Interlobe communication in multiple calcium-binding site mutants of Drosophila calmodulin

Protein Sci. 1996 Mar;5(3):468-77. doi: 10.1002/pro.5560050308.


We have generated mutants of Drosophila calmodulin in which pairs of calcium-binding sites are mutated so as to prevent calcium binding. In all sites, the mutation involves replacement of the -Z position glutamate residue with glutamine. Mutants inactivated in both N-terminal sites (B12Q) or both C-terminal sites (B34Q), and two mutants with one N- and one C-terminal site inactivated (B13Q and B24Q) were generated. The quadruple mutant with all four sites mutated was also studied. UV-difference spectroscopy and near-UV CD were used to examine the influence of these mutations upon the single tyrosine (Tyr-138) of the protein. These studies uncovered four situations in which Tyr-138 in the C-terminal lobe responds to a change to the calcium-binding properties of the N-terminal lobe. Further, they suggest that N-terminal calcium-binding events contribute strongly to the aberrant behavior of Tyr-138 seen in mutants with a single functional C-terminal calcium-binding site. The data also indicate that loss of calcium binding at site 1 adjusts the aberrant conformation of Tyr-138 produced by mutation of site 3 toward the wild-type structure. However, activation studies for skeletal muscle myosin light chain kinase (SK-MLCK) established that all of the multiple binding site mutants are poor activators of SK-MLCK. Thus, globally, the calcium-induced conformation of B13Q is not closer to wild type than that of either the site 1 or the site 3 mutant. The positioning of Tyr-138 within the crystal structure of calmodulin suggests that effects of the N-terminal lobe on this residue may be mediated via changes to the central linker region of the protein.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Binding Sites / genetics*
  • Calcium / metabolism*
  • Calcium / pharmacology
  • Calcium-Binding Proteins / genetics
  • Calcium-Binding Proteins / metabolism
  • Calmodulin / genetics*
  • Calmodulin / metabolism
  • Circular Dichroism
  • Cloning, Molecular
  • Drosophila / metabolism*
  • Enzyme Activation
  • Kinetics
  • Mutagenesis, Site-Directed / genetics
  • Myosin-Light-Chain Kinase / metabolism
  • Spectrophotometry, Ultraviolet
  • Tyrosine / chemistry


  • Calcium-Binding Proteins
  • Calmodulin
  • Tyrosine
  • Myosin-Light-Chain Kinase
  • Calcium