Investigating the structure of phosphatidylinositol 3,4,5-triphosphate and phosphatidylinositol 4,5-bisphosphate under Transmission Electron Microscopy and X-ray crystallography

Human liver receptor homolog-1 (hLRH-1), an orphan nuclear receptor coded by the NR5A2 gene, is crucial in regulating the balance of lipids, glucose, and cholesterol homeostasis. Previous studies have shown how the hLRH-1 ligand-binding domain (hLRH-1 LBD) binds phosphatidylinositol 3,4,5-triphosphate (PIP3) and phosphatidylinositol 4,5-bisphosphate (PIP2). However, whether the DNA-binding domain (DBD) and the LBD interact with each other in the full-length hLRH-1 when it is bound to PIP3 or PIP2 is still up for discussion. In this study, the structural interaction between the DBD and the LBD in full-length (FL) hLRH-1 when bound to PIP3 or PIP2 is examined. Considering the structure size, the PIP3 antibody (PIP3 AB) and PIP2 antibody (PIP2 AB) will be used. We hypothesize that the structures of PIP3 AB and PIP2 AB should be pentamers, and hLRH-1 will bind to all five on the outside, similar to a flower shape. Thus, the objective of this pilot experiment is to verify this hypothesis by examining the structure of PIP3 AB and PIP2 AB, involving protein purification, TEM imaging, X-ray crystallography, and co-incubation experiments, which provide a comprehensive approach to studying the interaction between hLRH-1 proteins and phosphatidylinositol antibodies. The results from these experiments will contribute to our understanding of the molecular mechanisms governing LRH-1 interaction with PIP3 and PIP2, potentially elucidating new insights into their functional roles and implications for therapeutic targeting.