Novel Insights into CRAC channels by Genetic Code Expansion

Calcium (Ca2+) ions are essential for a plethora of biological processes in the human body. We are particularly interested in a unique Ca2+ entry pathway the so-called Ca2+ release-activated Ca2+ ion channel (CRAC). It is constituted by two key players, STIM1 and Orai1, which are critical to human health, but also diseases such as severe combined immunodeficiencies. This makes them attractive targets for novel therapeutic applications, which, however, requires a detailed molecular understanding. Although our knowledge of CRAC channels is already very extensive, several aspects are still unclear, due to a lack of high spatiotemporal control. The focus of this project is to engineer a library of light-sensitive Orai1 proteins to understand the minimal requirements for Orai1 pore opening, to map novel functionally relevant binding pockets, to precisely control downstream signaling and to resolve drug binding pockets. To achieve these goals, we will use the innovative genetic code expansion technology together with functional, biochemical and structural studies. Genetic code expansion allows site-specific manipulation of protein function through the exchange of individual building blocks with novel biochemical and biophysical properties, of which light-sensitive ones are of particular interest. We anticipate that this will provide precise control over and unprecedented insights into working mechanisms and binding interfaces of CRAC channels at the molecular level in vitro and in vivo and in addition accurate control over downstream signaling processes in immune cells. This unique project enables high spatiotemporal, dynamic structure-function resolution of membrane proteins in the living cell. The direct and precise control of CRAC channel movements and binding pockets that we have achieved will open up new possibilities for rapid and targeted intervention in diseases associated with CRAC channels.