Interaction sites on phosphorylase kinase for calmodulin

Abstract

Holophosphorylase kinase was digested with Glu-C specific protease; from the peptide mixture calmodulin binding peptides were isolated by affinity chromatography and identified by N-terminal sequence analysis. Two peptides originating from the α subunit, having a high tendency to form a positively charged amphiphilic helix and containing tryptophane, were synthesized. Additionally, a homologous region of the β subunit and a peptide from the α subunit present in a region deleted in the α′ isoform were also selected for synthesis. Binding stoichiometry and affinity were determined by following the enhancement in tryptophane fluorescence occurring upon 1:1 complex formation between these peptides and calmodulin. Finally, Ca²⁺ binding to calmodulin in presence of peptides was measured. By this way, the peptides α 542–566, α 547–571, α 660–677 and β 597–614 have been found to bind specifically to calmodulin.

Together with previously predicted and synthesized calmodulin binding peptides four calmodulin binding regions have been characterized on each the α and β subunits. It can be concluded that endogenous calmodulin can bind to two calmodulin binding regions in γ as well as to two regions in α and β. Exogenous calmodulin can bind to two regions in α and in β. A binding stoichiometry of 0.8mol of calmodulin/αβγδ protomer of phosphorylase kinase has been determined by inhibiting the ubiquitination of calmodulin with phosphorylase kinase. Phosphorylase kinase is half maximally activated by 23nM calmodulin which is in the affinity range of calmodulin binding peptides from β to calmodulin. Therefore, binding of exogenous calmodulin to β activates the enzyme. A model for switching endogenous calmodulin between α, β and γ and modulation of ATP binding to α as well as Mg²⁺/ADP binding to β by calmodulin is presented.