title: Structural basis for RKIP binding with its substrate Raf1 kinase.
abstract: Raf1 kinase inhibitor protein (RKIP) negatively regulates the Raf1/MEK/ERK pathway which is vital for cell growth and differentiation. It is also a biomarker in clinical cancer diagnosis. RKIP binds to the N-terminus of Raf1 kinase but little is known about the structural basis of RKIP binding with Raf1. Here, we demonstrate that the N-terminus of human Raf1 kinase (hRaf11-147aa) binds with human RKIP (hRKIP) at its ligand-binding pocket, loop "127-149", and the C-terminal helix by NMR experiments. D70, D72, E83, Y120, and Y181 were further verified as the key residues participating in the interaction of hRKIP and hRaf11-147aa. G143-R146 fragment was also critical for hRKIP binding with hRaf11-147aa, for its deletion decreased the binding affinity around 300 times, from 154 to 0.46 mM(-1). Our results provide important structural clues for designing the lead compound that disrupts RKIP-Raf1 interaction.
authors.affiliation: Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, 422 Siming South Road, Xiamen, 361005, Fujian, China, wuzhihua890320@163.com.
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Wu
authors.forename: Cuiping
authors.initials: C
authors.lastname: Fu
authors.forename: Lina
authors.initials: L
authors.lastname: Shi
authors.forename: Lu
authors.initials: L
authors.lastname: Ruan
authors.forename: Donghai
authors.initials: D
authors.lastname: Lin
authors.forename: Chenyun
authors.initials: C
authors.lastname: Guo
mesh_terms: D001665:Binding Sites; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D049488:Phosphatidylethanolamine Binding Protein / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry* / Q000378:metabolism*; D017384:Sequence Deletion
chemical_list: C511482:PEBP1 protein, human; D049488:Phosphatidylethanolamine Binding Protein; D019908:Proto-Oncogene Proteins c-raf