Remodeling Ca2+ dynamics by targeting a promising E-box containing G-quadruplex at ORAI1 promoter in triple-negative breast cancer

•G4 motif regulating the expression profile of the ORAI1 transporter.•Structural analysis revealed a parallel G4 structure with high thermal stability.•Stable G4 formation alters Store Operated Calcium Entry in TNBC. ORAI1 is an intrinsic component of store-operated calcium entry (SOCE) that strictly regulates Ca2+ influx in most non-excitable cells. ORAI1 is overexpressed in a wide variety of cancers, and its signal transduction has been associated with chemotherapy resistance. There is extensive proteomic interaction of ORAI1 with other channels and effectors, resulting in various altered phenotypes. However, the transcription regulation of ORAI1 is not well understood. We have found a putative G-quadruplex (G4) motif, ORAI1-Pu, in the upstream promoter region of the gene, having regulatory functions. High-resolution 3-D NMR structure elucidation suggests that ORAI1-Pu is a stable parallel-stranded G4, having a long 8-nt loop imparting dynamics without affecting the structural stability. The protruded loop further houses an E-box motif that provides a docking site for transcription factors like Zeb1. The G4 structure was also endogenously observed using Chromatin Immunoprecipitation (ChIP) with anti-G4 antibody (BG4) in the MDA-MB-231 cell line overexpressing ORAI1. Ligand-mediated stabilization suggested that the stabilized G4 represses transcription in cancer cell line MDA-MB-231. Downregulation of transcription further led to decreased Ca2+ entry by the SOCE pathway, as observed by live-cell Fura-2 Ca2+ imaging. ORAI1 expression is downregulated in the presence of a stable G4 structure at the predicted ORAI1-Pu motif, which is formed at a regulatory docking site for transcription factors and RNA polymerase II. The G4 structure might act as a dynamic switch, as it is a transient structure and is regulated by various factors within a cell. Ligand-mediated stabilization of ORAI1-Pu via TMPyP4/ BRACO-19 indicated gain-of-function in ORAI1 expression. In MDA-MB-231, we observed greater fold endogenous binding of Zeb1 at the ORAI-Pu motif, compared to BG4, which binds to stable G4 structures. In MDA-MB-231, the ORAI1-Pu locus might be in a partially unfolded state or unfolded state, leading to over-expression of ORAI1. However, further studies can reveal the physiological dynamics of the structure and its functional effects on ORAI expression. Overexpression of ORAI1 increases its transport to the plasma membrane and increases SOCE on interaction with activ