by the cross-linked and cross-linked/trypsinized proteins are not significantly different from those for the control protein. This result indicates that one role for the central helix may be to serve as a flexible tether between the calmodulin lobes. This is consistent with a model calmodulin-enzyme complex in which the central helix is bent, and the two lobes exert a concerted effect. A detailed model of this type has been proposed for the calmodulin-myosin light chain kinase complex (Persechini, A. and Kretsinger, R.H. (1988) J. Cardiovasc. Pharmacol., in press).
D002147:Calmodulin / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D003432:Cross-Linking Reagents; D003460:Crystallization; D003545:Cysteine; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005033:Ethylmaleimide / Q000494:pharmacology; D005973:Glutamine; D009154:Mutation; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013912:Threonine; D014357:Trypsin / Q000378:metabolism
D002147:Calmodulin; D003432:Cross-Linking Reagents; D004220:Disulfides; D005973:Glutamine; D013912:Threonine; D009219:Myosin-Light-Chain Kinase; D014357:Trypsin; D003545:Cysteine; D005033:Ethylmaleimide