Organellar ion channels are essential for cell physiology. Their activities are often regulated by Ca2+ and H+, which are concentrated in many organelles. Here we report a novel structural element critical for Ca2+/pH dual regulation of TRPML1, a Ca2+ release channel crucial for endolysosomal functions. TRPML1 mutations cause mucolipidosis type IV (MLIV), a severe lysosomal storage disorder characterized by neurodegeneration, mental retardation and blindness. We obtained high-resolution crystal structures of a 213-amino acid luminal domain of human TRPML1 that harbors three missense MLIV-causing mutations. This domain forms a tetramer with a highly electronegative central pore formed by a novel luminal pore-loop. Cysteine crosslinking and cryo-EM confirm this structure in the full-length channel. Structure-function studies demonstrate that Ca2+ and H+ interact with the luminal pore to exert physiologically important regulation. The MLIV-causing mutations disrupt the luminal domain structure and cause TRPML1 mislocalization. Our study provides a structural underpinning for TRPML1's regulation, assembly and pathogenesis.

Cyclic nucleotide-gated (CNG) channels convert cyclic nucleotide binding and unbinding into electrical signals in sensory receptors and neurons. The molecular conformational changes underpinning ligand activation are largely undefined. We report both closed- and open-state atomic cryo-EM structures of a full-length C. elegans cGMP-activated channel TAX-4 reconstituted in lipid nanodiscs. These structures, together with computational and functional analyses and a mutant channel structure, reveal a double-barrier hydrophobic gate formed by two S6 amino acids in the central cavity. cGMP binding produces global conformational changes that open the cavity gate located ~52 Å away but do not alter the structure of the selectivity filter – the commonly presumed activation gate. Our work provides mechanistic insights into the allosteric gating and regulation of cyclic nucleotide-gated and -modulated channels and CNG channel-related channelopathies.

Holliday junction (HJ) is a hallmark intermediate in DNA recombination and must be processed by dissolution (for double HJ) or resolution to ensure genome stability. Although HJ resolvases have been identified in all domains of life, there is a long-standing effort to search in prokaryotes and eukarya for proteins promoting HJ migration. Here, we report the structural and functional characterization of a novel ATPase, Sulfolobus islandicus PilT N-terminal-domain-containing ATPase (SisPINA), encoded by the gene adjacent to the resolvase Hjc coding gene. PINA is conserved in archaea and vital for S. islandicus viability. Purified SisPINA forms hexameric rings in the crystalline state and in solution, similar to the HJ migration helicase RuvB in Gram-negative bacteria. Structural analysis suggests that ATP binding and hydrolysis cause conformational changes in SisPINA to drive branch migration. Further studies reveal that SisPINA interacts with SisHjc and coordinates HJ migration and cleavage.

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