Computational and site‐specific mutagenesis analyses of the asymmetric charge distribution on calmodulin

Abstract

Calmodulin's calculated electrostatic potential surface is asymmetrically distributed about the molecule. Concentrations of uncompensated negative charge are localized near certain alpha-helices and calcium-binding loops. Further calculations suggest that these charge features of calmodulin can be selectively perturbed by changing clusters of phylogenetically conserved acidic amino acids in helices to lysines. When these cluster charge reversals are actually produced by using cassette-based site-specific mutagenesis of residues 82-84 or 118-120, the resulting proteins differ in their interaction with two distinct calmodulin-dependent protein kinases, myosin light chain kinase and calmodulin-dependent protein kinase II. Each calmodulin mutant can be purified to apparent chemical homogeneity by an identical purification protocol that is based on conservation of its overall properties, including calcium binding. Although cluster charge reversals result in localized perturbations of the computed negative surface, single amino acid changes would not be expected to alter significantly the distribution of the negative surface because of the relatively high density of uncompensated negative charge in the region around residues 82-84 and 118-120. However, this does not preclude the possibility of single amino acid charge perturbations having a functional effect on the more intimate, catalytically active complex. The electrostatic surface of calmodulin described in this report may be a feature that would be altered only by cluster charge reversal mutations. Overall, the results suggest that the charge properties of calmodulin are one of several properties that are important for the efficient assembly of calmodulin-protein kinase signal transduction complexes in eukaryotic cells.

Interaction of smooth muscle caldesmon with calmodulin mutants

“…Many of the mutants have differences in charged amino acids which affect their ability to activate selected CaM-dependent enzymes such as MLCK [14,23,24], CaM-dependent protein kinase II [14], phosphorylase kinase [15], and plasma membrane Ca 2÷-ATPase [22]. In our study of CaM mutants we found that the results with the CaM mutants can be divided into three groups, as shown in Table 1.…”

“…Site-directed mutagenesis was used to generate the various mutant calmodulins [13,14]. The mutations are described in Table 1.…”

Primary Structures of Arabidopsis Calmodulin Isoforms Deduced from the Sequences of cDNA Clones

“…It is inferred from in vitro studies that interactions between CaM and its target enzymes involve structurally overlapping sites on the CaM molecule (29). It has also been speculated that selection pressure for CaM to remain highly conserved across phylogenetic lines is a consequence of tight substrate specificity for CaM-binding sites within CaM-dependent proteins.…”

Analysis of Phosphorylation and Mutation of Tyrosine Residues of Calmodulin on Its Activation of the Erythrocyte Ca2+‐transporting ATPase

“…Structural information has been derived from examination of calmodulin bound to peptides (Ikura et al, 1992;Meador et al, 1992Meador et al, , 1993 and by analysis of structural modifications of calmodulin on its activation of target enzymes (Craig et al, 1987;Persechini et al, 1989;Weber et al, 1989;Kosk-Kosicka and Bzdega, 1991 ;Cao et al, 1993 light-chain kinase (Ikura et al, 1992;Meador et al, 1992) and Ca*'/calmodulin-dependent protein kinase I1 (Meador et al, 1993). The crystal structure of calmodulin bound to Ca2'-ATPase is not known, but the complex appears to adopt a more extended conformation than the globular shape seen with the other peptides (Kataoka et al, 1991).…”