Synergizing Structure-Guided Photocaged Linear Diubiquitin-Dha with Biotinylated Linear-Fab for Time-Resolved Profiling of Interactors in Living Cells

Dynamically profiling of interactors of polyubiquitin (polyUb) chain topologies facilitates deciphering their functional specificity. Current strategies employing antibodies, synthetic antigen-binding fragments (sABs), or ″Affimers″ often lack temporal resolution and face challenges in detecting interactors of low-abundance polyUb chains, such as linear Ub (<0.2% of total Ub chains). Here, we structure-guided designed a photoactivatable OTULIN-specific inhibitor, linear-diUbp-Glu16(4-methoxy-7-nitroindoline, MNI)-dehydroalanine (Dha), which enables transient inhibition of this exclusively linear-specific deubiquitinase in living cells, thereby inducing rapid accumulation of linear Ub chains. Based on this tool, we developed a new strategy of synergizing the linear-diUbp-Glu16(MNI)-Dha with a biotinylated linear-antigen-binding fragment (Fab), which facilitated time-resolved profiling of linear polyubiquitination interactors in both TNF-α stimulated HeLa cells and macrophage colony-stimulating factor (M-CSF)-induced bone-marrow-derived macrophages. Our work also provides new opportunities for analyzing interactors of other low-abundance Ub chains, especially in hard-to-transfect cells, and highlights the effectiveness of chemical protein synthesis in developing advanced protein tools for biological discovery.