To determine which residues in E2s are important for lysine reactivity, the active site sequences of lysine-reactive E2s were aligned with that of UbcH7 (Fig 2a). Two residues in UbcH7 are distinctly different: D87 and D117 (in UbcH5c numbering) are proline and histidine, respectively, in UbcH7. To establish whether these residues contribute to lysine reactivity, each was mutated in UbcH5c and lysine reactivity was measured (Fig. 2b). The effect of substitution at position 87 ranges from no effect for the isosteric mutation D87N to complete loss of lysine reactivity for the charge-swapped D87K mutation. UbcH5c-D87E and -D87P have intermediate reactivities. Consistent with D87 playing a general role in lysine reactivity, mutation of the analogous residue in Ube2k (D94E) results in decreased lysine reactivity and impaired formation of free poly-Ub chains (Supplementary Fig. 4a,b). Substitution of D117 in UbcH5c with a histidine as found in UbcH7 greatly decreases lysine reactivity (Fig. 2b). A structurally analogous residue in the SUMO E2, (Ubc9-D127), has been shown to lower the pKa of a lysine approaching the active site¹⁸. The invariant active site asparagine residue (N77 in UbcH5c) is recognized for its role in isopeptide catalysis¹⁹, and its mutation to serine abolishes UbcH5c~Ub lysine reactivity in our assay. Identification of several residues that affect an E2’s intrinsic lysine reactivity suggests that the determinants are likely multi-factorial. Accordingly, we failed to convert UbcH7 into a lysine-reactive E2 by mutation (Supplementary Table).

To assess whether an E2’s intrinsic reactivity is predictive of its functional E3 interaction, UbcH5c mutants were assayed with RING and HECT-type E3s. Of the D87 mutants, only UbcH5c-D87N (which has wild-type lysine reactivity) is able to function with the RING E3 BRCA1/BARD1 in an auto-ubiquitination assay; the other substitutions are inactive (Fig. 2c). The observed loss of activity results from E2 catalytic defects, as NMR binding experiments confirm UbcH5c-D87 mutants bind BRCA1 comparable to UbcH5c-WT (Supplementary Fig. 5). UbcH5c-D117H, which has impaired lysine reactivity, retains some ability to transfer Ub to BRCA1 (Fig. 2c). Given its position at the active site, D117 may provide substrate-specific lysine reactivity as a gating residue. As expected, UbcH5c-N77S is inactive with BRCA1. Previous studies show that mutation of E2 residue N77 abolishes Ub-transfer with RING-type ligases Mdm2¹⁹, Rma1²⁰, CNOT4 and APC/C²¹ but retains activity with HECT-type ligases E6AP, KIAA10¹⁹, and NEDD4L²². This suggests that the catalytic requirements for trans-thiolation differ from those for isopeptide bond synthesis. Accordingly, the ability to form E3~Ub thioesters with the HECT E3 E6AP is unaffected in all UbcH5c mutants (Supplementary Fig. 6).Taken together, our results indicate that E2 lysine reactivity is a prerequisite for transfer with RING E3s. Furthermore, lysine-unreactive E2s such as UbcH5c-N77S, can be diagnostic for differentiating between RING- vs. HECT-type Ub transfer mechanisms.

