Minimale Bewegungen und Wirkstoffbindung von Orai-Isoformen

Calcium ions (Ca2+) play a central role in many processes in the human body, from immune responses to hormone release and muscle activity. Our work focuses on a very specific pathway that allows Ca2+ to enter cells: the Ca2+ release-activated Ca2+ channel (CRAC). These channels are built from two types of proteins, STIM and Orai. STIM proteins are found in the endoplasmic reticulum, while Orai proteins form the pore in the cell membrane through which Ca2+ enters. Although the important role of CRAC channels in regulating Ca2+ entry is well known, key mechanistic aspects remain unresolved, and addressing these gaps is critical for advancing future therapeutic strategies. This is especially true for the three Orai isoforms (Orai1-3), which differ in certain functional and structural properties and remain poorly understood, despite their comparable activation mechanism. The goal of this project is to uncover the pore-opening mechanisms of Orai1, the best-studied isoform, at the molecular level and to identify its drug-binding pockets. To this end, we build on our recent development of light-sensitive Orai1 variants. Using an advanced method called Genetic Code Expansion (GCE), we can substitute individual amino acids in Orai1 with unnatural amino acids (UAAs) that can form covalent bonds, either upon specific light irradiation (photocrosslinking UAAs) or through reaction with a specific nearby amino acid residue (chemical crosslinking UAAs). We expect that inserting these UAAs at key sites will promote interactions between transmembrane (TM) domains through covalent bonding, triggering local structural changes that may propagate throughout the entire Orai1 complex. This could eventually lock Orai1 in either a higher or lower active/inactive conformation. In addition, incorporating UAAs at specific sites and/or the formation of the covalent bond may also interfere with the action of certain drugs, enabling us to pinpoint drug-binding sites with a level of precision not previously possible. Once we uncover these mechanisms for Orai1, we will extend our work to the other two isoforms, Orai2 and Orai3, to reveal how their opening mechanisms and drug‑binding sites differ. To achieve these goals, we will employ the innovative technology of GCE in combination with functional studies and biochemical methods, supported by docking studies and molecular dynamics (MD) simulations. We expect that this project will provide unprecedented insight into Orai pore opening and drug-binding sites. Understanding these mechanisms will lay the foundation for developing highly specific Orai-targeted therapies for diseases in which Ca2+ signaling is disrupted.