Interlobe communication in multiple calcium-binding site mutants of Drosophila calmodulin.
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.
Mukherjea
P
P
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005-1892, USA.
Maune
J F
JF
Beckingham
K
K
D000818:Animals; D001665:Binding Sites / Q000235:genetics*; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002147:Calmodulin / Q000235:genetics* / Q000378:metabolism; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004330:Drosophila / Q000378:metabolism*; D004789:Enzyme Activation; D007700:Kinetics; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine / Q000737:chemistry
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D014443:Tyrosine; D009219:Myosin-Light-Chain Kinase; D002118:Calcium