A conserved asparagine plays a structural role in ubiquitin-conjugating enzymes

Ubiquitination of lysine side chains occurs in a cascade of enzymatic reactions catalyzed by an ubiquitin activating enzyme (E1), a ubiquitin conjugating enzyme (E2) and a ubiquitin ligase (E3) ^{1, 2}. A key step in ubiquitin transfer is the attack of the acceptor lysine on the E2~ubiquitin thioester to yield an isopeptide linkage between the ε-amine of lysine and the ubiquitin C terminus ^3–7. RING family E3 ligases stimulate this reaction by binding to the E2~Ub conjugate and positioning the ubiquitin in a “closed” conformation that favors catalysis ^8–10.By analogy with other ester-cleaving enzymes, the ubiquitination reaction was proposed to proceed through a tetrahedral intermediate with partial negative charge on the carbonyl oxygen that is stabilized by an “oxyanion hole” in the E2 active site ³. Structures of E2 enzymes showed a highly conserved active site asparagine (N79 in Ubc13, N77 in UBCH5B, N85 in Ubc9) as the only residue that could plausibly stabilize the oxyanion ^{3, 11}, although it has also been noted that this asparagine side chain may also play a structural role ^{4, 12}. While substitutions of the asparagine residue give rise to catalytic defects in E2 enzymes ^{3, 4, 6, 7}, these experiments do not differentiate between a structural role and a catalytic role for the asparagine in ubiquitin transfer. Moreover, the many structures of E2 enzymes reported since the initial proposal for the role of the asparagine show a nearly identical set of backbone contacts ^{3, 4, 6, 7, 12–14} (Supplementary Results, Supplementary Fig. 1) as does the structure of the SUMO E2, Ubc9, with glutamine in place of the conserved asparagine (N85Q) ⁴. Thus, while the importance of the conserved active site asparagine to E2 activity is undisputed, there is no direct evidence for its involvement in stabilizing a negative charge on the oxygen in the proposed tetrahedral intermediate.

We revisited the role of the conserved asparagine in ubiquitin transfer by studying the yeast E2 enzyme, Ubc13. Ubc13 catalyzes synthesis of K63-linked polyubiquitin by forming a heterodimer with a noncatalytic subunit Mms2 ¹⁵, which positions the substrate ubiquitin with its K63 side chain at the Ubc13 active site ¹². Ubc13/Mms2 catalyzes synthesis of unanchored K63 polyubiquitin in the absence of an E3 ¹⁵ but the RING domain of the E3 ligase, Rad5, stimulates this activity ¹⁶, thus making it possible to compare the E2-catalyzed reaction in the absence and presence of an E3. In light of recent reports that several RING E2 ligases stimulate E2 enzymes by a mechanism of conformational selection ^{8–10, 17}, we first verified that the Rad5 E3 has a corresponding effect on ubiquitination by the Ubc13/Mms2 heterodimer. We first identified a minimal well-behaved fragment containing the Rad5 RING domain (846–1169) that can stimulate free polyubiquitin chain synthesis by Ubc13/Mms2 to a degree comparable to the wild-type protein ¹⁶(Supplementary Fig. 2a, b). To address whether the RING domain of Rad5 promotes a catalytically competent conformation of charged Ubc13, we used a modification of the substrate partitioning experiment ^{18, 19}. Single-discharge assays were performed by mixing charged Ubc13~Ub/Mms2 with free acceptor ubiquitin in the presence of a large excess of DTT and cysteine, thus ensuring that the diubiquitin formed is only from a single encounter between the acceptor ubiquitin and Ubc13~Ub/Mms2. The results (Fig. 1a) show that ~30% of Ubc13~Ub/Mms2 formed diubiquitin on the first encounter, while the RING domain increases the active fraction of Ubc13~Ub/Mms2 to ~90%, consistent with the proposed role of the RING E3 in promoting the active E2~Ub conformation ^8–10. We also confirmed that the Rad5 RING binds 59-fold more tightly to Ubc13~Ub than to uncharged Ubc13 (Supplementary Fig. 3). These results are consistent with a role for the Rad5 RING domain in favoring a catalytically competent conformation of the charged Ubc13/Mms2 complex.

To explore the contribution of N79 to catalysis, we assayed diubiquitin formation by Ubc13/Mms2 in the presence and absence of Rad5 RING for Ubc13 mutants N79A, N79D, H79H, N79S and N79Q. While Ubc13 substitutions N79H, N79S and N79Q significantly decrease activity (Fig. 1b), the Rad5 RING-stimulated activity for these mutants was apparently similar to that of the wild-type enzyme under the conditions tested (Fig. 1b). The substitutions N79A and N79D have a particularly severe effect on Ubc13 activity, as previously reported ²⁰. However, Ubc13 with either of these substitutions is stimulated to comparable levels by the RING domain, although there is still a noticeable defect in chain formation. The N79A and N79D Ubc13 mutations exhibited significantly decreased catalytic rates in both the presence and absence of the Rad5 RING (Table 1), with both substitutions resulting in comparable values for k_cat/K_M in the presence of Rad5 (3 M⁻¹ s⁻¹ for N79A and 4 M⁻¹ s⁻¹ for N79D as compared to 22 M⁻¹ s⁻¹ for the wild-type enzyme). By contrast, the kinetic parameters for the conservative N79Q mutant were similar to the wild-type Ubc13, with a 6-fold decrease in the k_cat for the unstimulated reaction and a less than 2-fold decrease in the k_cat for the stimulated reaction (Table 1). The surprising similarity in the rates of diubiquitin formation between the alanine and aspartate substitutions is inconsistent with the proposed role for this residue in oxyanion stabilization, since the negatively charged side chain of aspartate should be more detrimental than alanine to transition state formation.

