Doctoral Dissertations
Date of Award
12-1996
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Major
Biochemistry and Cellular and Molecular Biology
Major Professor
Daniel M. Roberts
Committee Members
Cynthia Peterson, John Koontz, Elizabeth Howell, Solon Georghiou
Abstract
The methyltransferase that catalyzes the trimethylation of lysine 115 in calmodulin has been purified from sheep brain. The enzyme is a monomer with an apparent molecular weight of 38,000 based on gel filtration chromatography and SDS-polyacrylamide electrophoresis. In the presence of calcium the methyltransferase exhibited a Km of 100 nM for unmethylated calmodulin and a kcat of 0.0278 sec-1. The enzyme was able to use calcium-depleted calmodulin as a substrate albeit with less efficiency. The methylation of calcium- depleted calmodulin was inhibited by increases in ionic strength whereas calcium-saturated calmodulin was not affected. Conversely, a calmodulin binding peptide based on the calmodulin-dependent protein kinase II sequence and the naphthalene sulfonamide W-7 inhibit the calmodulin methyltransferase/calmodulin interaction in the presence of calcium but not in the absence of calcium. This suggests a difference in the mode of interaction of calcium-saturated and calcium-depleted calmodulins with the enzyme.
The oxidation of the methionines of calmodulin by performic acid treatment decreases the ability of the methyltransferase to recognize and methylate calmodulin. This data, and the peptide and W-7 inhibitor data, suggest that the integrity of the hydrophobic core/cleft is essential for enzyme binding. Removal of the NH₂-terminal lobe (residues 1- 77) does not affect the ability of the calmodulin methyl transferase to recognize and methylate lysine-115. Thus, the determinants for calmodulin methyltransferase binding reside solely in the COOH-terminal lobe of calmodulin.
Previous work showed that mutant Paramecium with a calmodulin mutant with an isoleucine-to-threonine change at residue 136 showed reduced endogenous level of lysine-115 methylation (Lukas et al., 1989). In this study, a calmodulin with a val to thr-136 substitution (V136T CaM) has been generated and expressed in E. coli to study its regulatory properties and its kinetics as a substrate for the calmodulin- methyltransferase. The rate of V136T calmodulin methylation by the calmodulin methyltransferase was indistinguishable from wild type VU-1 calmodulin in saturating (1 mM) calcium. However, unlike wild type VU-1 calmodulin, V136T calmodulin was incapable of methylation in the absence of calcium (1 mM EGTA). Calcium binding measurements revealed a decreased calcium-binding affinity of V136T calmodulin compared to wild- type calmodulin. Lower calcium-binding affinities were also reflected by an approximate 10-fold shift in the K0.5 values for calcium-dependent activation of NAD kinase (shifted from 1.1 to 9.1 µM Ca2+) and calcium-dependence of methylation (from 0.71 to 7.2 µM Ca2+).
Tyrosine-138 fluorescence spectra showed a different quantum yield between the mutant (q = 0.048) and wild-type (q = 0.037) calmodulins in the absence of calcium, suggesting a different environment of tyr-138 between the two calmodulins in their calcium-depleted forms. This is supported by the electrophoresis profile of V136T calmodulin on nondenaturing gels. The tyrosine fluorescence spectra of the two calmodulins in the presence of saturating calmodulin were identical (q = 0.70 to 0.71), however a higher concentration of calcium was required to titrate the fluorescence spectrum of V136T calmodulin. Overall, the results suggest that the mutation in this highly conserved position in the COOH- terminal hydrophobic cleft results in a lower calcium binding affinity which results in a calmodulin-dependent enzymes, and lower ability to activate a lower ability to be recognized by the methyltransferase under physiological calcium concentrations.
To investigate further the structural requirements of the calmodulin/methyltransferase interaction, an attempt was made to engineer a methylation sequence within a symmetrical position of the amino terminal lobe. An EKL sequence was introduced at position 41-43 of the NH2-terminal lobe (corresponding to position 114-116 of the COOH-terminal lobe) of VU-3 calmodulin which has an arginine residue at position 115. Although the NH2-terminal lobe of the mutant calmodulin [QNP (41-43) EKL/K115R] possessed the sequence of the loop (residues 112-117, LGEKLT) that is methylated in the COOH- terminal lobe, it was not methylated by the methyltransferase. The finding suggests that other residues unique to the COOH- terminal lobe are necessary for binding and recognition by the enzyme.
A calmodulin-DHFR sandwich fusion protein was generated by insertion of VU-1 calmodulin into the ẞ-bulge region of DHFR to observe the effects of structurally constraining the calmodulin structure. The calcium binding properties of the sandwich protein were almost identical to VU-1 calmodulin. However, NAD kinase and PDE activator properties of the sandwich protein were lower than that of VU-1 calmodulin, and the sandwich protein was poorly methylated by the methyltransferase, particularly in the presence of trimethoprim. Overall, the data suggest that the insertion of calmodulin into the DHFR structure has little effect on calcium binding by the individual lobes of calmodulin, but may constrain the lobes in a manner that results in altered interaction with the calmodulin-dependent proteins, and severely influences recognition by the methyltransferase.
Recommended Citation
Han, Chang-Hoon, "Isolation of the calmodulin N-methyltransferase and characterization of its interaction with calmodulin. " PhD diss., University of Tennessee, 1996.
https://trace.tennessee.edu/utk_graddiss/9755