Abstract
S100A4 is a homodimeric protein that belongs to the S100 subfamily of EF hand of Calcium binding protein. Calcium bounded S100A4 is known to activate the RAGE receptor and stimulate both ERK and NF-κB signaling. RAGE receptor and S100A4 protein involved in a wide range of inflammation-related pathological states, such as vascular diseases, diabetes, neurodegeneration and cancer. As RAGE receptor and S100A4 play an important role in tumor formation, it is clear that preventing the formation of RAGE-S100A4 multi-protein complex is an effective strategy to inhibit various cancers. In this study, we elucidated the structural interactions between mS100A4 and RAGE V-domain. We employed a variety of biophysical techniques, including fluorescence spectroscopy, multidimensional NMR spectroscopy, HADDOCK, mutagenesis study and functional assay to characterize the interactions between mS100A4 and RAGE V-domain. The binding constant was determined from fluorescence titration. The binding interfaces upon complex formation of mS100A4 and RAGE V mapping from 15N-1H HSQC titrations. Further HADDOCK modeling and mutagenesis study and functional assay indicated the role of important residues of mS100A4 protein for RAGE V-domain protein interactions. In this study, we also identified amlexanox as a small molecule that can bind to mS100A4 and inhibit the interactions between mS100A4 and the RAGE V-domain, according to our HADDOCK binding model and functional assay studies. The present study describes the binding properties of mS100A4-Amlexanox and provides knowledge of the binding sites at the molecular level, which facilitate the design of better drugs to disrupt the mS100A4-RAGE pathway and treat various diseases, such as cancer, metastasis, and diabetes.