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Altered methylation substrate kinetics and calcium binding of a calmodulin with a Val136-->Thr substitution
1Department of Biochemistry, Cellular and Molecular Biology, The University of Tennessee, Knoxville 37996-0840, USA.
Abstract:
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.
Insights
A mutation in calmodulin (Thr136) impairs calcium binding and alters its structure, reducing methylation at physiological calcium levels. This affects calmodulin
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Chemistry
Background:
- Calmodulin (CaM) undergoes post-translational modification, including trimethylation on Lys115, catalyzed by a specific CaM methyltransferase.
- A Paramecium cam2 mutant (Ile136 to Thr) exhibits reduced Lys115 methylation, suggesting a role for this residue in the methylation process.
Purpose of the Study:
- To investigate the impact of the Ile136Thr substitution on CaM structure, function, and interaction with CaM methyltransferase.
- To characterize the biochemical properties of a recombinant Thr136-substituted CaM ([Thr136]CaM) expressed in E. coli.
Main Methods:
- Expression of unmethylated [Thr136]CaM in Escherichia coli.
- In vitro analyses of enzyme activation, calcium binding, and methylation kinetics.
- Non-denaturing electrophoresis and Tyr138 spectroscopy to assess structural conformations.
Main Results:
- [Thr136]CaM exhibited wild-type enzyme activation and steady-state kinetics with CaM methyltransferase at saturating calcium.
- Key defects observed: inability to be methylated in the absence of calcium and impaired calcium binding.
- A ~10-fold shift in Ca2+ dependence for enzyme activation (K0.5: 1.1 µM to 9.1 µM) and methylation (K0.5: 0.71 µM to 7.2 µM) was noted.
- Structural differences in calcium-depleted states between wild-type and [Thr136]CaM were suggested by spectroscopic and electrophoretic data.
Conclusions:
- The mutation at position 136 in the conserved COOH-terminal hydrophobic core reduces CaM's calcium-binding affinity.
- Altered calcium-depleted structure of [Thr136]CaM leads to decreased methylation efficiency at physiological calcium concentrations.
- The Ile136 residue is crucial for maintaining CaM's structural integrity and efficient calcium-dependent methylation.