We determined structures of the wild-type human PANX1 (PANX1(WT)) in the presence of EDTA, Ca 2+ or K + . These structures are indistinguishable, suggesting that Ca 2+ or K + is unlikely to directly activate PANX1. To study the location and role of the CTT and the N-terminal helix (NTH), we cleaved the CTT from the PANX1(WT) and from an NTH-truncation mutant using caspase 7, and determined their structures (PANX1(ΔCTT) and PANX1(ΔNTH/ΔCTT), respectively). We also determined structures of PANX1 bound to CBX (CBX-PANX1(ΔCTT) and CBX-PANX1(ΔNTH/ ΔCTT)). To study the role of N-glycosylation of PANX1, we investigated the structure of a glycosylation-deficient mutant (N255A), which yielded both gap junctions (PANX1(N255A) Gap ) and hemichannels (PANX1(N255A) Hemi ). The structural determination is detailed in the Methods, and the results are summarized in Extended Data Tables 1-3. The structure of PANX1(ΔCTT) has the highest overall quality (map available at the Electron Microscopy Data Bank (EMD-21589) and model at the Protein Data Bank (6WBG)), and is therefore the reference structure for the discussion throughout the text unless otherwise noted.The PANX1 channel forms a heptamer that contains (from top to bottom) an extracellular domain (ECD), a transmembrane domain (TMD) and an intracellular domain (ICD), with the unstructured CTT

SUMMARY The UbiB protein kinase-like (PKL) family is widespread—comprising one-quarter of microbial PKLs and five human homologs—yet its biochemical activities remain obscure. COQ8A (ADCK3) is a mammalian UbiB protein associated with ubiquinone (CoQ) biosynthesis and an ataxia (ARCA2) through unclear means. We show that mice lacking COQ8A develop a slowly progressive cerebellar ataxia linked to Purkinje cell dysfunction and mild exercise intolerance, recapitulating ARCA2. Interspecies biochemical analyses show that COQ8A and yeast Coq8p specifically stabilize a CoQ biosynthesis complex through unorthodox PKL functions. While COQ8 was predicted to be a protein kinase, we demonstrate that it lacks canonical protein kinase activity in trans. Instead, COQ8 has ATPase activity and interacts with lipid CoQ intermediates—functions that are likely conserved across all domains of life. Collectively, our results lend insight into the molecular activities of the ancient UbiB family and elucidate the biochemical underpinnings of a human disease.

The activity of the proton-activated chloride channel (PAC) is widespread and is involved in acid-induced cell death and tissue injury 1 2 , 3 . Its molecular identity has recently been identified as a novel and evolutionarily conserved protein family 4 , 5 . We present two cryo-EM structures of human PAC in a high-pH resting closed state and a low-pH proton-bound non-conducting state. PAC is a trimer; each subunit consists of a transmembrane domain (TMD) formed by two helices, TM1–2, and an extracellular domain (ECD). We observed striking conformational changes in the ECD–TMD interface and the TMD when the pH drops from 8 to 4. The rearrangement of the ECD–TMD interface is characterized by the movement of histidine-98, which is, upon acidification, decoupled from the resting position and inserted into an acidic pocket that is about 5-Å away. Within the TMD, TM1 undergoes a rotational movement, switching its interaction partner from the cognate to the adjacent TM2. The anion selectivity of PAC is determined by the positively charged lysine-319 on TM2. Replacement of lysine-319 by a glutamate converts PAC to a cation-selective channel. Our data provide the first glimpse of the molecular assembly of PAC, and a basis for understanding the mechanism of proton-dependent activation.

The zebrafish kidney marrow is considered to be the organ of definitive hematopoiesis, analogous to the mammalian bone marrow. We have sequenced 26,143 ESTs and isolated 304 cDNAs with putative full-length ORF from a zebrafish kidney marrow cDNA library. The ESTs formed 7,742 assemblies, representing both previously identified zebrafish ESTs (56%) and recently discovered zebrafish ESTs (44%). About 30% of these EST assemblies have orthologues in humans, including 1,282 disease-associated genes in the Online Mendelian Inheritance in Man (OMIM) database. Comparison of the effective and regulatory molecules related to erythroid functions across species suggests a good conservation from zebrafish to human. Interestingly, both embryonic and adult zebrafish globin genes showed higher homology to the human embryonic globin genes than to the human fetal͞adult ones, consistent with evo-devo correlation hypothesis. In addition, conservation of a whole set of transcription factors involved in globin gene switch suggests the regulatory network for such remodeling mechanism existed before the divergence of the teleost and the ancestor of mammals. We also carried out whole-mount mRNA in situ hybridization assays for 493 cDNAs and identified 80 genes (16%) with tissue-specific expression during the first five days of zebrafish development. Twenty-six of these genes were specifically expressed in hematopoietic or vascular tissues, including three previously unidentified zebrafish genes: coro1a, nephrosin, and dab2. Our results indicate that conserved genetic programs regulate vertebrate hematopoiesis and vasculogenesis, and support the role of the zebrafish as an important animal model for studying both normal development and the molecular pathogenesis of human blood diseases.