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Nuclear magnetic resonance (NMR) is an effective, commonly used experimental approach to screen small organic molecules against a protein target. A very popular method consists of monitoring the changes of the NMR chemical shifts of the protein nuclei upon addition of the small molecule to the free protein. Multidimensional NMR experiments allow the interacting residues to be mapped along the protein sequence. A significant amount of human effort goes into manually tracking the chemical shift variations, especially when many signals exhibit chemical shift changes and when many ligands are tested. Some computational approaches to automate the procedure are available, but none of them as a web server. Furthermore, some methods require the adoption of a fairly specific experimental setup, such as recording a series of spectra at increasing small molecule:protein ratios. In this work, we developed a tool requesting a minimal amount of experimental data from the user, implemented it as an open-source program, and made it available as a web application. Our tool compares two spectra, one of the free protein and one of the small molecule:protein mixture, based on the corresponding peak lists. The performance of the tool in terms of correct identification of the protein-binding regions has been evaluated on different protein targets, using experimental data from interaction studies already available in the literature. For a total of 16 systems, our tool achieved between 79% and 100% correct assignments, properly identifying the protein regions involved in the interaction.

The Src homology 2 (SH2) domain is present in many proteins that bind to phosphotyrosine (pY) post translational modifications in partner proteins to trigger downstream signalling. Since pY-driven protein-protein interactions can sustain disease, SH2 domains have long been the focus of small-molecule ligand discovery efforts, but have been stymied by the poor drug-like properties of phosphate and its mimetics. Here, we have used structure-based design to target the SH2 domain of the suppressor of cytokine signalling 2 (SOCS2) – the substrate recognition subunit of a Cullin5 RING E3 ligase. Starting from the highly ligand-efficient pY amino acid, a fragment growing approach improved binding affinities in the nanomolar range. During the course optimization campaign, in one of our co-crystal structures we observed serendipitous modification of Cys111 at a flexible variable loop distal from the phosphate binding site. This inspired the rational design of a cysteine-directed electrophilic covalent inhibitor MN551. A prodrug strategy using a pivaloyloxymethyl (POM) protecting group aided cell permeability and its rapid unmasking inside the cell enabled efficient and selective covalent engagement of SOCS2. We qualify MN551 and its masked analogue MN714 as covalent inhibitors of SOCS2, that could find attractive applications as chemical probes to understand the biology of SOCS2 and its CRL5 complex, and as covalent E3 ligase handles in proteolysis targeting chimera (PROTACs) to induce targeted protein degradation. More broadly, this work provides a blueprint for developing and exploiting pY-based small molecules to target other SH2 domains.

Cellular processes can be regulated at multiple levels, including transcriptional, post-transcriptional, and post-translational mechanisms. We have recently shown that the small, noncoding vault RNA1–1 negatively riboregulates p62 oligomerization in selective autophagy through direct interaction with the autophagic receptor. This function is highly specific for this Pol III transcript, but the determinants of this specificity and a mechanistic explanation of how vault RNA1–1 inhibits p62 oligomerization are lacking. Here, we combine biochemical and functional experiments to answer these questions. We show that the PB1 domain and adjacent linker region of p62 (aa 1–122) are necessary and sufficient for specific vault RNA1–1 binding, and we identify lysine 7 and arginine 21 as key hinges for p62 riboregulation. Chemical structure probing of vault RNA1–1 further reveals a central flexible loop within vault RNA1–1 that is required for the specific interaction with p62. Overall, our data provide molecular insight into how a small RNA riboregulates protein–protein interactions critical to the activation of specific autophagy.

Macroautophagy ensures the clearance of intracellular substrates ranging from single ubiquitinated proteins to large proteotoxic aggregates and defective organelles. The selective autophagy receptor p62 binds these targets and recruits them to double-membrane vesicles, which fuse with lysosomes to degrade their content. We recently uncovered that p62 function is riboregulated by the small non-coding vault RNA1-1. Here, we present detailed insight into the underlying mechanism. We show that the PB1 domain and adjacent linker region of p62 (aa 1-122) are necessary and sufficient for specific vault RNA1-1 binding, and identify lysine 7 and arginine 21 as key hinges for p62 riboregulation. Chemical structure probing of vault RNA1-1 further reveals a central flexible loop within the RNA that mediates the specific p62 interaction. Our data define molecular determinants that govern mammalian autophagy via the p62-vault RNA1-1 riboregulatory pair.

TRIM proteins constitute a large, diverse and ancient protein family which play a key role in processes including cellular differentiation, autophagy, apoptosis, DNA repair, and tumour suppression. Mostly known and studied through the lens of their ubiquitination activity as E3 ligases, it has recently emerged that many of these proteins are involved in direct RNA binding through their NHL or PRY/SPRY domains. We summarise the current knowledge concerning the mechanism of RNA binding by TRIM proteins and its biological role. We discuss how RNA-binding relates to their previously described functions such as E3 ubiquitin ligase activity, and we will consider the potential role of enrichment in membrane-less organelles.

RIG-I is a viral RNA sensor that induces the production of type I interferon (IFN) in response to infection with a variety of viruses. Modification of RIG-I with K63-linked poly-ubiquitin chains, synthesised by TRIM25, is crucial for activation of the RIG-I/MAVS signalling pathway. TRIM25 activity is targeted by influenza A virus non-structural protein 1 (NS1) to suppress IFN production and prevent an efficient host immune response. Here we present structures of the human TRIM25 coiled-coil-PRYSPRY module and of complexes between the TRIM25 coiled-coil domain and NS1. These structures show that binding of NS1 interferes with the correct positioning of the PRYSPRY domain of TRIM25 required for substrate ubiquitination and provide a mechanistic explanation for how NS1 suppresses RIG-I ubiquitination and hence downstream signalling. In contrast, the formation of unanchored K63-linked poly-ubiquitin chains is unchanged by NS1 binding, indicating that RING dimerisation of TRIM25 is not affected by NS1., NS1 of influenza A virus inhibits TRIM25 activity, which is an E3 ligase important for induction of the interferon response. Here, Koliopoulos et al. present structures of TRIM25 and NS1 and show how NS1 binding interferes with substrate recognition of TRIM25.