Structure of a Conjugating Enzyme-Ubiquitin Thiolester Intermediate Reveals a Novel Role for the Ubiquitin Tail

Abstract

and oncogenesis [1]. The common denominator of these processes is the initial C-terminal activation of ubiquitin The University of Alberta Edmonton, Alberta T6G 2H7 (Ub) by the activating enzyme (E1) followed by its subsequent transfer to a Ub-conjugating enzyme (E2) as a Canada 3 Department of Biochemistry and R.S. covalent E2-Ub thiolester intermediate. At this point, the mechanism of ubiquitination appears to diverge along McLaughlin Macromolecular Structure Facility either of two lines. In one case, Ub is transferred directly from the E2 to the lysine of a target protein in a reaction The University of Western Ontario London, Ontario N6A 5C1 that is facilitated by a Ub protein ligase (E3). In the other case, Ub is first transferred from the E2 to the active site Canada cysteine of an E3 as an E3-Ub thiolester intermediate, whereupon it is then transferred to the target protein. In either event, degradation of the target by the 26S Summary proteasome is facilitated by the assembly of a multi-Ub chain on the target in which the C terminus of each Ub Background: Ubiquitin-conjugating enzymes (E2s) are is linked to Lys48 (K48) of its neighbor. central enzymes involved in ubiquitin-mediated protein The ability of E2 proteins to orchestrate ubiquitination degradation. During this process, ubiquitin (Ub) and the through their interactions with Ub, E1, E3, and target E2 protein form an unstable E2-Ub thiolester intermediproteins makes them central players of the ubiquitin casate prior to the transfer of ubiquitin to an E3-ligase procade. All E2 proteins consist of a catalytic domain (150 tein and the labeling of a substrate for degradation. A residues) that includes the active site cysteine used to series of complex interactions occur among the target form the E2-Ub thiolester complex. Furthermore, X-ray substrate, ubiquitin, E2, and E3 in order to efficiently crystallographic structures of the catalytic domains from facilitate the transfer of the ubiquitin molecule. However, the E2 proteins Ubc1 (vide infra), Ubc2 [2], Ubc7 [3], and due to the inherent instability of the E2-Ub thiolester, Ubc9 [4] from Saccharomyces cerevisiae have shown the structural details of this complex intermediate are that this region is structurally conserved. Four ␣ helices not known. (␣1-␣4) essentially form one face of the protein, while a 4 strand antiparallel ␤ sheet (␤1-␤4) is found on the Results: A three-dimensional model of the E2-Ub thibackside of the enzyme between helices ␣1 and ␣2 in olester intermediate has been determined for the catathe sequence. The thiolester-forming cysteine is located lytic domain of the E2 protein Ubc1 (Ubc1 ⌬450 ) and ubion a relatively unstructured region (L2) ranging from 20 quitin from S. cerevisiae. The interface of the E2-Ub to 30 residues in length and linking ␤4 and ␣2. Recent intermediate was determined by kinetically monitoring X-ray crystallographic studies have shown that two thiolester formation by 1 H-15 N HSQC spectra by using structurally unrelated E3 proteins, E6AP and cCbl, intercombin...

Mechanistic insight into the allosteric activation of a ubiquitin-conjugating enzyme by RING-type ubiquitin ligases

“…Although our results do not support a mechanism that employs strong affinity between CNOT4N and ubiquitin, we cannot rule out the possibility that CNOT4N, when bound to UbcH5b, directly contacts the ubiquitin molecule of the UbcH5b thioester, and thus facilitates ubiquitin release. Consistent with this notion, for several E2s, the 3 10 helix immediately C-terminal to their active site provides a major contacting surface for the thioester-bound ubiquitin (33,34). Two allosteric residues identified in this study, I88 and L89, are located in the 3 10 helix.…”

Ubiquitin Manipulation by an E2 Conjugating Enzyme Using a Novel Covalent Intermediate

“…In addition, residual Ub correlations are present in the 1 H-15 N HSQC spectrum of the thioester (Fig. 4C) are common to those reported to undergo a decrease in peak intensity in the Ub-Ubc1 thioester intermediate (27), indicating that the interacting surface in Ubc1 is similar to that of the thioester intermediate. The interacting residues in both Ubc1 and ubiquitin in the disulfide complex are similar to those observed in the thioester, indicating that the disulfide complex mimics the thioester intermediate, and a minimal influence on the E2 surface is created by the presence of a charged carboxylate group near the active site.…”

“…To date, the best structural analysis of the ubiquitin-E2 thioester complex has been found from peak intensity changes in NMR experiments upon thioester formation. This has indicated that the surface on ubiquitin at the protein-protein interface encompasses residues Val 70 -Gly 76 and Arg 48 (27). In the current work, the ubiquitin-E2 disulfide is the first thioester intermediate synthesized that mimics this surface.…”

“…NMR spectroscopy was used to identify residues in ubiquitin or Ubc1 that decreased in intensity during formation of the thioester. However, the stability of the thioester (ϳ1 h) did not allow sequence-specific assignment of 1 H-15 N resonances in the Ub-E2 thioester complex thereby limiting this approach (27). Because Ub Cys -Ubc1 is orders of magnitude more stable than its thioester counterpart this has now allowed the 1 H, 13 C, and 15 N resonance (backbone) assignment of the 33-kDa Ub Cys -Ubc1 complex using conventional triple resonance techniques.…”

A unique E1-E2 interaction required for optimal conjugation of the ubiquitin-like protein NEDD8

“…In recent years, several general principles have emerged for protein-protein interactions in UBL transfer cascades [15][16][17][18][19][20][21][22][23][24][25][26] . Nonetheless, the selective coordination of enzymes within a particular UBL's cascade remains incompletely understood.…”