title: Crystal structure of the Ca²⁺/calmodulin-dependent protein kinase kinase in complex with the inhibitor STO-609.
abstract: Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) kinase (CaMKK) is a member of the CaMK cascade that mediates the response to intracellular Ca(2+) elevation. CaMKK phosphorylates and activates CaMKI and CaMKIV, which directly activate transcription factors. In this study, we determined the 2.4 Å crystal structure of the catalytic kinase domain of the human CaMKKβ isoform complexed with its selective inhibitor, STO-609. The structure revealed that CaMKKβ lacks the αD helix and that the equivalent region displays a hydrophobic molecular surface, which may reflect its unique substrate recognition and autoinhibition. Although CaMKKβ lacks the activation loop phosphorylation site, the activation loop is folded in an active-state conformation, which is stabilized by a number of interactions between amino acid residues conserved among the CaMKK isoforms. An in vitro analysis of the kinase activity confirmed the intrinsic activity of the CaMKKβ kinase domain. Structure and sequence analyses of the STO-609-binding site revealed amino acid replacements that may affect the inhibitor binding. Indeed, mutagenesis demonstrated that the CaMKKβ residue Pro(274), which replaces the conserved acidic residue of other protein kinases, is an important determinant for the selective inhibition by STO-609. Therefore, the present structure provides a molecular basis for clarifying the known biochemical properties of CaMKKβ and for designing novel inhibitors targeting CaMKKβ and the related protein kinases.
authors.affiliation: RIKEN Systems and Structural Biology Center, Yokohama 230-0045, Japan.
authors.forename: Mutsuko
authors.initials: M
authors.lastname: Kukimoto-Niino
authors.forename: Seiko
authors.initials: S
authors.lastname: Yoshikawa
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Takagi
authors.forename: Noboru
authors.initials: N
authors.lastname: Ohsawa
authors.forename: Yuri
authors.initials: Y
authors.lastname: Tomabechi
authors.forename: Takaho
authors.initials: T
authors.lastname: Terada
authors.forename: Mikako
authors.initials: M
authors.lastname: Shirouzu
authors.forename: Atsushi
authors.initials: A
authors.lastname: Suzuki
authors.forename: Suni
authors.initials: S
authors.lastname: Lee
authors.forename: Toshimasa
authors.initials: T
authors.lastname: Yamauchi
authors.forename: Miki
authors.initials: M
authors.lastname: Okada-Iwabu
authors.forename: Masato
authors.initials: M
authors.lastname: Iwabu
authors.forename: Takashi
authors.initials: T
authors.lastname: Kadowaki
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Minokoshi
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
mesh_terms: D000595:Amino Acid Sequence; D001562:Benzimidazoles / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D053644:Naphthalimides / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D001562:Benzimidazoles; D053644:Naphthalimides; D020033:Protein Isoforms; D047428:Protein Kinase Inhibitors; C458525:STO 609; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase