Cefotetan-bound human RKIP involves in Ras/Raf1/MEK/ERK signaling pathway

Cefotetan is widely used to treat bacterial infections in the clinic owing to its broad spectrum of antibacterial activity. In the present study, we demonstrate that cefotetan can bind to the conserved ligand-binding pocket of human Raf1 kinase inhibitory protein (hRKIP), which acts as a negative regulator of the Ras/Raf1/MEK/ERK signaling pathway. The cefotetan-bound hRKIP adopts a rigid structure with insufficient space for binding Raf1 kinase, thereby reliving the inhibitory activity of hRKIP in the Ras/Raf1/MEK/ERK signaling pathway and enhancing the phosphorylation level of ERK. Both NMR titration and molecular docking approaches show that several residues (P74, Y81, W84, P111, P112, K113, S142, G143, D144, W173, P178, Y181 and L184) play crucial roles in hRKIP binding cefotetan. NMR dynamics analysis reveals that the binding of cefotetan with hRKIP promotes ps-ns internal motion but reduces μs-ms conformational exchange for residues in the cefotetan-binding pocket of hRKIP. Our results not only disclose the structural basis of cefotetan upregulating the Ras/Raf1/MEK/ERK signaling pathway but also benefit developing novel drugs against diseases caused by the impaired Ras/Raf1/MEK/ERK pathway.