Engineering a Ubiquitin Ligase Reveals Conformational Flexibility Required for Ubiquitin Transfer

Protein ubiquitination regulates numerous cellular functions in eukaryotes. The prevailing view about the role of RING or U-box ubiquitin ligases (E3) is to provide precise positioning between the attached substrate and the ubiquitin-conjugating enzyme (E2). However, the mechanism of ubiquitin transfer remains obscure. Using the carboxyl terminus of Hsc70-interacting protein as a model E3, we show herein that although U-box binding is required, it is not sufficient to trigger the transfer of ubiquitin onto target substrates. Furthermore, additional regions of the E3 protein that have no direct contact with E2 play critical roles in mediating ubiquitin transfer from E2 to attached substrates. By combining computational structure modeling and protein engineering approaches, we uncovered a conformational flexibility of E3 that is required for substrate ubiquitination. Using an engineered version of the carboxyl terminus of Hsc70-interacting protein ubiquitin ligase as a research tool, we demonstrate a striking flexibility of ubiquitin conjugation that does not affect substrate specificity. Our results not only reveal conformational changes of E3 during ubiquitin transfer but also provide a promising approach to custom-made E3 for targeted proteolysis.Protein modification by ubiquitin and ubiquitin-like proteins is a common mechanism through which numerous cellular pathways are regulated (1). The canonical cascade of ubiquitination involves the action of three enzymes, termed E1, E2, and E3, which activate and then conjugate ubiquitin to its substrates (2, 3). The E3 ligase catalyzes the final step in ubiquitin transfer in a substrate-specific manner. Despite advances in understanding the enzymatic cascade of ubiquitination, the mechanism of ubiquitin transfer to the substrate remains an outstanding issue (4). In particular, the role of E3 ubiquitin ligases and how they adapt to progressively modified substrates to maintain specific ubiquitin chain topology is still a mystery.The known E3s belong to three protein families: HECT, RING, and U-box. HECT domain enzymes form a covalent intermediate with ubiquitin before the final transfer of ubiquitin to substrates. In contrast, RING and U-box E3s have been suggested to function as adaptors that position the substrate in close proximity to the E2-ubiquitin thioester (E2-Ub) (5). It has become common “wisdom” that the substrate has to be precisely positioned to get ubiquitinated (6). The positioning hypothesis originally predicted that E3 substrates would have a specific ubiquitination site. However, the absence of “consensus” ubiquitination sites has become apparent in an increasing list of E3 substrates (7–9). In addition, the crystal structures of several ubiquitination machinery components have revealed a puzzling gap (～50 Å) between the substrate binding sites and the E2 active sites (10, 11). This raises a fundamental question in ubiquitin transfer. How does the ubiquitin molecule shuttle from the E2 to substrates? Though several interesting models for ubiquitin transfer have been proposed, only limited explicit experimental evidence support these models (4).We used carboxyl terminus of Hsc70-interacting protein (CHIP)³ as a model E3 system to investigate the role of substrate positioning in its ubiquitination. CHIP is a protein quality control E3 that consists of an NH₂-terminal tetratricopeptide repeat (TPR) domain, a helical linker domain, and a COOH-terminal U-box domain (12, 13). The TPR domain of CHIP binds directly to EEVD motifs located at the COOH termini of Hsc/Hsp70 and Hsp90, whereas the U-box domains possess ubiquitin ligase activity. CHIP recruits E2 enzymes of the Ubc4/5 family to ubiquitinate misfolded proteins that occupy the chaperone substrate-binding sites, thus remodeling the chaperones from protein-refolding complexes to complexes that promote degradation (14). Using the chaperone as an adaptor, CHIP targets a variety of substrates for ubiquitination (15). In the absence of substrates, CHIP is also able to ubiquitinate the bound chaperones (16). Thus, there is apparent substrate diversity for CHIP-mediated ubiquitination. Insights into the mechanism of action of CHIP have been provided by an x-ray crystal structure which reveals a remarkable, highly asymmetric dimer (25). Here, we demonstrate the existence of intrinsic structural flexibility in the CHIP homodimer that is required for substrate polyubiquitination. The flexible orientation allows CHIP to accommodate substrates with different sizes and structures. Mutations that restrict the flexibility of CHIP markedly decrease substrate ubiquitination, whereas maintaining flexibility enables us to rebuild a functional ubiquitin ligase with altered substrate specificity. Our results provide evidence for the importance of structural flexibility in E3 ligases, which we propose is of general importance to orchestrate progressive ubiquitin conjugation on substrates.