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.