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Updated: Mar 21, 2026
Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET
Published on: October 9, 2021
Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2
Lisa Gerner1, Steffi Munack1, Koen Temmerman2
1Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, Forskningsparken, University of Oslo, Oslo University Hospitals, 0349 Oslo, Norway.
Abstract:
Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) has been implicated in the regulation of metabolic activity in cancer and immune cells, and affects whole-body metabolism by regulating ghrelin-signalling in the hypothalamus. This has led to efforts to develop specific CaMKK2 inhibitors, and STO-609 is the standardly used CaMKK2 inhibitor to date. We have developed a novel fluorescence-based assay by exploiting the intrinsic fluorescence properties of STO-609. Here, we report an in vitro binding constant of KD ∼17 nM between STO-609 and purified CaMKK2 or CaMKK2:Calmodulin complex. Whereas high concentrations of ATP were able to displace STO-609 from the kinase, GTP was unable to achieve this confirming the specificity of this association. Recent structural studies on the kinase domain of CaMKK2 had implicated a number of amino acids involved in the binding of STO-609. Our fluorescent assay enabled us to confirm that Phe(267) is critically important for this association since mutation of this residue to a glycine abolished the binding of STO-609. An ATP replacement assay, as well as the mutation of the 'gatekeeper' amino acid Phe(267)Gly, confirmed the specificity of the assay and once more confirmed the strong binding of STO-609 to the kinase. In further characterising the purified kinase and kinase-calmodulin complex we identified a number of phosphorylation sites some of which corroborated previously reported CaMKK2 phosphorylation and some of which, particularly in the activation segment, were novel phosphorylation events. In conclusion, the intrinsic fluorescent properties of STO-609 provide a great opportunity to utilise this drug to label the ATP-binding pocket and probe the impact of mutations and other regulatory modifications and interactions on the pocket. It is however clear that the number of phosphorylation sites on CaMKK2 will pose a challenge in studying the impact of phosphorylation on the pocket unless the field can develop approaches to control the spectrum of modifications that occur during recombinant protein expression in Escherichia coli.
Insights
Researchers developed a novel fluorescence assay using STO-609 to study Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2). This assay confirms STO-609
Area of Science:
- Biochemistry and Molecular Biology
- Enzymology
- Signal Transduction
Background:
- Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) is a key regulator of cellular metabolism in cancer and immune cells.
- CaMKK2 influences whole-body metabolism via hypothalamic ghrelin signaling, prompting the development of specific inhibitors.
- STO-609 is the established inhibitor for CaMKK2, necessitating methods to study its interaction with the enzyme.
Purpose of the Study:
- To develop a novel fluorescence-based assay for studying CaMKK2.
- To characterize the binding interaction between the CaMKK2 inhibitor STO-609 and CaMKK2.
- To investigate the role of specific amino acids, including the gatekeeper residue Phe(267), in STO-609 binding.
Main Methods:
- Development of a fluorescence assay utilizing the intrinsic fluorescence of STO-609.
- In vitro binding studies to determine the dissociation constant (KD) of STO-609 with purified CaMKK2 and CaMKK2:Calmodulin complex.
- ATP and GTP competition assays, site-directed mutagenesis (Phe(267)Gly), and characterization of phosphorylation sites.
Main Results:
- A high-affinity binding interaction between STO-609 and CaMKK2 was established, with KD ∼17 nM.
- Binding specificity was confirmed by ATP displacement and GTP's inability to displace STO-609.
- Mutation of Phe(267) to Gly significantly abolished STO-609 binding, highlighting its critical role.
- Novel phosphorylation sites on CaMKK2, particularly in the activation segment, were identified.
Conclusions:
- The intrinsic fluorescence of STO-609 enables its use as a probe for the CaMKK2 ATP-binding pocket.
- The developed assay effectively probes the impact of mutations on inhibitor binding.
- Further research is needed to address challenges in studying phosphorylation effects on the ATP-binding pocket due to multiple phosphorylation sites.
