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

Abstract

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.

Keywords: BLI; ERK phosphorylation; NMR; cefotetan; hRKIP.

Figure 1

Effects of cefotetan on the Ras/Raf1/MEK/ERK signaling pathway in human HEK293T cells and HeLa cells (A,B) The phosphorylation level of ERK in HEK293T cells incubated with cefotetan. (C,D) The phosphorylation level of ERK in HeLa cells incubated with cefotetan. (E,G) The phosphorylation levels of ERK in both hRKIP-transferred HEK293T cells and hRKIP-transferred HeLa cells. WT, wild-type cells transfected with pCDNA3.0 plasmid; RKIP, cells transfected with pCDNA3.0-hRKIP plasmid. (H,I) The phosphorylation level of ERK in hRKIP-transferred HEK293T cells incubated with cefotetan. (J,K) The phosphorylation level of ERK in hRKIP-transferred HeLa cells incubated with cefotetan. Cefotetan was dissolved in DMSO, and the control lanes were DMSO only with 0 μM cefotetan. Data are shown as the mean±SD ( n=3 or 4). * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

Figure 2

Determination of affinities of binding between hRKIP and cefotetan by BLI and ITC assays (A) The BLI experiment was performed on the ForteBio Octet Red system. The association at each cefotetan concentration was detected for 900 s, while the dissociation was monitored for 1800 s. (B) The ITC experiment was conducted on a MicroCal ITC200. Cefotetan (5 mM) was titrated stepwise into 280 μL of 30 μM hRKIP solution by 20 injections (2 μL aliquots at 120-s intervals).

Figure 3

Identification of binding sites of cefotetan on hRKIP (A) Overlaid 1H- 15N HSQC spectra were recorded on 15N-labelled hRKIP before and after cefotetan titration. Black: hRKIP before cefotetan addition; Red: hRKIP after cefotetan titration with a molar ratio of 1:5. (B) Chemical shift perturbations of nonpro residues in hRKIP after cefotetan titration. Solid line: the averaged chemical shift perturbation; dashed line: one standard deviation (SD) plus the average. (C) The identified binding sites of cefotetan on hRKIP. (D) The structural model of the hRKIP-cefotetan complex established by HADDOCK. The 3D structure of cefotetan is displayed as sticks in rainbow. (E) Schematic diagram of intermolecular interactions in the structural model of the hRKIP-cefotetan complex produced by LigPlot.

Figure 4

Comparison of dynamic properties between cefotetan-bound hRKIP and free hRKIP derived from NMR relaxation measurements (A) Backbone amide relaxation parameters R 1, R 2 and { 1H}- 15N NOE values versus residue number. Black: free hRKIP; Red: cefotetan-bound hRKIP. (B) Dynamics parameters S 2 and R ex obtained from model-free analyses. Black: free hRKIP; Red: cefotetan-bound hRKIP. (C,D) Residues with decreased S 2 values in the binding pocket of cefotetan-bound hRKIP (D) relative to free hRKIP (C) are displayed in red balls. (E,F) Residues with significant conformational exchange in the binding pocket of free hRKIP (E) and cefotetan-bound hRKIP (F) are displayed in yellow balls.