UC Berkeley

Leukocyte-endothelial adhesion and firm adhesion are chief steps in leukocyte recruitment cascade and immune system response to inflammation. Irregularities in these processes have been related to several human diseases including arthritis, cancer, and atherosclerosis. Given the fundamental importance of integrin activation and Ca2+ signaling in adhesion assembly and strengthening, understanding the molecular mechanisms of kindlin-mediated integrin activation and STIM-mediated Orai gating have potentially important implications in designing new therapeutic interventions. This study employs molecular dynamics and bioinformatic techniques to unravel detailed molecular interactions in these two activation processes.

First, we analyzed the effect of kindlin cooperation with talin for integrin activation. Integrins are transmembrane proteins that mediate the signaling between the cytoplasm and extracellular matrix to promote cell adhesion. In order to initiate signaling, integrins must be activated from a low-affinity state to a high-affinity conformation for binding to extracellular molecules. Talin and kindlin are two cytoplasmic proteins that bind to integrin and modulate its affinity for extracellular ligands. Although the molecular details of talin-mediated integrin activation are known, the mechanism of kindlin involvement in this process remains elusive. Here, we carried out, for the first time, a comprehensive molecular dynamics study of integrin activation mediated through simultaneous interaction of talin and kindlin with its b subdomain. We showed that kindlin modifies the molecular mechanisms of inside-out activation by enhancing talin interaction with the membrane proximal region of integrin and decreasing the crossing angle between transmembrane helices of integrin, which eventually results in parallelization of integrin dimer.

Second, we investigated the molecular details of STIM-Orai interaction and its downstream consequences on the Orai channel gating. The immune response is triggered by a decline in Ca2+ concentration within the endoplasmic reticulum (ER) that is followed by the opening of the calcium release-activated channels (CRAC), thus increasing the intracellular Ca2+ concentration. Stromal interaction molecule (STIM) has been identified as a Ca+2 sensor in the ER activating the CRAC channel via physical interaction with Orai, the pore unit of the CRAC channel. In this study, we showed that the forces from STIM binding allosterically propagate through Orai helices, which lead to opening of the basic region of the pore. Moreover, we demonstrated important interactions that maintain the open structure of the Orai channel.

Overall, this study provides valuable insights into the molecular mechanisms that contribute to the activation of mechanosensitive proteins involved in leukocyte adhesion and firm adhesion.