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Bragginton, E.

Publications and source records attributed to Bragginton, E..

2 recordsLinked to original sources
bioRxiv2024

Structure and dynamics of Burkholderia pseudomallei OXA-57, a distinctive low-efficiency class D β-lactamase with carbapenem-hydrolyzing activity

The Gram-negative bacterium Burkholderia pseudomallei causes the severe disease melioidosis. {beta}-Lactams, including carbapenems, are the primary treatment, but are susceptible to chromosomal {beta}-lactamases, including the class D enzyme OXA-57. Here we show recombinant OXA-57 is active towards penicillins and first-generation cephalosporins, slowly hydrolyzes carbapenems including imipenem and meropenem, but is inactive towards oxyimino-cephalosporins (e.g., ceftazidime). Unlike many OXA enzymes, OXA-57 is sensitive to the mechanism-based inhibitor clavulanic acid, but less so to the diazabicyclooctane avibactam and not to the cyclic boronate vaborbactam. Crystal structures of covalent OXA-57:avibactam and OXA-57:meropenem complexes reveal limited hydrogen- bonding interactions with bound ligands. In molecular dynamics simulations, bound meropenem is mobile, while the water necessary for deacylation has only limited active site access. These observations are consistent with the low level of meropenem turnover, supporting proposals that OXA {beta}-lactamases generally possess limited carbapenemase activity, and highlight the potential importance of OXA-57 in B. pseudomallei {beta}-lactam resistance. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/630153v1_ufig1.gif" ALT="Figure 1000"> View larger version (24K): org.highwire.dtl.DTLVardef@d635d7org.highwire.dtl.DTLVardef@168d67borg.highwire.dtl.DTLVardef@1d95733org.highwire.dtl.DTLVardef@1ebeabb_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗
bioRxiv2024

MDC1 mediates Pellino recruitment to sites of DNA double-strand breaks

Ubiquitylation is critically implicated in the recognition and repair of DNA double-strand breaks. The adaptor protein MDC1 mediates the recruitment of the key DNA damage responsive E3 ubiquitin ligase RNF8 to the break sites. It does so by directly interacting with RNF8 in a phosphorylation-dependent manner that involves the RNF8 FHA domain, thus initiating targeted chromatin ubiquitylation at the break sites. Here, we report that MDC1 also directly binds to two additional E3 ubiquitin ligases, Pellino1 and 2, which were recently implicated in the DNA damage response. Through a combination of biochemical, biophysical and X-ray crystallographic approaches, we reveal the molecular details of the MDC1-Pellino complexes. Furthermore, we show that in mammalian cells, MDC1 mediates Pellino recruitment to sites of DNA double-strand breaks by a direct phosphorylation-dependent interaction between the two proteins. Taken together, our findings provide new molecular insights into the ubiquitylation pathways that govern genome stability maintenance.

cell biology↗