Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a member of the Ca2+/calmodulin-dependent kinase (CaMK) family involved in adiposity regulation, glucose homeostasis and cancer, and is a potential target for therapeutic intervention. This upstream activator of CaMKI, CaMKIV and AMP-activated protein kinase is negatively regulated by phosphorylation, which triggers an association with the scaffolding protein 14-3-3. Mechanistically, 14-3-3 proteins constrain the conformation, occlude sequence-specific and structural features, promote protein-protein interactions, or prevent the aggregation of their binding partners. Studies have shown that CaMKKs bind to various 14-3-3 protein isoforms and that the conserved motif containing phosphorylated Ser74 in CaMKK1 (corresponding to Ser100 in CaMKK2) functions as the primary 14-3-3 binding site. Furthermore, a second 14-3-3 binding motif containing phosphorylated Ser475 in CaMKK1 (Ser511 in CaMKK2) has also been suggested. Although the putative C-terminal 14-3-3 binding motif (sequence RSLpSer511AP) is a canonical “mode I” 14-3-3 binding site (RXX(pS/pT)XP, wherein pS/pT is phosphoserine or phosphothreonine and X is any residue), the N-terminal motif (sequence RKLpS100LQE) contains a Gln residue at the position +2 relative to the phosphorylated residue pSer100. Bioinformatics survey of 14-3-3 binding sites revealed that Gln is seldom found at +2 because the Pro residue and, to a lesser extent, Ser, Gly and Asp also, are frequently found at this position.
To elucidate the structural basis of interactions between 14-3-3 proteins and the 14-3-3 binding motifs of CaMKK2, we solved the crystal structures of phosphopeptides pepS100 (sequence RKLpSLQER) and pepS511 (sequence RSLpSAPGN) bound to 14-3-3z and 14-3-3g, respectively. The 14-3-3 isoforms were selected based on quality of the resulting crystals. The crystal structures were solved by molecular replacement using the structures of 14-3-3z (PDB ID: 4FJ3) and 14-3-3g (PDB ID: 2B05) as search models, and refined at a resolution of 2.68 and 2.84 Å, respectively. The final electron densities allowed us to build seven residues (KLpSLQER) of pepS100 and six residues (RSLpSAP) of pepS511. The crystal structures showed that both phosphopeptides interact with the amphipathic groove of 14-3-3 similarly to other 14-3-3 complexes. Nevertheless, in the case of the N-terminal motif, the interaction between the side-chain of Gln at the position +2 relative to pSer100 and the phosphate group appears to change the direction of the polypeptide chain. Interestingly, the superimposition of this structure with that of the ternary complex between the phosphopeptide derived from the C-terminus of plant plasma membrane H+-ATPase, plant 14-3-3C, and fusicoccin showed that the fusicoccin binding cavity stays empty due to the abrupt change in the direction of the C-terminal part of pepS100. Therefore, this protein-protein interaction might be stabilized by small-molecule compounds, as previously reported for other 14-3-3 complexes, which is a potential strategy to inhibit the CaMKK activity.
This study was supported by the Czech Science Foundation (Projects 16-02739S) and the Initial Training Network, funded by the H2020 Marie Curie Actions of the European Commission under Grant Agreement 675179.