Middleton, A. J. | Search bioRxiv

bioRxiv2024

Engineered Ubiquitin Variants Mitigate Pathogenic Bacterial Ubiquitin Ligase Function

During infection some pathogenic gram-negative bacteria, such as Salmonella, manipulate the host ubiquitination system through the delivery of secreted effectors known as novel E3 ubiquitin ligases (NELs). Despite the presence of NELs amongst these well-studied bacterial species, their unique structure has limited the tools that are available to probe their molecular mechanisms and explore their therapeutic potential. In this work, we report the identification of two high affinity engineered ubiquitin variants that can modulate the activity of the Salmonella enterica serovar Typhimurium encoded NEL, SspH1. We show that these ubiquitin variants suppress SspH1-mediated toxicity phenotypes in Saccharomyces cerevisiae. Additionally, we provide microscopic and flow cytometric evidence that SspH1-mediated toxicity is caused by interference with S. cerevisiae cell cycle progression that can be suppressed in the presence of ubiquitin variants. In vitro ubiquitination assays revealed that these ubiquitin variants increased the amount of SspH1-mediated ubiquitin chain formation. Interestingly, despite the increase in ubiquitin chains, we observe a relative decrease in the formation of SspH1-mediated K48-linked ubiquitin chains on its substrate, PKN1. Taken together our findings suggest that SspH1 toxicity in S. cerevisiae occurs through cell cycle interference and that an engineered ubiquitin variant approach can be used to identify modulators of bacterially encoded ubiquitin ligases. Author SummaryNovel E3 ligases (NELs) are a family of secreted effectors found in various pathogenic gram- negative bacteria. During infection these effectors hijack vital host ubiquitin signaling pathways to aid bacterial invasion and persistence. Despite interacting with a protein as highly conserved as ubiquitin, they have a distinct architecture relative to the eukaryotic E3 enzymes. This unique architecture combined with the indispensable role ubiquitin signaling plays in host cell survival has made hindering the contribution of NELs to bacterial infections a difficult task. Here, we applied protein engineering technology to identify two ubiquitin variants (Ubvs) with high affinity for SspH1, a Salmonella-encoded NEL. We provide evidence that these high affinity Ubvs suppress a known SspH1-meidated toxicity phenotype in the eukaryotic model system Saccharomyces cerevisiae. We also show that this suppression occurs without interfering with host ubiquitin signaling. Furthermore, we demonstrate the ability of a Ubv to modulate the activity of SspH1 in vitro, ultimately altering the lysine linkages found in SspH1-mediated ubiquitination. To our knowledge, this is the first evidence that an engineered ubiquitin variant approach can be implemented to modulate the activity of a family of previously untargetable bacterial-encoded E3 ligases.

microbiology↗

bioRxiv2024

Structural and biophysical characterisation of ubiquitin variants that specifically inhibit the ubiquitin conjugating enzyme Ube2d2

The ubiquitin conjugating E2 enzymes play a central role in ubiquitin transfer. Disruptions to the ubiquitin system are implicated in multiple diseases and as a result, molecules that modulate the activity of the ubiquitin system are of interest. E2 enzyme function is reliant on interactions with partner proteins and disruption of these is an effective way of modulating activity. Here, we report the discovery of ubiquitin variants (UbVs) that inhibit the E2 enzyme, Ube2d2 (UbcH5b). The six UbVs identified inhibit ubiquitin chain building, and structural and biophysical characterisation of two of these demonstrate they bind to Ube2d2 with low micromolar affinity and high specificity within the Ube2d family of E2 enzymes. Both characterised UbVs bind at a site that overlaps with E1 binding, while the more inhibitory UbV blocks a critical non-covalent ubiquitin binding site on the E2 enzyme. The discovery of novel protein-based ubiquitin derivatives that inhibit protein-protein interactions is an important step towards discovery of small molecules that inhibit the activity of E2 enzymes. Furthermore, the specificity of the UbVs within the Ube2d family suggests that it may be possible to develop tools to selectively inhibit highly related E2 enzymes.

biochemistry↗

bioRxiv2021

Identification of ubiquitin variants that inhibit the E2 ubiquitin conjugating enzyme, Ube2k

Transfer of ubiquitin to substrate proteins regulates most processes in eukaryotic cells. E2 enzymes are a central component of the ubiquitin machinery, and generally determine the type of ubiquitin signal generated and thus the ultimate fate of substrate proteins. The E2, Ube2k, specifically builds degradative ubiquitin chains on diverse substrates. Here we have identified protein-based reagents, called ubiquitin variants (UbVs), that bind tightly and specifically to Ube2k. Crystal structures reveal that the UbVs bind to the E2 enzyme at a hydrophobic cleft that is distinct from the active site and previously identified ubiquitin binding sites. We demonstrate that the UbVs are potent inhibitors of Ube2k and block both ubiquitin charging of the E2 enzyme, and E3-catalysed ubiquitin transfer. The binding site of the UbVs suggests they directly clash with the ubiquitin activating enzyme, while potentially disrupting interactions with E3 ligases via allosteric effects. Our data reveal the first protein-based inhibitors of Ube2k and unveil a hydrophobic groove that could be an effective target for inhibiting Ube2k and other E2 enzymes.

biochemistry↗