infects half of the world's population, and strains that encode the type IV secretion system for injection of the oncoprotein CagA into host gastric epithelial cells are associated with elevated levels of cancer. CagA translocation into host cells is dependent on interactions between the adhesin protein HopQ and human CEACAMs. Here, we present high-resolution structures of several HopQ-CEACAM complexes and CEACAMs in their monomeric and dimeric forms establishing that HopQ uses a coupled folding and binding mechanism to engage the canonical CEACAM dimerization interface for CEACAM recognition. By combining mutagenesis with biophysical and functional analyses, we show that the modes of CEACAM recognition by HopQ and CEACAMs themselves are starkly different. Our data describe precise molecular mechanisms by which microbes exploit host CEACAMs for infection and enable future development of novel oncoprotein translocation inhibitors and -specific antimicrobial agents.

A broad range of proteins employ nucleotide flipping to recognize specific sites in nucleic acids, including DNA glycosylases, which remove modified nucleobases to initiate base excision repair. Deamination, a pervasive mode of damage, typically generates lesions that are recognized by glycosylases as being foreign to DNA. However, deamination of 5-methylcytosine (mC) generates thymine, a canonical DNA base, presenting a challenge for damage recognition. Nevertheless, repair of mC deamination is important because the resulting G•T mispairs cause C→T transition mutations, and mC is abundant in all three domains of life. Countering this threat are three types of glycosylases that excise thymine from G•T mispairs, including thymine DNA glycosylase (TDG). These enzymes must minimize excision of thymine that is not generated by mC deamination, in A•T pairs and in polymerase-generated G•T mispairs. TDG preferentially removes thymine from DNA contexts in which cytosine methylation is prevalent, including CG and one non-CG site. This remarkable context specificity could be attained through modulation of nucleotide flipping, a reversible step that precedes base excision. We tested this idea using fluorine NMR and DNA containing 2′-fluoro-substituted nucleotides. We find that dT nucleotide flipping depends on DNA context and is efficient only in contexts known to feature cytosine methylation. We also show that a conserved Ala residue limits thymine excision by hindering nucleotide flipping. A linear free energy correlation reveals that TDG attains context specificity for thymine excision through modulation of nucleotide flipping. Our results provide a framework for characterizing nucleotide flipping in nucleic acids using 19 F NMR.

Human cells express the UDG superfamily of glycosylases, which excise uracil (U) from the genome. The three members of this structural superfamily are uracil DNA glycosylase (UNG/UDG), single-strand selective monofunctional uracil DNA glycosylase (SMUG1), and thymine DNA glycosylase (TDG). We previously reported that UDG is efficient at removing U from DNA packaged into nucleosome core particles (NCP) and is minimally affected by the histone proteins when acting on an outward-facing U in the dyad region. In an effort to determine whether this high activity is a general property of the UDG superfamily of glycosylases, we compare the activity of UDG, SMUG1, and TDG on a U:G wobble base pair using NCP assembled from Xenopus laevis histones and the Widom 601 positioning sequence. We found that while UDG is highly active, SMUG1 is severely inhibited on NCP and this inhibition is independent of sequence context. Here we also provide the first report of TDG activity on an NCP, and found that TDG has an intermediate level of activity in excision of U and is severely inhibited in its excision of T. These results are discussed in the context of cellular roles for each of these enzymes.

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Helicobacter pylori infects half of the world's population and strains that encode the cag type IV secretion system for injection of the oncoprotein CagA into host gastric epithelial cells are associated with elevated levels of cancer. CagA translocation into host cells is dependent on interactions between the H. pylori adhesin protein HopQ and human CEACAMs. Here, we present high-resolution structures of numerous HopQ-CEACAM complexes and CEACAMs in their monomeric and dimeric forms establishing that HopQ uses a coupled folding and binding mechanism to engage the canonical CEACAM dimerization interface for CEACAM recognition. By combining mutagenesis with biophysical and functional analyses, we show that the modes of CEACAM recognition by HopQ and CEACAMs themselves are starkly different. Our data describe precise molecular mechanisms by which microbes exploit host CEACAMs for infection and enable future development of novel oncoprotein translocation inhibitors and H. pylori-specific antimicrobial agents.