Crystal Structure of the Ca2+/Calmodulin-dependent Protein Kinase Kinase in Complex with the Inhibitor STO-609

Abstract

Ca2؉ /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 CaMKK␤ isoform complexed with its selective inhibitor, STO-609. The structure revealed that CaMKK␤ lacks the ␣D helix and that the equivalent region displays a hydrophobic molecular surface, which may reflect its unique substrate recognition and autoinhibition. Although CaMKK␤ 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 CaMKK␤ 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 CaMKK␤ 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 CaMKK␤ and for designing novel inhibitors targeting CaMKK␤ and the related protein kinases.

Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2

“…The tryptic peptides were analyzed using an Ultimate 3000 nano-UHPLC system connected to a Q Exactive mass spectrometer (ThermoElectron, Bremen, Germany). 2ZV2) [13]. Based on the structure of STO-609 we speculated that it could have intrinsic fluorescent properties and during the course of this study this has been confirmed in a recent publication, reporting background fluorescence that interfered with the imaging experiments [17].…”

“…1B) accompanied by a 10-fold enhancement of the quantum yield upon saturation (Fig. 1C); two observations that strongly indicate that STO-609 binding to CaMKK2 results in shielding of the drug/chromophore from the aqueous environment, supporting the reported mainly hydrophobic interaction [13].…”

“…We used the data analysis program GraphPad Prism version 6.00 for Mac (GraphPad Software, www.graphpad.com) to analyse the generated data points to obtain a value for the ‫ܭ‬ assuming only a single binding site (N=1). To analyze the fluorescence binding data we applied a 1 : 1 binding model in accordance with the 1 : 1 molecular stoichiometry of binding between CaMKK2 and STO-609 as observed in the crystal structure [13].…”

“…These physiological associations have led to considerable interest over the last decade in the development of CaMKK2 inhibitors and the most specific example to date, called STO-609 (PubChem CID: 3467590), is known to be a selective ATP-competitive CaMKK inhibitor [12,13]. STO-609 is a naphthoyl fused benzimidazole cell-permeable compound reported to have a low nanomolar IC 50 value as determined by in vitro kinase assays against CaMKKs [12].…”

“…Consequently in order to progress CaMKK2 inhibitors into a clinical setting additional drug development is required. Despite the intense interest in the function of this kinase only two structures have yet been determined; the inhibitor-kinase domain complex [13] and a peptide-derived from the kinase in complex with calmodulin (CaM) [15]. Consequently in order to accelerate the development of new CaMKK2 inhibitors…”