Engineering a Ubiquitin Ligase Reveals Conformational Flexibility Required for Ubiquitin Transfer

Abstract

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 (ϳ50 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 tra...

A Bipartite Interaction between Hsp70 and CHIP Regulates Ubiquitination of Chaperoned Client Proteins

“…We combined our new structure with recent structures of full-length Hsp70 family members in ADP-bound or ATP-bound conformations (Bertelsen et al, 2009; Kityk et al, 2012), RING or U-box domains complexed with E2~Ubiquitin conjugates (Dou et al, 2012; Plechanovová et al, 2012; Pruneda et al, 2012) and our previously determined structure of a UbcH5b bound to the U-box domain of CHIP (Xu et al, 2008a)(Figure 6). We utilized the asymmetric crystal structure of the CHIP dimer (Zhang et al , 2005), the only currently available structure of full-length CHIP, although there is substantial evidence that CHIP is structurally dynamic and adopts other conformations (Graf et al, 2010; Qian et al, 2009; Xu et al, 2006). The asymmetric CHIP dimer does not clash with the chaperone, regardless of which of its TPR domains serves as the docking site for Hsp70 ADP or Hsp70 ATP (Figure 6).…”

“…We used a crystal structure of near-full-length (murine) CHIP in these models (Zhang et al, 2005); in this structure, CHIP is an asymmetric dimer with different relative positions of the TPR and U-box domains in each protomer. However, several studies provided evidence that CHIP adopts other conformations, and that CHIP dynamics and conformational changes strongly influence ubiquitination (Graf et al, 2010; Qian et al, 2009). The CHIP dimer could alternate between asymmetric conformations (Qian et al, 2009; Xu et al, 2006), modulating the distance between TPR and U-box domains and allowing the Hsp70-SBD to approach the E2~Ub conjugate thioester more closely than in our current models.…”

Monitoring cotranslational protein folding in mammalian cells at codon resolution

“…To probe the folding status of FRB domain, we took advantage of its binding partner FKBP (FK506 binding protein). The dimerization of FKBP and FRB relies on their 3D structures and the presence of rapamycin or rapalog (12,13) ( Fig. 1).…”

Isoform-Specific Regulation of a Steroid Hormone Nuclear Receptor by an E3 Ubiquitin Ligase inDrosophila melanogaster

“…Also, the interaction specificity among the EcR isoforms is not dependent on the differential N termini (AF1 fragment), probably because it requires a 3D feature that only the entire protein can exhibit. Indeed, E3 enzymes frequently use these high-order structural motifs to recognize their substratum (Qian et al 2009). …”