Covert Changes in CaMKII Holoenzyme Structure Identified for Activation and Subsequent Interactions

Biophys J. 2015 May 5;108(9):2158-70. doi: 10.1016/j.bpj.2015.03.028.

Abstract

Between 8 to 14 calcium-calmodulin (Ca(2+)/CaM) dependent protein kinase-II (CaMKII) subunits form a complex that modulates synaptic activity. In living cells, the autoinhibited holoenzyme is organized as catalytic-domain pairs distributed around a central oligomerization-domain core. The functional significance of catalytic-domain pairing is not known. In a provocative model, catalytic-domain pairing was hypothesized to prevent ATP access to catalytic sites. If correct, kinase-activity would require catalytic-domain pair separation. Simultaneous homo-FRET and fluorescence correlation spectroscopy was used to detect structural changes correlated with kinase activation under physiological conditions. Saturating Ca(2+)/CaM triggered Threonine-286 autophosphorylation and a large increase in CaMKII holoenzyme hydrodynamic volume without any appreciable change in catalytic-domain pair proximity or subunit stoichiometry. An alternative hypothesis is that two appropriately positioned Threonine-286 interaction-sites (T-sites), each located on the catalytic-domain of a pair, are required for holoenzyme interactions with target proteins. Addition of a T-site ligand, in the presence of Ca(2+)/CaM, elicited a large decrease in catalytic-domain homo-FRET, which was blocked by mutating the T-site (I205K). Apparently catalytic-domain pairing is altered to allow T-site interactions.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural

MeSH terms

  • Amino Acid Sequence
  • Calcium / metabolism
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2 / chemistry*
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2 / metabolism
  • Catalytic Domain*
  • HEK293 Cells
  • Holoenzymes / chemistry
  • Holoenzymes / metabolism
  • Humans
  • Molecular Sequence Data
  • Protein Binding
  • Threonine / chemistry
  • Threonine / metabolism

Substances

  • Holoenzymes
  • Threonine
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2
  • Calcium