Stabilization of Protein–Protein Interactions between CaMKK2 and 14–3–3 by Fusicoccins

Abstract

Ca 2+ /calmodulin-dependent protein kinase kinase 2 (CaMKK2) regulates several key physiological and pathophysiological processes, and its dysregulation has been implicated in obesity, diabetes, and cancer. CaMKK2 is inhibited through phosphorylation in a process involving binding to the scaffolding 14−3−3 protein, which maintains CaMKK2 in the phosphorylation-mediated inhibited state. The previously reported structure of the N-terminal CaMKK2 14−3−3-binding motif bound to 14− 3−3 suggested that the interaction between 14−3−3 and CaMKK2 could be stabilized by small-molecule compounds. Thus, we investigated the stabilization of interactions between CaMKK2 and 14−3−3γ by Fusicoccin A and other fusicoccanes diterpene glycosides that bind at the interface between the 14−3− 3 ligand binding groove and the 14−3−3 binding motif of the client protein. Our data reveal that two of five tested fusicoccanes considerably increase the binding of phosphopeptide representing the 14−3−3 binding motif of CaMKK2 to 14−3−3γ. Crystal structures of two ternary complexes suggest that the steric contacts between the C-terminal part of the CaMKK2 14−3−3 binding motif and the adjacent fusicoccane molecule are responsible for differences in stabilization potency between the study compounds. Moreover, our data also show that fusicoccanes enhance the binding affinity of phosphorylated full-length CaMKK2 to 14−3−3γ, which in turn slows down CaMKK2 dephosphorylation, thus keeping this protein in its phosphorylation-mediated inhibited state. Therefore, targeting the fusicoccin binding cavity of 14−3−3 by small-molecule compounds may offer an alternative strategy to suppress CaMKK2 activity by stabilizing its phosphorylation-mediated inhibited state.

The 14-3-3 Proteins as Important Allosteric Regulators of Protein Kinases

“…As such, the interaction between CaMKK2 and 14-3-3 may be stabilized by small-molecule compounds, as previously described for other 14-3-3 complexes [34,57,58,76]. Indeed, the recently published study confirmed that fusicoccins can stabilize protein-protein interactions between CaMKK2 and 14-3-3 [56]. On the other hand, structure of the diphosphorylated pSer 100 -pSer 511 peptide (PDB ID: 6EF5) did not show the interaction between Gln 102 and pSer 100 , thus suggesting that the proline-like conformation of the polypeptide chain can also be formed without that interaction [74].…”

“…Because the 14-3-3 protein is a physiological inhibitor of ASK1, the ASK1:14-3-3 and ASK2:14-3-3 complexes are potential targets for therapeutic intervention as an alternative or complimentary strategy in suppressing the ASK1 activity. Recent advances in the development of small molecule compounds aimed at stabilizing 14-3-3 protein-kinase interactions [21,[55][56][57][58] have demonstrated the feasibility of this approach.…”

Fragment-based exploration of the 14-3-3/Amot-p130 interface

“…2 A and B). Crystal structures of 14-3-3 bound to other interaction partners have shown how such a pocket can potentially accommodate the natural product Fusicoccin-A (FC-A) or its derivatives, increasing the stability of the ternary complex ( Stevers et al., 2016 ; Würtele et al., 2003 ; Anders et al., 2013 ; Bier et al., 2016 ; Wolter et al., 2020 ; Lentini Santo et al., 2020 ). An initial exploration of the pocket was performed by using FC-A as a tool compound and a small selection of FC-like compounds, at single-dose in an FP assay.…”

Increased CaMKK2 expression is an adaptive response that maintains the fitness of tumor-infiltrating natural killer cells

“…The contrasting effects of CaMKK2 inhibitors and LDDs on NK cell survival suggest a distinct “scaffold” function for CaMKK2. Several proteins are known to bind CaMKK2, including the eponymous calmodulin activator and the inhibitory 14-3-3γ protein 1,48 . Interestingly, CaMKK2 serves an important scaffold function in hepatocellular carcinoma, nucleating the assembly of a CaMKIV, mTOR, and S6K complex that promotes tumor proliferation 49 .…”