To explore whether the effects of N79 mutations on ubiquitin chain formation could be explained by a structural role for this residue, we determined the crystal structure of Ubc13 with an N79A substitution at a resolution of 2.65 Å (Fig. 2a and Supplementary Table 1). The overall structures of wild type (PDB code: 2GMI) and mutant Ubc13 ^N79A are similar, with an RMSD in C_α positions of 0.9 Å. Strikingly, there is no electron density corresponding to residues 119–122, indicating that they are disordered (Fig. 2b). In addition, residues 116–118 are not well-ordered, with an absence of clear side chain density for N116 and N118 and high B factors for backbone atoms (Fig. 2b). The global changes in the active site loop that result from the N79A mutation are consistent with a structural role for the N79 side chain in maintaining the structure of the active site loop by hydrogen bonding to backbone atoms of N116 (Fig. 2a and Supplementary Fig. 1) in the loop. Consistent with this, a recently reported structure of the BIRC7 RING bound to a UBCH5B~Ub oxyester in which UBCH5B contains the corresponding alanine substitution (N77A) showed that this loop has incomplete electron density and high B factors, indicating that it is similarly not well-ordered ¹⁰, whereas this loop is well-ordered in structure of wild-type UBCH5B ^{6, 21}.

The structural effect of the N79A mutation in Ubc13 raised the possibility that the decrease in enzymatic activity may derive from the disruption of the active site loop structure. In the structure of the RNF4 RING bound to a UBCH5A~Ub conjugate ⁸, this loop forms one side of a groove where the ubiquitin C-terminal tail lies (Supplementary Fig. 1). Mutations in this loop have previously been shown to disrupt activity of other E2 enzymes: substitutions of the residue corresponding to D119 significantly disrupts activity of Ubc9 ⁴ and UBCH5A ⁸, while N114 of UBCH5A (N116 in Ubc13) is also important for activity due to its direct hydrogen bonding interaction with the donor ubiquitin ⁸. We tested the contributions of Ubc13 loop residues to activity and found that N116A, N116L, D119N or L121S substitutions markedly decrease diubiquitin formation (Fig. 2c and Supplementary Fig. 5), whereas S114A, P117A or N123A substitutions showed no significant defects (Fig. 2c)¹². Since the region of the loop containing N116 is displaced from its wild-type position (Fig. 2a,b) and D119 and L121 are completely disordered in the structure of Ubc13^N79A, these structural defects can account for the loss of enzymatic activity due to the N79A mutation without the need to invoke a catalytic role for this residue. We note that our results do not exclude the possibility that the amino group of N79 may also hydrogen bond with the carbonyl of the ubiquitin C-terminal residue G76 in the thioester or in the product complex, as has been observed in the structure of Ubc9 bound to a SUMO-RanGAP product ¹⁴ and in the RNF4-UBCH5A^C88K~Ub structure ⁸.

The nature of the transition state and how it is stabilized by the E2 during the ubiquitin transfer reaction remain open questions. However, we note that immobilization of the thioester alone can provide significant rate enhancement. Bruice and Pandit^{22, 23} showed that reducing the conformational freedom of an ester and the nucleophile increased the rate of ester hydrolysis nearly 53,000-fold. In the case of E2~Ub conjugates, the E3-induced binding of the donor ubiquitin to the E2 positions the donor ubiquitin tail in the active site groove and thereby immobilizes the E2~Ub thioester ^{8, 10}. The increased rate of E2~Ub oxyester and thioester hydrolysis in the presence of the RING domain ^{17, 24} can similarly be accounted for by the immobilization of the thioester or ester bond. By stabilizing the loop flanking the donor ubiquitin tail (Supplementary Fig. 1e), the conserved asparagine may indirectly aid in immobilizing the thioester. Consistent with this, the C-terminal tail of the donor ubiquitin is well ordered in the structure of UBCH5A~Ub bound to RNF4⁸, whereas the two C-terminal glycines of the donor ubiquitin in the UBCH5B^N77A~Ub conjugate have high B factors¹⁰. The additional proposed role of an aspartate in the active site loop in suppressing the pK_a of the attacking lysine ⁴ provides further rate enhancement. The role of the conserved asparagine in maintaining the structure of the active site loop is thus critical to E2 activity.