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J. Biol. Chem., Vol. 266, Issue 32, 21488-21495, 11, 1991
MF VanBerkum and AR Means
TaM-BMI is a genetically engineered chimeric protein consisting of the
first 55 amino acids of cardiac troponin C (but with the normally inactive
first Ca2+ binding domain reactivated by site- directed mutagenesis)
ligated to the last three domains of chicken calmodulin (George, S.E.,
VanBerkum, M.F., Ono, T., Cook, R., Hanley, R.M., Putkey, J.A., and Means,
A. R. (1990) J. Biol. Chem. 265, 9228-9235). This protein binds chicken
smooth muscle myosin light chain kinase (smMLCK) but fails to activate the
enzyme, thus functioning as a potent competitive inhibitor (Ki = 66 nM). We
have created 29 mutants of calmodulin designed to identify the minimal
number of alterations which must be introduced in the first domain to
convert the protein to a competitive inhibitor of smMLCK. Alterations of
three amino acids predicted to lie on the external surface of calmodulin
(E14A, T34K, S38M) recapitulated the phenotype of TaM-BMI and exhibited a
Ki of 38 nM. Both the triple mutant and TaM-BMI activated phosphodiesterase
and bound a synthetic peptide analog of the calmodulin binding region of
smMLCK with an affinity similar to that of native calmodulin (Kact and Kd
values of approximately 2 and 3 nM respectively). When a synthetic peptide
analog of the myosin light chain phosphorylation site was used as substrate
rather than the 20-kDa light chains, TaM-BMI and the triple mutant were
partial agonists: the Km for peptide substrate was increased 100- and
60-fold, and catalytic activity was 45 and 60%, respectively, relative to
calmodulin. These data suggest TaM-BMI and E14A/T34K/S38M may interact with
the calmodulin binding domain of smMLCK in a manner similar to calmodulin.
However, alterations in electrostatic and hydrophobic interactions created
by the three amino acid substitutions prevent the conformational change in
the enzyme usually produced by calmodulin binding. Lack of such changes
results in loss of catalytic activity and light chain binding.
Additionally, our results show that altering only 3 amino acids residues
converts calmodulin to an enzyme-selective antagonist, thus demonstrating
the ability to separate calmodulin binding to smMLCK from calmodulin-
induced activation of the enzyme.
Three amino acid substitutions in domain I of calmodulin prevent the activation of chicken smooth muscle myosin light chain kinase
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
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