The structural effects of endogenous and exogenous Ca2+/calmodulin on phosphorylase kinase.
The activity of phosphorylase b kinase (PbK) is stimulated by Ca2+ ions, which act through its endogenous calmodulin subunit (delta), and further stimulated by the Ca2+-dependent binding of exogenous calmodulin (delta'). In contrast to their highly characterized effects on activity, little is known regarding the structural effects on the (alphabetagammadelta)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 effectors influence the structure of the catalytic gamma subunit and the interactions among all of the subunits. As determined by reductive methylation and carboxymethylation, Ca2+ increased the accessibility of the gamma subunit; it also increased the formation by phenylenedimaleimide of an alphagammagamma conjugate that is characteristic of activated conformations of PbK (Nadeau, O. W., Sacks, D. M., and Carlson, G. M. (1997) J. Biol. Chem. 272, 26196-26201); however, Ca2+ also had structural effects that were clearly distinct from other activators. Moreover, similar structural effects of Ca2+ 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-linked conjugates formed in the presence of Ca2+ by o-phenylenedimaleimide or m-maleimidobenzoyl-N-hydroxysuccinimide ester showed that the binding of Ca2+ to the delta subunit triggers changes in the interactions among all subunits, including between protomers, indicating an extensive communication network throughout the PbK complex. Most of the structural effects of delta'/Ca2+ were qualitatively similar to, but quantitatively greater than, the effects of Ca2+ alone; but delta'/Ca2+ also had distinct effects, especially involving cross-linking of the delta subunit.
Nadeau
O W
OW
Department of Biochemistry, College of Medicine, the University of Tennessee, Memphis, Tennessee 38163, USA.
Sacks
D B
DB
Carlson
G M
GM
D000818:Animals; D002118:Calcium / Q000494:pharmacology / Q000502:physiology*; D002147:Calmodulin / Q000494:pharmacology / Q000502:physiology*; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000737:chemistry; D004789:Enzyme Activation; D008301:Maleimides / Q000737:chemistry; D010764:Phosphorylase Kinase / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D013388:Succinimides / Q000737:chemistry
D002147:Calmodulin; D003432:Cross-Linking Reagents; D008301:Maleimides; D013388:Succinimides; C013097:3-maleimidobenzoyl N-hydroxysuccinimide; D010764:Phosphorylase Kinase; D002118:Calcium