3.4 ISX9 enhances the interaction of LRP6-ICD with Axin1 through covalently binding to the N-terminal region of Axin
To explore the mechanism through which ISX9 potentiates the protein-protein interaction between LRP6 and Axin1, a microscale thermophoresis (MST) analysis was performed to determine whether ISX9 could directly bind to LRP6 and Axin1. We expressed and purified the three truncated forms of Axin1, Axin1-300 (aa 1–300), Axin301-521 (aa 301-521) and Axin522-862 (aa 522-862), and LRP6-ICD domain as His-tagged fusion proteins. The results showed the titration of ISX9 to fluorescently labeled Axin1-300, yielded dose-dependent upward-shifted thermophoresis profiles with a Kd value of 595 nM (Figure 4A), suggesting the direct binding between ISX9 and Axin1-300. However, no detectable binding was observed between ISX9 and Axin301-521(Figure 4B), Axin522-862 (Figure 4C) or LRP6-ICD (Figure 4D). The fluorescence emission spectra also showed that ISX9 could interact with purified Axin1-300 protein (Figure 4E).
Next, mass spectrometry was used to verify ISX9 binding mode with Axin1-300. The molecular weight of recombinant Axin1-300 is about 31417.38 Da. We detected an extra peak at 31650.81 Da after incubating purified Axin1-300 fragment with ISX9 (233.44 Da) (Figure 4F), indicating that covalent binding was formed between ISX9 and Axin1-300.