Stewart, M. J. | bioRxiv Science

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↗

bioRxiv2020

Substrate-dependent accessibility changes in the Na+-dependent C4-dicarboxylate transporter, VcINDY, suggest differential substrate effects in a multistep mechanism.

Members of the divalent anion sodium symporter (DASS) family (SLC13 in humans) play critical roles in metabolic homeostasis, influencing many processes including fatty acid synthesis, insulin resistance, adiposity, and lifespan determination. DASS transporters catalyse the Na+-driven concentrative uptake of Krebs cycle intermediates and sulfate into cells; disrupting their function can protect against age-related metabolic diseases and can extend lifespan. An inward-facing crystal structure and an outward-facing model of a bacterial DASS family member, VcINDY from Vibrio cholerae, predict an elevator-like transport mechanism involving a large rigid body movement of the substrate binding site. How substrate binding influences the conformational state of VcINDY is currently unknown. Here, we probe the interaction between substrate binding and VcINDY conformation using a site-specific alkylation strategy to probe the solvent accessibility of several broadly distributed positions in VcINDY in the presence and absence of substrates (Na+ and succinate). Our findings reveal that accessibility to all positions tested can be modulated by the presence of substrates, with the majority becoming less accessible in the presence of Na+. Mapping these solvent accessibility changes onto the known structures of VcINDY suggests that Na+ binding drives the transporter into an as-yet-unidentified intermediate state. We also observe substantial, separable effects of Na+ and succinate binding at several amino acid positions suggesting distinct effects of the two substrates. Furthermore, analysis of a solely succinate-sensitive residue indicates that VcINDY binds its substrate with a low affinity and proceeds via an ordered process in which one or more Na+ ions must bind prior to succinate. These findings provide insight into the mechanism of VcINDY, which is currently the only structural-characterised representative of the entire DASS family.

biochemistry↗