Structural Characterization of the Recombinant Human Fibroblast Growth Factor Receptor 2b Kinase Domain Upon Interaction with Flavonoids

Fibroblast growth factor receptor 2b (FGFR2b) plays a crucial role in cell signaling pathway, regulating several key biological processes, including cellular differentiation and proliferation. Different types of cancers, including breast cancer, have also been associated with various genetic alterations in the kinase domain of FGFR2b. The present study was conducted to shed new light on a possible mechanism, by which flavonoids alter FGF cell signaling. Recombinant pLEICS-01 expression vectors containing the gene encoding the FGFR2b kinase domain were transformed to E. coli BL21 (DE3). Expression of the recombinant protein was then induced with IPTG. Next, the protein was purified by affinity chromatography. The recombinant protein functionality was assessed using PAGE, by analyzing interactions of the protein with the wild type and the mutant SH2 domains of phospholipase C-γ (PLC-γ). Finally, stability and structural changes of the kinase domain were studied upon interaction with gallic acid, naringenin, quercetin and catechin. The PAGE analysis clearly demonstrated that the recombinant protein interacts only with the wild type SH2 domains of PLC-γ, suggesting the kinase domain is functional. The results also showed that presence of the flavonoids cause a red-shift in the intrinsic fluorescence emission spectra of the kinase domain, suggesting a change in the overall structure of the kinase domain. Further structural studies, using a comprehensive step chemical denaturation analysis, conspicuously showed that the presence of the flavonoids significantly changed the tertiary structure of the kinase domain. The comprehensive structural analyses revealed that the flavonoids significantly impinge on the tertiary structure of the kinase domain thereby suppressing fibroblast growth factor signaling.