Altered methylation substrate kinetics and calcium binding of a calmodulin with a Val136-->Thr substitution.
Calmodulin is trimethylated on Lys115 by a specific calmodulin methyltransferase. Previously, it was shown that the cam2 mutant (Ile136-->Thr) of Paramecium has a decreased level of methylated Lys115 [Lukas, T. J., Friedman, M. W., Kung, C. & Watterson, D. M. (1989) Proc. Natl Acad. Sci. USA 86, 7331-7335]. To investigate how this substitution affects calmodulin structure, function and recognition by the calmodulin methyltransferase, a calmodulin with a Thr136 substitution ([Thr136]calmodulin) was expressed in Escherichia coli in an unmethylated form for in vitro enzyme activator, calcium binding and methylation kinetic analyses. [Thr136]calmodulin was indistinguishable from wild-type calmodulin in saturating (1 mM) calcium in its ability to activate calmodulin-dependent enzymes and in its steady-state kinetic properties with isolated calmodulin methyltransferase. However, [Thr136]calmodulin did show two defects: a complete inability to be methylated in the absence of calcium; and defective calcium binding. As a result, an approximate 10-fold shift in the K0.5 values for calcium dependence of enzyme activation (shifted from 1.1 microM to 9.1 microM of Ca2+ for NAD kinase) and methylation (from 0.71 microM to 7.2 microM of Ca2+ in 0.15 M K+, 2 mM Mg2+) were observed. Non-denaturing electrophoresis and Tyr138 spectroscopic measurements suggest a difference in the conformation of the calcium-depleted structures of normal calmodulin and [Thr136]calmodulin. Overall, the results suggest that the mutation in this conserved position in the COOH-terminal hydrophobic core lowers calcium-binding affinity and alters the calcium-depleted structure leading to decreased methylation at physiological Ca2+ concentrations.
Han
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Department of Biochemistry, Cellular and Molecular Biology, The University of Tennessee, Knoxville 37996-0840, USA.
Roberts
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016054:DNA, Protozoan / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D016805:Paramecium tetraurelia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity
D002147:Calmodulin; D016054:DNA, Protozoan; D011994:Recombinant Proteins; D008780:Methyltransferases; C026647:calmodulin methyltransferase; D002118:Calcium