title: Covert Changes in CaMKII Holoenzyme Structure Identified for Activation and Subsequent Interactions.
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.
authors.affiliation: Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, Maryland.
authors.forename: Tuan A
authors.initials: TA
authors.lastname: Nguyen
authors.affiliation: Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, Maryland.
authors.forename: Pabak
authors.initials: P
authors.lastname: Sarkar
authors.affiliation: Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, Maryland.
authors.forename: Jithesh V
authors.initials: JV
authors.lastname: Veetil
authors.affiliation: Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, Maryland.
authors.forename: Kaitlin A
authors.initials: KA
authors.lastname: Davis
authors.affiliation: Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, Maryland.
authors.forename: Henry L
authors.initials: HL
authors.lastname: Puhl
authors.affiliation: Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, Maryland. Electronic address: stevevog@mail.nih.gov.
authors.forename: Steven S
authors.initials: SS
authors.lastname: Vogel
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D057809:HEK293 Cells; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D013912:Threonine / Q000737:chemistry / Q000378:metabolism
chemical_list: D020035:Holoenzymes; D013912:Threonine; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D002118:Calcium