A conserved catalytic residue in the ubiquitin-conjugating enzyme family

Abstract

Ubiquitin (Ub) regulates diverse functions in eukaryotes through its attachment to other proteins. The defining step in this protein modification pathway is the attack of a substrate lysine residue on Ub bound through its C-terminus to the active site cysteine residue of a Ub-conjugating enzyme (E2) or certain Ub ligases (E3s). So far, these E2 and E3 cysteine residues are the only enzyme groups known to participate in the catalysis of conjugation. Here we show that a strictly conserved E2 asparagine residue is critical for catalysis of E2- and E2/RING E3-dependent isopeptide bond formation, but dispensable for upstream and downstream reactions of Ub thiol ester formation. In contrast, the strictly conserved histidine and proline residues immediately upstream of the asparagine are dispensable for catalysis of isopeptide bond formation. We propose that the conserved asparagine side chain stabilizes the oxyanion intermediate formed during lysine attack. The E2 asparagine is the first non-covalent catalytic group to be proposed in any Ub conjugation factor.

Fig. 1. Chemical and structural issues in the catalysis of Ub conjugation. (A) Model for catalysis of isopeptide bond formation in Ub chain synthesis (:B denotes a general base). (B) E2 active site sequences (blue, conserved asparagine; orange, active site cysteine). (C) Representative interactions of conserved asparagine in unliganded E2s, taken from the crystal structure of Ubc13 (Van Demark et al., 2001).

Fig. 2. E2 asparagine mutations do not impair E2 folding, E2–Ub formation or HECT E3–Ub formation. (A) Binding of wild-type Ubc13 (Ubc13-wt) and Ubc13-N79Q proteins to H₁₀-Mms2 immobilized on Ni beads (Coomassie-stained gel). Successive lanes show proportional loading of the starting material (SM), unbound fraction (FT), washes with 0.1 M (w) and 0.4 M NaCl (W), and EDTA strip (EL). There is no binding of Ubc13 to the beads in the absence of Mms2 (data not shown). (B) Steady-state level of E2–[¹²⁵I]Ub (autoradiograph). Assays were quenched as indicated (E2–Ub is labile to β-mercaptoethanol, βME). Small panel, Coomassie staining of Ubc13, showing that differences in E2–Ub levels reflect variations in [Ubc13]. (C) The level of Ubc13–Ub formed after 2 min of reaction (representing the initial rate) was determined at varying [E1]. (D) Binding of in vitro-translated E2s to immobilized GST–AO7 RING domain (autoradiograph). UbcH5B and GST are positive and negative controls, respectively. (E) E6-AP-HECT–Ub thiol ester formation (autoradioagraph). Assays with the indicated version of UbcH5A and [¹²⁵I]Ub were quenched with or without β-mercaptoethanol as indicated.

Fig. 3. E2 asparagine mutations impair E2/UEV- and E2-catalyzed isopeptide bond synthesis. (A) Qualitative assay of K63-Ub₂ synthesis catalyzed by Mms2/Ubc13 (Coomassie-stained gel). (B) Quantitative assay of K63-Ub₂ synthesis by Mms2/Ubc13. [¹²⁵I]Ub was employed; the Ub₂ product was quantitated by band excision and gamma counting. Percent values refer to relative slopes of the lines. (C) The Ubc13–Ub thiol ester was pre-formed in a short pulse incubation with E1 and [¹²⁵I]Ub. Lysine was then added to initiate the chase (see Materials and methods). The plot shows the decay of the respective E2–Ub thiol esters. (D) E2-25K-catalyzed synthesis of K48-Ub₂ [assayed as in (B)]. (E) Stat1 sumoylation catalyzed by Ubc9 (–, no E1/E2; N, Ubc9-wt; Q, Ubc9-N85Q). A western blot was developed with Stat1 antibodies. In the lanes labeled 1×, the [Ubc9] was 0.2 µM. In the lanes labeled 0.1×, [Ubc9] was 20 nM. PIASxα (0.3 µM) was included in the 0.1× assays. Similar results were obtained in an assay with in vitro-translated Stat1 (data not shown).

Fig. 4. Conserved E2 histidine and proline are dispensable for catalysis of isopeptide bond formation. (A and B) Pulse–chase assays of Ub₂ synthesis were conducted with the indicated Ubc13 proteins. The assays were carried out as in Figure 3C, except that wild-type Ub (117 µM) served as the acceptor during the chase, producing K63-Ub₂ as the product. (A) Comparison of Ubc13-wt and Ubc13-H77A; (B) (a separate experiment) compares Ubc13-wt and Ubc13-N79Q. (C) Steady-state K63-Ub₂ synthesis. Assays comparing Ubc13-wt and Ubc13-P78A were carried out as in Figure 3B.

Fig. 5. Effects of E2 asparagine mutations on E3-dependent isopeptide bond synthesis. (A) Mdm2-dependent ubiquitylation of p53 was assayed with UbcH5B (5B, positive control), no E2 (–, negative control), or two different concentrations (1×, 2×) of wild-type UbcH5A (N) or UbcH5A-N77Q (Q). A western blot was developed with p53 antibodies. (B) Mdm2 autoubiquitylation was monitored with radiolabeled Ub [autoradiograph; annotation is the same as in (A)]. The blot shown in (A) was also stripped and re-probed with an Mdm2 antibody to confirm that autoubiquitylation was impaired by the N77Q mutation (data not shown). (C) AO7-catalyzed autoubiquitylation was assayed [autoradiograph, annotation is the same as in (A)]. (D) Ubc13-N79A cannot support DNA damage tolerance in vivo. A yeast mms2Δubc13Δ strain (Hofmann and Pickart, 1999) was transformed with empty vectors (diamonds), plasmids expressing Mms2 and HA-tagged wild-type Ubc13 (squares) or plasmids expressing Mms2 and HA-tagged Ubc13-N79A (circles). Inset, Ubc13-N79A is highly expressed (western blot developed with HA antibodies). Asterisk, cross-reacting band (loading control; the wild-type lane is underloaded). (E) KIAA10 (HECT) E3-dependent synthesis of K48-linked polyUb chains (Coomassie-stained gel). E3 was omitted from lanes 1 and 5 (negative controls). Numbers denote chains consisting of the indicated number of Ub units. Asterisk denotes bovine serum albumin (carrier protein).

Fig. 6. Model for oxyanion stabilization by conserved E2 asparagine residue. Ubc1 in the model of the Ubc1–Ub thiol ester (Hamilton et al., 2001) was replaced by Ubc13 and the N79 side chain was modeled in a conformation suitable for oxyanion stabilization.