Carbon monoxide dehydrogenase (CODH)-catalyzed oxidation of CO to CO 2 provides a promising means of removal of toxic and waste CO from industrial ue gas despite of the lack of active and stable enzymes in the atmosphere. Herein we present rationally and selectively redesigned ChCODH-II (Carboxydothermus hydrogenoformans) variants by engineering gas tunnels in order for O 2 -tolerant CODHs to catalyze e cient CO oxidation under oxygen (O 2 ). Using the redesigned ChCODH-II A559W and A559H variants showing 42-and 128-fold elevation of O 2 tolerance, respectively, complete CO removal was achieved under a near-atmospheric condition. Moreover, these variants e ciently removed CO from industrial ue gas (Linz-Donawiz converter Gas: LDG) discharged from a steel mill despite the high O 2 level (13.4%) during successful and repeated reuse after immobilized on Ni-NTA agarose beads. Our study will provide insights into redesigning the transformation of O 2 -sensitive CODHs into tolerant enzymes for use as workhorses for conversion of toxic or waste gases into safe or value-added chemicals. MainLarge amounts of CO pollutants are emitted from natural sources as well as man-made processes 1 (e.g. annually over 219 billion Nm 3 of CO-containing ue gases from POSCO) 2,3 . Carbon monoxide (CO), the most abundant air pollutant found in the atmosphere other than CO 2 according to the OECD database (https://stats.oecd.org, air emissions source in 2017), can provide su cient carbon and energy sources for converting waste gas to fuels and chemicals through a clean and sustainable method. To convert

Connexin 36 (Cx36) is responsible for signal transmission in electrical synapses by forming interneuronal gap junctions. Despite the critical role of Cx36 in normal brain function, the molecular architecture of the Cx36 gap junction channel (GJC) is unknown. Here, we determine cryo-electron microscopy structures of Cx36 GJC at 2.2–3.6 Å resolutions, revealing a dynamic equilibrium between its closed and open states. In the closed state, channel pores are obstructed by lipids, while N-terminal helices (NTHs) are excluded from the pore. In the open state with pore-lining NTHs, the pore is more acidic than those in Cx26 and Cx46/50 GJCs, explaining its strong cation selectivity. The conformational change during channel opening also includes the α-to-π-helix transition of the first transmembrane helix, which weakens the protomer-protomer interaction. Our structural analyses provide high resolution information on the conformational flexibility of Cx36 GJC and suggest a potential role of lipids in the channel gating.

G-protein coupled receptors (GPCRs) and ion channels serve as key molecular switches through which extracellular stimuli are transformed into intracellular effects, and it has long been postulated that ion channels are direct effector molecules of the alpha subunit of G-proteins (Gα). However, no complete structural evidence supporting the direct interaction between Gα and ion channels is available. Here, we present the cryo-electron microscopy structures of the human transient receptor potential canonical 5 (TRPC5)-Gαi3 complexes with a 4:4 stoichiometry in lipid nanodiscs. Remarkably, Gαi3 binds to the ankyrin repeat edge of TRPC5 ~ 50 Å away from the cell membrane. Electrophysiological analysis shows that Gαi3 increases the sensitivity of TRPC5 to phosphatidylinositol 4,5-bisphosphate (PIP2), thereby rendering TRPC5 more easily opened in the cell membrane, where the concentration of PIP2 is physiologically regulated. Our results demonstrate that ion channels are one of the direct effector molecules of Gα proteins triggered by GPCR activation–providing a structural framework for unraveling the crosstalk between two major classes of transmembrane proteins: GPCRs and ion channels.

Although both the p53 and forkhead box (FOX) family proteins are key transcription factors associated with cancer progression, their direct relationship is unknown. Here, we found that FOX family proteins bind to the non-canonical homotypic cluster of the p53 promoter region (TP53). Analysis of crystal structures of FOX proteins (FOXL2 and FOXA1) bound to the p53 homotypic cluster indicated that they interact with a 2:1 stoichiometry accommodated by FOX-induced DNA allostery. In particular, FOX proteins exhibited distinct dimerization patterns in recognition of the same p53-DNA; dimer formation of FOXA1 involved protein–protein interaction, but FOXL2 did not. Biochemical and biological functional analyses confirmed the cooperative binding of FOX proteins to the TP53 promoter for the transcriptional activation of TP53. In addition, up-regulation of TP53 was necessary for FOX proteins to exhibit anti-proliferative activity in cancer cells. These analyses reveal the presence of a discrete characteristic within FOX family proteins in which FOX proteins regulate the transcription activity of the p53 tumor suppressor via cooperative binding to the TP53 promoter in alternative dimer configurations.

E3 ubiquitin ligase Mdm2 facilitates β-arrestin ubiquitination, leading to the internalization of G protein–coupled receptors (GPCRs). In this process, β-arrestins bind to Mdm2 and recruit it to the receptor; however, the molecular architecture of the β-arrestin-Mdm2 complex has not been elucidated yet. Here, we identified the β-arrestin-binding region (ABR) on Mdm2 and solved the crystal structure of β-arrestin1 in complex with Mdm2 ABR peptide. The acidic residues of Mdm2 ABR bind to the positively charged concave side of the β-arrestin1 N-domain. The C-tail of β-arrestin1 is still bound to the N-domain, indicating that Mdm2 binds to the inactive state of β-arrestin1, whereas the phosphorylated C-terminal tail of GPCRs binds to activate β-arrestins. The overlapped binding site of Mdm2 and GPCR C-tails on β-arrestin1 suggests that the binding of GPCR C-tails might trigger the release of Mdm2. Moreover, hydrogen/deuterium exchange experiments further show that Mdm2 ABR binding to β-arrestin1 induces the interdomain interface to be more dynamic and uncouples the IP 6 -induced oligomer of β-arrestin1. These results show how the E3 ligase, Mdm2, interacts with β-arrestins to promote the internalization of GPCRs.