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Abstract One of the major challenges in evaluating the suitability of potential ∼700 E3 ligases for target protein degradation (TPD) is the lack of binders specific to each E3 ligase. Here we apply genetic code expansion (GCE) to encode a tetrazine-containing non-canonical amino acid (Tet-ncAA) site-specifically into the E3 ligase, which can be conjugated with strained trans-cyclooctene (sTCO) tethered to a neo-substrate protein binder by click chemistry within living cells. The resulting E3 ligase minimally modified and functionalized in an E3-ligand free (ELF) manner, can be evaluated for TPD of the neo-substrate. We demonstrate that CRBN encoded with clickable Tet-ncAA, either in the known immunomodulatory drug (IMiD)-binding pocket or across surface, can be covalently tethered to sTCO-linker-JQ1 and recruit BRD2/4 for CRBN mediated degradation, indicating the high plasticity of CRBN for TPD. The degradation efficiency is dependent on location of the Tet-ncAA encoding on CRBN as well as the length of the linker, showing the capability of this approach to map the surface of E3 ligase for identifying optimal TPD pockets. This ELF-degrader approach has the advantages of not only maintaining the native state of E3 ligase, but also allowing the interrogation of E3 ligases and target protein partners under intracellular conditions and can be applied to any known E3 ligase. 

Quantitative labeling of biomolecules is necessary to advance areas of antibody–drug conjugation, super-resolution microscopy imaging of molecules in live cells, and determination of the stoichiometry of protein complexes. Bio-orthogonal labeling to genetically encodable noncanonical amino acids (ncAAs) offers an elegant solution; however, their suboptimal reactivity and stability hinder the utility of this method. Previously, we showed that encoding stable 1,2,4,5-tetrazine (Tet)-containing ncAAs enables rapid, complete conjugation, yet some expression conditions greatly limited the quantitative reactivity of the Tet-protein. Here, we demonstrate that reduction of on-protein Tet ncAAs impacts their reactivity, while the leading cause of the unreactive protein is near-cognate suppression (NCS) of UAG codons by endogenous aminoacylated tRNAs. To overcome incomplete conjugation due to NCS, we developed a more catalytically efficient tRNA synthetase and developed a series of new machinery plasmids harboring the aminoacyl tRNA synthetase/tRNA pair (aaRS/tRNA pair). These plasmids enable robust production of homogeneously reactive Tet-protein in truncation-free cell lines, eliminating the contamination caused by NCS and protein truncation. Furthermore, these plasmid systems utilize orthogonal synthetic origins, which render these machinery vectors compatible with any common expression system. Through developing these new machinery plasmids, we established that the aaRS/tRNA pair plasmid copy-number greatly affects the yields and quality of the protein produced. We then produced quantitatively reactive soluble Tet-Fabs, demonstrating the utility of this system for rapid, homogeneous conjugations of biomedically relevant proteins. 

ABSTRACT The dinucleotide second messenger c-di-GMP has emerged as a central regulator of reversible cell attachment during bacterial biofilm formation. A prominent cell adhesion mechanism first identified in pseudomonads combines two c-di-GMP-mediated processes: transcription of a large adhesin and its cell surface display via posttranslational proteolytic control. Here, we characterize an orthologous c-di-GMP effector system and show that it is operational in Vibrio cholerae , where it regulates two distinct classes of adhesins. Through structural analyses, we reveal a conserved autoinhibition mechanism of the c-di-GMP receptor that controls adhesin proteolysis and present a structure of a c-di-GMP-bound receptor module. We further establish functionality of the periplasmic protease controlled by the receptor against the two adhesins. Finally, transcription and functional assays identify physiological roles of both c-di-GMP-regulated adhesins in surface attachment and biofilm formation. Together, our studies highlight the conservation of a highly efficient signaling effector circuit for the control of cell surface adhesin expression and its versatility by revealing strain-specific variations. IMPORTANCE Vibrio cholerae , the causative agent of the diarrheal disease cholera, benefits from a sessile biofilm lifestyle that enhances survival outside the host but also contributes to host colonization and infectivity. The bacterial second messenger c-di-GMP has been identified as a central regulator of biofilm formation, including in V. cholerae ; however, our understanding of the pathways that contribute to this process is incomplete. Here, we define a conserved signaling system that controls the stability of large adhesion proteins at the cell surface of V. cholerae , which are important for cell attachment and biofilm formation. Insight into the regulatory circuit underlying biofilm formation may inform targeted strategies to interfere with a process that renders this bacterium remarkably adaptable to changing environments.