THE STRUCTURAL EFFECTS OF ENDOGENOUS AND EXOGENOUS CA2+ CALMODULIN ONPHOSPHORYLASE-KINASE/

The activity of phosphorylase b kinase (PbK) is stimulated by Ca2+ ion s, which act through its endogenous calmodulin subunit (delta), and fu rther stimulated by the Ca2+-dependent binding of exogenous calmodulin (delta'). In contrast to their highly characterized effects on activi ty, little is known regarding the structural effects on the (alpha bet a gamma delta)(4) PbK. holoenzyme induced by Ca2+ and delta'/Ca2+, We have used mono- and bifunctional chemical modifiers as conformational probes to compare how the two effecters influence the structure of the catalytic gamma subunit and the interactions among all of the subunit s. As determined by reductive methylation and carboxymethylation, Ca2 increased She accessibility of the gamma subunit; it also increased t he formation by phenylenedimaleimide of an alpha gamma gamma conjugate that is characteristic of activated conformations of PbK (Nadeau, O.W ., Sacks, D.M., and Carlson, G.M. (1997) J. Biol. Chem:. 272, 26196-26 201); however; Ca2+ also had structural effects that were clearly dist inct fi om other activators, Moreover, similar structural effects of C a2+ were observed with PbK that had been activated by phosphorylation, consistent with the fact that such activation does not eliminate the catalytic dependence of the enzyme on Ca2+, Our results suggest tiers of conformational transitions in the activation of PbK, with the most fundamental being induced by Ca2+. Analysis of the various cross-linke d conjugates formed in the presence of Ca2+ by o-phenylenedimaleimide or m-maleimidobenzoyl-N-hydroxysuccinimide ester showed that the bindi ng of Ca2+ to the delta subunit triggers changes in the interactions a mong all subunits, including between protomers, indicating an extensiv e communication network throughout the PbK complex. Most of the struct ural effects of delta'/Ca2+ were qualitatively similar to, but quantit atively greater than, the effects of Ca2+ alone; but delta'/Ca2+ also had distinct effects, especially involving cross-linking of the delta subunit.