FC0090
MAPK kinase 7 (MKK7)  -  JNK1

Biological function
MKK7 activates the JNK pathway, which primarily regulates stress and inflammatory responses.

Domain organization/sequence features
Molecular recognition occurs through docking sites in the disordered regulatory domains of MKKs that is composed of two to three basic residues followed by a short spacer of one to six residues and finally a hydrophobic-X-hydrophobic submotif. MKK7 is the only MKK, which contains three motifs within its regulatory domain. JNK binds to three distinct docking sites in MKK7: D1 (25-34 AA), D2 (37-47 AA), D3 (70-76 AA).

Structural evidence
HSQC intensities decrease towards the C terminus, indicating that residues outside the canonical motif contact the MAPK surface. NMR relaxation rates (R2 values) indicate conformational exchange of MKK7 on the microsecond to millisecond timescale. CEST and RD experiments also show conformational exchange for residues in and around D2 and D3 motifs. In the crystal structure of the D2 docking site of MKK7 with JNK1, alternative conformations were observed. These resemble the active and auto-inhibited conformations of JNK1, indicating functional relevance.

Biochemical evidence
The affinity for the extended D3 site (48-100 AA) is K_D=11μM, whereas for D3 K_D=49μM, indicating the contribution of residues outside the canonical docking motif.

Structure/Mechanism
The MKK7 peptide inserts into three pockets on the surface of JNK1. In subunits A, B, and C, the peptide is anchored to the hydrophobic pockets via L43, L45, and L47, whereas the positively charged arginines make looser and less well-defined contacts with the surface of JNK1. In an alternative binding mode R40 contacts D326 and E329 on the surface of JNK1 and P41, L45, and L47 occupy the three pockets, resulting in increased flexibility at the level of T42 and L43 and consequently missing electron density for these two residues. The main binding mode of the MKK7 peptide resembles that of the docking site motif of the JNK substrate NFAT4, whereas the alternative binding mode resembles the binding mode of the docking site motif of the scaffold protein JIP1.

Mechanism category
polymorphic (D2 alternative conformations), tethering (D3 flanking)

Significance
Fuzziness contributes to different apparent binding mechanisms and different interaction kinetics for the distinct JNK1 interaction sites. Exchange of alternative conformations in the bound form may tune the balance between auto-inhibited and active state.