Dual oxidases (DUOXs) produce hydrogen peroxide by transferring electrons from intracellular NADPH to extracellular oxygen. They are involved in many crucial biological processes and human diseases, especially in thyroid diseases. DUOXs are protein complexes co-assembled from the catalytic DUOX subunits and the auxiliary DUOXA subunits and their activities are regulated by intracellular calcium concentrations. Here, we report the cryo-EM structures of human DUOX1-DUOXA1 complex in both high-calcium and low-calcium states. These structures reveal the DUOX1 complex is a symmetric 2:2 hetero-tetramer stabilized by extensive inter-subunit interactions. Substrate NADPH and cofactor FAD are sandwiched between transmembrane domain and the cytosolic dehydrogenase domain of DUOX. In the presence of calcium ions, intracellular EF-hand modules might enhance the catalytic activity of DUOX by stabilizing the dehydrogenase domain in a conformation that allows electron transfer.

TRPC5 channel is a non-selective cation channel that participates diverse physiological processes. TRPC5 inhibitors show promise in the treatment of anxiety disorder, depression and kidney disease. However, the binding sites and inhibitory mechanism of TRPC5 inhibitors remain elusive. Here we present the cryo-EM structures of human TRPC5 in complex with two distinct inhibitors, namely clemizole and HC-070, to the resolution of 2.7 Å. The structures reveal that clemizole binds inside the voltage sensor-like domain of each subunit. In contrast, HC-070 is wedged between adjacent subunits and replaces the glycerol group of a putative DAG molecule near the extracellular side. Moreover, we found mutations in the inhibitor binding pockets altered the potency of inhibitors. These structures suggest that both clemizole and HC-070 exert the inhibitory functions by stabilizing the ion channel in a non-conductive closed state. These results pave the way for further design and optimization of inhibitors targeting human TRPC5.

Phagocyte oxidase plays an essential role in the first line of host defense against pathogens. It oxidizes intracellular NADPH to reduce extracellular oxygen to produce superoxide anions that participate in pathogen killing. The resting phagocyte oxidase is a heterodimeric complex formed by two transmembrane proteins NOX2 and p22. Despite the physiological importance of this complex, its structure remains elusive. Here we reported the cryo-EM structure of the functional human NOX2-p22 complex in nanodisc in the resting state. NOX2 shows a canonical 6-TM architecture of NOX and p22 has four transmembrane helices. M3, M4, and M5 of NOX2 and M1 and M4 helices of p22 are involved in the hetero-dimer formation. DH domain of NOX2 in the resting state is not optimally docked onto the transmembrane domain (TMD), leading to inefficient electron transfer and NADPH binding. Structural analysis suggests that the cytosolic factors might activate the NOX2-p22 complex by stabilizing the dehydrogenase domain (DH) in a productive docked conformation.