Study of the interaction between an intrinsically disordered region of the scaffold protein JIP-2 and JNK-1 kinase

Abstract

[eng] Scaffold proteins play key roles in assembling signaling molecules, facilitating their localization, and coordinating feedback signals for pathway regulation. JIP-2, a scaffold protein in the MAP kinase pathway, warrants exploration for its interaction with JNK-1 kinase, crucial in various cell signaling pathways and acknowledged for its interaction with the D-motif of the JIP-1 scaffold protein. This research targets the 100-240aa region of JIP-2, with the presence of a D-motif, to confirm and understand the interaction with JNK-1. Employing recombinant techniques using Escherichia coli as host microorganisms, JIP2 fragment, along with the JNK-1 and the TEV protease, were obtained through expression plasmids. The purification of the recombinant proteins was conducted through affinity chromatography using a nickel resin, due to its affinity for the histidinetag expressed alongside the recombinant proteins of interests. Furthermore, due to the instability of the JNK-1 enzyme, the production and purification protocols were optimized to ensure an ample quantity of the protein for subsequent studies. The AlphaFold AI tool was used to predict the interaction between JIP-2 and JNK-1 and associated residues, to compare theoretical and experimental outcomes. Experimental verification involved SDS-PAGE electrophoresis and analysis of previously available JIP2 NMR data (2D- HSQCs, and 3D- HNCO, HNCACO, HNCOCACB and iHNCACB) for assignment and NMR titration with JNK-1. Furthermore, chemical shifts and JIP-2 sequence were used to predict secondary structures and potential correlations with complex formation. Interpretation of NMR data substantiated complex formation, confirming D-motif interaction, like JIP-1. The region lacks secondary structure tendency, except at the C-terminal end with a slightly alpha helix tendency and a possible binding to JNK-1 via a non-D-motif. Nevertheless, further substantiation through subsequent studies is required. Understanding the interaction is vital to enhance knowledge of key signaling pathways in neurodegenerative and cardiovascular diseases, cancer, and other conditions where these proteins are present or involved.