Abstract
Signaling specificity in the mitogen-activated protein kinase (MAPK) pathways is controlled by disordered domains of the MAPK kinases (MKKs) that specifically bind to their cognate MAPKs via linear docking motifs. MKK7 activates the c-Jun N-terminal kinase (JNK) pathway and is the only MKK containing three motifs within its regulatory domain. Here, we characterize the conformational behavior and interaction mechanism of the MKK7 regulatory domain. Using NMR spectroscopy, we develop an atomic resolution ensemble description of MKK7, revealing highly diverse intrinsic conformational propensities of the three docking sites, suggesting that prerecognition sampling of the bound-state conformation is not prerequisite for binding. Although the different sites exhibit similar affinities for JNK1, interaction kinetics differ considerably. Importantly, we determine the crystal structure of JNK1 in complex with the second docking site of MKK7, revealing two different binding modes of the docking motif correlating with observations from NMR exchange spectroscopy. Our results provide unique insight into how signaling specificity is regulated by linear motifs and, in general, into the role of conformational disorder in MAPK signaling.
Highlights
Signaling specificity in the mitogen-activated protein kinase (MAPK) pathways is controlled by disordered domains of the Mitogen-activated protein kinases (MAPKs) kinases (MKKs) that bind to their cognate MAPKs via linear docking motifs
Signaling specificity is controlled by intrinsically disordered N-terminal regulatory domains of the MAPK kinases (MKKs) that selectively bind to their cognate MAPKs [4,5,6,7,8]
We study the interaction between MKK7 and JNK1 by NMR and isothermal titration calorimetry (ITC), providing a comprehensive picture of the affinity, stoichiometry, and kinetics of the JNK1–MKK7 complex
Summary
Signaling specificity in the mitogen-activated protein kinase (MAPK) pathways is controlled by disordered domains of the MAPK kinases (MKKs) that bind to their cognate MAPKs via linear docking motifs. MKK7 is the only human MKK for which three putative docking sites (D1, D2, and D3) are present within its 100-aa regulatory domain (Fig. 1 A and B), posing a number of questions concerning the affinity, stoichiometry, and kinetics as well as the structure and dynamics of the MKK7–JNK signaling complex. We determine the crystal structure of JNK1 in complex with the second MKK7 docking site By combining these data with experimental NMR measurements of the same complex, we provide insight into the dynamic nature of the interaction between the two proteins, suggesting conformational exchange between distinct MKK7 binding modes. The atomic resolution description of full-length MKK7 obtained here extends our view beyond the folded, catalytic domains of the MAPK family and highlights potential new avenues for the discovery of drugs targeting this protein family
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