Characterization of the STIM1-Orai1 activation-interface

Store operated calcium entry represents a fundamental way of calcium uptake and is essential for many cellular processes but also for the adaptive immune system. This activation pathway mainly involves two key player proteins and is initiated by the depletion of internal calcium stores of the cell. The first is represented by STIM1, which serves as a calcium sensor and prolongs the signal of store depletion to its counterpart, the plasma membrane resident ion channel Orai1. The activation of STIM1 is controlled by conformational rearrangements of the protein that lead to the exposure of cytosolic domains which are buried and shielded in the resting state. These domains are now able to interact with Orai1in a direct way resulting in the gating of the ion channel. Despite intensive studies the underlying activation mechanism and their complex interplay are still far away from fully understood. In this project we take advantage of a novel approach termed FRET-derived Interaction in a Restricted Environment (FIRE) which will enable to dissect the interplay of domain interactions on a molecular level. We will focus on the characterization of homo- as well as heteromeric interactions of distinct alpha helical segments within STIM1 and Orai1 that constitute the STIM1-Orai1 activation- interface. Characterization of this STIM1-Orai1 activation-interface may lead to novel concepts of CRAC channel blockers essential in the therapy of immune diseases.

Store operated Ca2+ entry represents a fundamental way of calcium uptake and is essential for many cellular processes. This activation pathway is initiated by the depletion of internal endoplasmic reticulum (ER) Ca2+ stores and mainly involves two key proteins. STIM1 serves as an ER located Ca2+ sensor and is tightly connected to microtubules in resting cells. Store depletion results in the unleash from these cytoskeleton structures and triggers its redistribution into the cell periphery. There it contributes to the stable formation of ER-PM junctions and communicates the signal of store depletion to the plasma membrane resident Ca2+ channel Orai1. On a more detailed molecular level these activation process comprises a series of sequential steps that also involves conformational rearrangements of certain cytosolic regions within STIM1. This leads to the exposure of a crucial domain named CAD or SOAR, which is mainly comprised of 4 alpha helical domains (1, 2, 3, 4). The activation of Orai1 requires a series of sequential binding steps ranging from the initial primary binding to the final gating of the channel. The former is determined by the longer alpha helical structure 1 whereas the latter is accomplished by the two shorter segments 2 and 3. The most important results in this project on the STIM1-Orai1 interaction interface can be seen in the identification of the small 3 domain to represent a key player for Orai1 channel gating. A systematic screen enabled us to narrow down these gating site and highlight a single amin, which is indispensable for the transduction of the gating signal. Furthermore, a combined functional as well as biochemical approach was realized to identify its counterpart within Orai1. The so uncovered the interaction of the STIM1 3 domain to a region within Orai1 located at the cytosolic extension of transmembrane helix 3 (TM3) might represent a binding interface that is key for CRAC channel gating and therefore offers a new starting point for future CRAC channel studies.