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Calcium homeostasis modulator 1 (CALHM1) is a voltage-dependent channel involved in neuromodulation and gustatory signaling. Despite recent progress in the structural biology of CALHM1, insights into functional regulation, pore architecture, and channel blockade remain limited. Here we present the cryo-EM structure of human CALHM1, revealing an octameric assembly pattern similar to the non-mammalian CALHM1s and the lipid-binding pocket conserved across species. We demonstrate by MD simulations that this pocket preferentially binds a phospholipid over cholesterol to stabilize its structure and regulate the channel activities. Finally, we show that residues in the amino-terminal helix form the channel pore that ruthenium red binds and blocks.

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Membrane transport is a fundamental means to control basic cellular processes such as apoptosis, inflammation, and neurodegeneration and is mediated by a number of transporters, pumps, and channels. Accumulating evidence over the last half century has shown that a type of so-called "large-pore channel" exists in various tissues and organs in gap-junctional and non-gap-junctional forms in order to flow not only ions but also metabolites such as ATP. They are formed by a number of protein families with little or no evolutionary linkages including connexin, innexin, pannexin, leucine-rich repeat-containing 8 (LRRC8), and calcium homeostasis modulator (CALHM). This review summarizes the history and concept of large-pore channels starting from connexin gap junction channels to the more recent developments in innexin, pannexin, LRRC8, and CALHM. We describe structural and functional features of large-pore channels that are crucial for their diverse functions on the basis of available structures.

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The early phase of protein drug development has traditionally focused on target binding properties leading to a desired mode of therapeutic action. As more protein therapeutics pass through the development pipeline; however, it is clear that non-optimal biophysical properties can emerge, particularly as proteins are formulated at high concentrations, causing aggregation or polyreactivity. Such late-stage "developability" problems can lead to delay or failure in traversing the development process. Aggregation propensity is also correlated with increased immunogenicity, resulting in expensive, late-stage clinical failures. Using nucleases-directed integration, we have constructed large mammalian display libraries where each cell contains a single antibody gene/cell inserted at a single locus, thereby achieving transcriptional normalization. We show a strong correlation between poor biophysical properties and display level achieved in mammalian cells, which is not replicated by yeast display. Using two well-documented examples of antibodies with poor biophysical characteristics (MEDI-1912 and bococizumab), a library of variants was created based on surface hydrophobic and positive charge patches. Mammalian display was used to select for antibodies that retained target binding and permitted increased display level. The resultant variants exhibited reduced polyreactivity and reduced aggregation propensity. Furthermore, we show in the case of bococizumab that biophysically improved variants are less immunogenic than the parental molecule. Thus, mammalian display helps to address multiple developability issues during the earliest stages of lead discovery, thereby significantly de-risking the future development of protein drugs.

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Pannexins are large-pore forming channels responsible for ATP release under a variety of physiological and pathological conditions. Although predicted to share similar membrane topology with other large-pore forming proteins such as connexins, innexins, and LRRC8, pannexins have minimal sequence similarity to these protein families. Here, we present the cryo-EM structure of a frog pannexin 1 (Panx1) channel at 3.0 Å. We find that Panx1 protomers harbor four transmembrane helices similar in arrangement to other large-pore forming proteins but assemble as a heptameric channel with a unique constriction formed by Trp74 in the first extracellular loop. Mutating Trp74 or the nearby Arg75 disrupt ion selectivity, whereas altering residues in the hydrophobic groove formed by the two extracellular loops abrogates channel inhibition by carbenoxolone. Our structural and functional study establishes the extracellular loops as important structural motifs for ion selectivity and channel inhibition in Panx1.

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An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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The biological membranes of many cell types contain large-pore channels through which a wide variety of ions and metabolites permeate. Examples include connexin, innexin and pannexin, which form gap junctions and/or bona fide cell surface channels. The most recently identified large-pore channels are the calcium homeostasis modulators (CALHMs), through which ions and ATP permeate in a voltage-dependent manner to control neuronal excitability, taste signaling and pathologies of depression and Alzheimer's disease. Despite such critical biological roles, the structures and patterns of their oligomeric assembly remain unclear. Here, we reveal the structures of two CALHMs, chicken CALHM1 and human CALHM2, by single-particle cryo-electron microscopy (cryo-EM), which show novel assembly of the four transmembrane helices into channels of octamers and undecamers, respectively. Furthermore, molecular dynamics simulations suggest that lipids can favorably assemble into a bilayer within the larger CALHM2 pore, but not within CALHM1, demonstrating the potential correlation between pore size, lipid accommodation and channel activity.

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In a previous paper (Syrjänen et al., 2014), we reported the first structural characterisation of a synaptonemal complex (SC) protein, SYCP3, which led us to propose a model for its role in chromosome compaction during meiosis. As a component of the SC lateral element, SYCP3 has a critical role in defining the specific chromosome architecture required for correct meiotic progression. In the model, the reported compaction of chromosomal DNA caused by SYCP3 would result from its ability to bridge distant sites on a DNA molecule with the DNA-binding domains located at each end of its strut-like structure. Here, we describe a single-molecule assay based on optical tweezers, fluorescence microscopy and microfluidics that, in combination with bulk biochemical data, provides direct visual evidence for our proposed mechanism of SYCP3-mediated chromosome organisation.

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In all bacterial species examined thus far, small regulatory RNAs (sRNAs) contribute to intricate patterns of dynamic genetic regulation. Many of the actions of these nucleic acids are mediated by well-characterized chaperones such as the Hfq protein, but genetic screens have also recently identified the 3'-to-5' exoribonuclease polynucleotide phosphorylase (PNPase) as an unexpected stabilizer and facilitator of sRNAs in vivo. To understand how a ribonuclease might mediate these effects, we tested the interactions of PNPase with sRNAs and found that the enzyme can readily degrade these nucleic acids in vitro but, nonetheless, copurifies from cell extracts with the same sRNAs without discernible degradation or modification to their 3' ends, suggesting that the associated RNA is protected against the destructive activity of the ribonuclease. In vitro, PNPase, Hfq, and sRNA can form a ternary complex in which the ribonuclease plays a nondestructive, structural role. Such ternary complexes might be formed transiently in vivo, but could help to stabilize particular sRNAs and remodel their population on Hfq. Taken together, our results indicate that PNPase can be programmed to act on RNA in either destructive or stabilizing modes in vivo and may form complex, protective ribonucleoprotein assemblies that shape the landscape of sRNAs available for action.

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The synaptonemal complex (SC) is an evolutionarily-conserved protein assembly that holds together homologous chromosomes during prophase of the first meiotic division. Whilst essential for meiosis and fertility, the molecular structure of the SC has proved resistant to elucidation. The SC protein SYCP3 has a crucial but poorly understood role in establishing the architecture of the meiotic chromosome. Here we show that human SYCP3 forms a highly-elongated helical tetramer of 20 nm length. N-terminal sequences extending from each end of the rod-like structure bind double-stranded DNA, enabling SYCP3 to link distant sites along the sister chromatid. We further find that SYCP3 self-assembles into regular filamentous structures that resemble the known morphology of the SC lateral element. Together, our data form the basis for a model in which SYCP3 binding and assembly on meiotic chromosomes leads to their organisation into compact structures compatible with recombination and crossover formation.

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