DNA charge transport chemistry offers a means of long range, rapid redox signaling. Here we demonstrate that the [4Fe4S] cluster in human DNA primase can utilize this chemistry to coordinate the first steps of replication. Through DNA electrochemistry, we find that a change in oxidation state of the [4Fe4S] cluster acts as a switch for DNA binding. Single-atom mutations that inhibit this charge transfer, moreover, hinder primase initiation without affecting primase structure or polymerization. Generating a single base mismatch in the growing primer duplex, which attenuates DNA charge transport, inhibits primer truncation. Thus redox signaling by [4Fe4S] clusters using DNA charge transport regulates primase binding to DNA and illustrates chemistry that may efficiently drive substrate handoff between polymerases during DNA replication.

SignificanceRedox switching driven by [4Fe4S] cluster cofactors modulates DNA binding affinity in proteins, providing a rapid, efficient method of substrate binding and dissociation. Our study establishes an essential redox switch with an aromatic pathway through the yeast DNA primase; a single-residue mutation at position 397 along this redox pathway causes [4Fe4S] cluster degradation and is lethal in yeast.

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Helicobacter pylori colonizes the human stomach and contributes to the development of gastric cancer and peptic ulcer disease. H. pylori secretes a pore-forming toxin called vacuolating cytotoxin A (VacA), which contains two domains (p33 and p55) and assembles into oligomeric structures. Using single particle cryo-electron microscopy, we have determined low-resolution structures of a VacA dodecamer and heptamer, as well as a 3.8 Å structure of the VacA hexamer. These analyses show that VacA p88 consists predominantly of a right-handed beta-helix that extends from the p55 domain into the p33 domain. We map the regions of p33 and p55 involved in hexamer assembly, model how interactions between protomers support heptamer formation, and identify surfaces of VacA that likely contact membrane. This work provides structural insights into the process of VacA oligomerization and identifies regions of VacA protomers that are predicted to contact the host cell surface during channel formation.

The Receptor for Advanced Glycation End products (RAGE) is a pattern recognition receptor that signals for inflammation via the NF‐κB pathway. RAGE has been pursued as a potential target to suppress symptoms of diabetes and is of interest in a number of other diseases associated with chronic inflammation, such as inflammatory bowel disease and bronchopulmonary dysplasia. Screening and optimization have previously produced small molecules that inhibit the activity of RAGE in cell‐based assays, but efforts to develop a therapeutically viable direct‐binding RAGE inhibitor have yet to be successful. Here, we show that a fragment‐based approach can be applied to discover fundamentally new types of RAGE inhibitors that specifically target the ligand‐binding surface. A series of systematic assays of structural stability, solubility, and crystallization were performed to select constructs of the RAGE ligand‐binding domain and optimize conditions for NMR‐based screening and co‐crystallization of RAGE with hit fragments. An NMR‐based screen of a highly curated ~14 000‐member fragment library produced 21 fragment leads. Of these, three were selected for elaboration based on structure‐activity relationships generated through cycles of structural analysis by X‐ray crystallography, structure‐guided design principles, and synthetic chemistry. These results, combined with crystal structures of the first linked fragment compounds, demonstrate the applicability of the fragment‐based approach to the discovery of RAGE inhibitors.