The calcium release-activated calcium channel Orai regulates Ca2+ entry into non-excitable cells and is required for proper immune function. While the channel typically opens following Ca2+ release from the endoplasmic reticulum, certain pathologic mutations render the channel constitutively open. Previously, using one such mutation (H206A), we obtained low (6.7 Å) resolution X-ray structural information on Drosophila melanogaster Orai in an open conformation (Hou, Burstein, & Long, 2018). Here, we present a structure of this open conformation at 3.3 Å resolution using fiducial-assisted cryo-EM. The improved structure reveals the conformations of amino acids in the open pore, which dilates by outward movements of subunits. A ring of phenylalanine residues repositions to expose previously shielded glycine residues to the pore without significant rotational movement of the associated helices. Together with other hydrophobic amino acids, the phenylalanines act as the channel's gate. Structured M1-M2 turrets, not evident previously, form the channel's extracellular entrance.

Although cyclophilins are attractive targets for probing biology and therapeutic intervention, no subtype-selective cyclophilin inhibitors have been described. We discovered novel cyclophilin inhibitors from the in vitro selection of a DNA-templated library of 256,000 drug-like macrocycles for cyclophilin D (CypD) affinity. Iterated macrocycle engineering guided by ten X-ray co-crystal structures yielded potent and selective inhibitors (half maximal inhibitory concentration (IC50) = 10 nM) that bind the active site of CypD and also make novel interactions with non-conserved residues in the S2 pocket, an adjacent exo-site. The resulting macrocycles inhibit CypD activity with 21- to >10,000-fold selectivity over other cyclophilins and inhibit mitochondrial permeability transition pore opening in isolated mitochondria. We further exploited S2 pocket interactions to develop the first cyclophilin E (CypE)-selective inhibitor, which forms a reversible covalent bond with a CypE S2 pocket lysine, and exhibits 30- to >4,000-fold selectivity over other cyclophilins. These findings reveal a strategy to generate isoform-selective small-molecule cyclophilin modulators, advancing their suitability as targets for biological investigation and therapeutic development.

The human genome encodes more than 500 different protein kinases: signaling enzymes with tightly regulated activity. Enzymatic activity within the conserved kinase domain is influenced by numerous regulatory inputs including the binding of regulatory domains, substrates, and the effect of post-translational modifications such as autophosphorylation. Integration of these diverse inputs occurs via allosteric sites that relate signals via networks of amino acid residues to the active site and ensures controlled phosphorylation of kinase substrates. Here, we review mechanisms of allosteric regulation of protein kinases and recent advances in the field.

Objective Volunteerism represents an important mechanism to promote resilience, empathy, and general well-being in medical students, a group that stands to benefit. Medical students report feelings of fatigue, burnout, exhaustion, and stress that correlates with poor academic performance, and significant decline in empathy over the 3 rd year of both MD and DO programs. Volunteer motivations have been shown to mediate participant well-being. The relationship between medical student volunteer motivations and specific outcomes during the COVID-19 pandemic has not been addressed. Methods We characterized features of medical student volunteers during the COVID-19 pandemic in 2020, including volunteering motivation using the Volunteer Functions Inventory, the types of activities in which they participated, and the physical, psychosocial, and emotional outcomes they experienced following volunteering. Results Altruistic and humanitarian values–centric motivation predicts positive volunteering outcomes including increased resilience, ability to deal with disappointment and loss, and ability to cope with the COVID-19 pandemic. Values-centric motivation also increases volunteer empathy independent of educational stage. Values-centric participants were more likely to select volunteering activities with patient contact, which promotes student empathy and resilience. Conversely, career-centric motivation does not predict positive outcomes. These students are more likely to engage in research-oriented activities. Conclusions The efficacy of integrating volunteerism into medical school curricula may be limited by professional pressure that manifests as career-oriented motivation. We propose that practical integration should promote altruistic and humanitarian values–centric participant orientation to the volunteering process, which is associated with enhanced recruitment, preservation of empathy, and additional positive volunteering outcomes of interest.

The calcium release-activated calcium channel Orai regulates Ca2+ entry into non-excitable cells and is required for proper immune function. The channel typically opens following the release of Ca2+ from the endoplasmic reticulum. Certain pathologic mutations render the channel constitutively open. Here, using one such mutation (H206A), we present a cryo-EM structure of Orai from Drosophila melanogaster in an open conformation at 3.3 Å resolution. Comparison with previous closed structures reveals that opening occurs through the outward movements of M1 helices that dilate the central pore. Repositionings of a ring of phenylalanine residues (F171) expose previously shielded glycine residues (G170) to the channel pore, despite the absence of significant rotational movement of the associated pore-lining helices. This phenylalanine ring and two rings of flanking hydrophobic amino acids act as a hydrophobic gate to control ion permeation. Extracellular M1-M2 turrets, not evident from previous Orai structures, form an electronegative pore entrance.Single sentence summaryA structure of the Ca2+ channel Orai in an open conformation provides insight into the opening mechanism of the channel and its role in regulating selective Ca2+ entry into immune and other non-excitable cells.