14-3-3 proteins inactivate DAPK2 by promoting its dimerization and protecting key regulatory phosphosites

Death-associated protein kinase 2 (DAPK2) is a CaM-regulated Ser/Thr protein kinase, involved in apoptosis, autophagy, granulocyte differentiation and motility regulation, whose activity is controlled by autoinhibition, autophosphorylation, dimerization and interaction with scaffolding proteins 14-3-3. However, the structural basis of 14-3-3-mediated DAPK2 regulation remains unclear. Here, we structurally and biochemically characterize the full-length human DAPK2:14-3-3 complex by combining several biophysical techniques. The results from our X-ray crystallographic analysis revealed that Thr369 phosphorylation at the DAPK2 C terminus creates a high-affinity canonical mode III 14-3-3-binding motif, further enhanced by the diterpene glycoside Fusicoccin A. Moreover, concentration-dependent DAPK2 dimerization is disrupted by Ca2+/CaM binding and stabilized by 14-3-3 binding in solution, thereby protecting the DAPK2 inhibitory autophosphorylation site Ser318 against dephosphorylation and preventing Ca2+/CaM binding. Overall, our findings provide mechanistic insights into 14-3-3-mediated DAPK2 inhibition and highlight the potential of the DAPK2:14-3-3 complex as a target for anti‐inflammatory therapies.

O There are 4 unique types of molecules in this entry. The entry contains 7579 atoms, of which 0 are hydrogens and 0 are deuteriums.
In the tables below, the ZeroOcc column contains the number of atoms modelled with zero occupancy, the AltConf column contains the number of residues with at least one atom in alternate conformation and the Trace column contains the number of residues modelled with at most 2 atoms.

Mol Chain Residues
Atoms ZeroOcc AltConf Trace 3 Residue-property plots i O These plots are drawn for all protein, RNA, DNA and oligosaccharide chains in the entry. The rst graphic for a chain summarises the proportions of the various outlier classes displayed in the second graphic. The second graphic shows the sequence view annotated by issues in geometry and electron density. Residues are color-coded according to the number of geometric quality criteria for which they contain at least one outlier: green = 0, yellow = 1, orange = 2 and red = 3 or more. A red dot above a residue indicates a poor t to the electron density (RSRZ > 2). Stretches of 2 or more consecutive residues without any outlier are shown as a green connector. Residues present in the sample, but not in the model, are shown in grey.
There are no chirality outliers.
There are no planarity outliers. Continued on next page...

Mol Chain Non-H H(model) H(added) Clashes Symm-Clashes
The all-atom clashscore is dened as the number of clashes found per 1000 atoms (including hydrogen atoms). The all-atom clashscore for this structure is 3.
All (48)  There are no Ramachandran outliers to report.

Protein sidechains i O
In the following table, the Percentiles column shows the percent sidechain outliers of the chain as a percentile score with respect to all X-ray entries followed by that with respect to entries of similar resolution.
The Analysed column shows the number of residues for which the sidechain conformation was analysed, and the total number of residues.
Mol Chain Analysed Rotameric Outliers Percentiles Mol Chain Res Type Atoms There are no ring outliers.
3 monomers are involved in 3 short contacts: Mol Chain Res Type Clashes Symm-Clashes The following is a two-dimensional graphical depiction of Mogul quality analysis of bond lengths, bond angles, torsion angles, and ring geometry for all instances of the Ligand of Interest. In addition, ligands with molecular weight > 250 and outliers as shown on the validation Tables will also be included. For torsion angles, if less then 5% of the Mogul distribution of torsion angles is within 10 degrees of the torsion angle in question, then that torsion angle is considered an outlier.
Any bond that is central to one or more torsion angles identied as an outlier by Mogul will be highlighted in the graph. For rings, the root-mean-square deviation (RMSD) between the ring in question and similar rings identied by Mogul is calculated over all ring torsion angles. If the average RMSD is greater than 60 degrees and the minimal RMSD between the ring in question and any Mogul-identied rings is also greater than 60 degrees, then that ring is considered an outlier. The outliers are highlighted in purple. The color gray indicates Mogul did not nd sucient equivalents in the CSD to analyse the geometry. The following is a graphical depiction of the model t to experimental electron density of all instances of the Ligand of Interest. In addition, ligands with molecular weight > 250 and outliers as shown on the geometry validation Tables will also be included. Each t is shown from dierent orientation to approximate a three-dimensional view.
Electron density around FSC A 301: 2mF o -DF c (at 0.7 rmsd) in gray mF o -DF c (at 3 rmsd) in purple (negative) and green (positive) 6.5 Other polymers i O There are no such residues in this entry.