Replacement of Lys-75 of calmodulin affects its interaction with smooth muscle caldesmon

The interaction of smooth muscle caldesmon with synthetic calmodulin (SynCa m) and its five mutants with replacement of Lys-75 was analyzed by means of intrinsic Trp fluorescence, zero-length crosslinking and by caldesmon-indu ced inhibition of actomyosin ATPase activity. SynCam and its double mutant with replacement K75P and simultaneous insertion of KGK between residues 80 and 81 have a comparably low affinity to caldesmon and the probability of crosslinking of this mutant to caldesmon was the lowest among all mutants a nalyzed. SynCam and its double mutant (K75P+KCK) induced nearly complete re version of caldesmon inhibition of actomyosin ATPase activity with half-max imal reversion achieved at about 1 muM. Two mutants, K75A and K75V, with pa rtially stabilized less positive central domain have higher affinity to cal desmon. These mutants induce 80-85% reversion of caldesmon inhibition of ac tomyosin ATPase and the half-maximal reversion was achieved at about 0.3-0. 4 muM. Two last mutants, K75P and K75E, with distorted central domain have high affinity to caldesmon and the probability of crosslinking of K75P to c aldesmon was the highest among calmodulin mutants tested. These mutants ind uced complete reversion of caldesmon inhibition with half-maximal effect ob served at 0.3-0.4 muM We suggest that the length, flexibility and charge of the central domain affect binding of calmodulin mutants and their ability to reverse caldesmon-induced inhibition of actomyosin ATPase activity. (C) 2001 Elsevier Science B.V. All rights reserved.