Mutation of Tyr¹³⁸ Disrupts the Structural Coupling between the Opposing Domains in Vertebrate Calmodulin^†

We have used circular dichroism and frequency-domain fluorescence spectroscopy to determine how the site-specific substitution of Tyr¹³⁸ with either Phe¹³⁸ or Gln¹³⁸ affects the structural coupling between the opposing domains of calmodulin (CaM). A double mutant was constructed involving conservative substitution of Tyr⁹⁹ → Trp⁹⁹ and Leu⁶⁹ → Cys⁶⁹ to assess the structural coupling between the opposing domains, as previously described [Sun, H., Yin, D., and Squier, T. C. (1999) Biochemistry 38, 12266−12279]. Trp⁹⁹ acts as a fluorescence resonance energy transfer (FRET) donor in distance measurements to probe the conformation of the central helix. Cys⁶⁹ provides a reactive group for the covalent attachment of 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS), which functions as a FRET acceptor and permits the measurement of the rotational dynamics of the amino-terminal domain. These CaM mutants demonstrate normal calcium-dependent gel-mobility shifts and changes in their near-UV CD spectra, have similar secondary structures to wild-type CaM following calcium activation, and retain the ability to fully activate the plasma membrane Ca-ATPase. The global folds, therefore, of both the carboxyl- and amino-terminal domains in these CaM mutants are similar to that of wild-type CaM. However, in comparison to wild-type CaM, the substitution of Tyr¹³⁸ with either Phe¹³⁸ or Gln¹³⁸ results in (i) alterations in the average spatial separation and increases in the conformational heterogeneity between the opposing globular domains and (ii) the independent rotational dynamics of the amino-terminal domain. These results indicate that alterations in either the hydrogen bond between Tyr¹³⁸ and Glu⁸² or contact interactions between aromatic amino acid side chains have the potential to initiate the structural collapse of CaM normally associated with target protein binding and activation.