Although many E2s possess an intrinsic reactivity for lysine, most do not transfer Ub to protein substrates independent of an E3. To explore the contribution of E3s to E2 reactivity, we examined the intrinsic reactivity of E2~Ub in the presence of RING or HECT E3s. Interaction with BRCA1/BARD1 (RING) or E6AP (HECT) does not change the intrinsic reactivity of UbcH5c and UbcH7 with free amino acids (Supplementary Fig. 7a, b). However, BRCA1/BARD1 enhances UbcH5c~Ub lysine reactivity compared to a reaction containing the E2-binding mutant BRCA1-I26A^3,15 (Fig. 2d). Intriguingly, the lysine reactivity of UbcH5c-D87E, which retains both its ability to bind BRCA1/BARD1 and some intrinsic lysine reactivity, is not enhanced by BRCA1 (Fig. 2d). These results suggest that RING-binding to a lysine-reactive E2~Ub results in a thioester with enhanced reactivity to lysine and residues such as D87 in UbcH5c couple E3-binding and E2 activation. Notably, E6AP does not enhance UbcH5c~Ub lysine reactivity (Supplementary Fig. 8b), highlighting a mechanistic difference between RINGs and HECTs. In matched experiments with UbcH7, neither BRCA1/BARD1 nor E6AP enhance reactivity toward free cysteine (Supplementary Fig 8a, c.)

Besides HECT-type ligases, UbcH7 is reported to function with members of the RING E3 family known as RBRs that includes Parkin and HHARI^5,6. This activity runs contrary to our conclusion that UbcH7 lacks lysine reactivity and is consequently restricted to Ub transfer involving trans-thiolation chemistry. Therefore, we examined the Ub ligase mechanism for HHARI and Parkin. The minimal ligase (RBR) domains of HHARI_R1-IBR-R2 and Parkin_R1-IBR-R2 (Supplementary Fig. 9 for schematic of constructs) show comparable auto-ubiquitination activity with either UbcH7 or UbcH5c (Fig. 3a,b). In contrast to our results with BRCA1/BARD1, UbcH5c-N77S exhibits Ub transfer with HHARI_R1-IBR-R2 and Parkin_R1-IBR-R2 (Fig. 3a, b). As UbcH5c-N77S activity is restricted to HECT-type ligases, its activity with Parkin and HHARI suggests that these ligases do not function via a typical RING-type mechanism.

A hallmark of HECT-type Ub transfer is the formation of an obligate E3~Ub thioester intermediate. Reactions designed to trap a HHARI~Ub conjugate were conducted on ice and quenched with SDS-loading buffer with or without reducing agent (βME) to distinguish reducible thioester-linked Ub from non-reducible isopeptide-linked Ub. A βME-sensitive band corresponding to the molecular weight of HHARI~Ub was detected at 10, 20, and 30 seconds post addition of pre-charged UbcH7~Ub (Fig. 3c). We next sought to determine the position of the HHARI active site cysteine. The N-terminal canonical RING1 of HHARI has been shown to be the principal E2-binding region²³, although RING2 is also required for ligase activity⁵. Cysteine 357 in RING2 is highly conserved across RBR ligases (Fig. 4a). C357 is not a Zn²⁺-liganding residue, and mutation of C357 does not destabilize the RING2 structure (Supplementary Fig. 10 and Ref. 5), but does abolish the ability of HHARI to transfer Ub (Fig. 4b and Ref. ⁵). This suggests C357 may have a catalytic function. Although HHARI_R1-IBR-R2-C357A showed no ligase activity in an auto-ubiquitination assay, HHARI_R1-IBR-R2-C357S generated a single monoubiquitinated species. An analogous oxyester-linked Ub product can be generated for E2s whose active site cysteines have been mutated to serines^{20, 22, 24}. Consistent with its identity as an oxyester (vs. isopeptide) bond, the Ub-adduct on HHARI_R1-IBR-R2-C357S is labile to alkaline treatment (Supplementary Fig. 11). Formation of an oxyester is unique to C357 as serine substitution of other conserved cysteines (both Zn⁺²- and non Zn⁺²-liganding) in RING2 does not stall the E3 at a single-Ub adduct but rather impairs (C375S) or abolishes (C367S, C372S) ligase activity (Supplementary Fig. 12). Similarly, the Parkin mutant C431S (analogous to HHARI C357) eliminates E3 ligase activity (Fig. 4c). We were unable to trap a Parkin_R1-IBR-R2-C431S Ub-adduct under our reaction conditions. However, we note that the Parkin mutation C431F has been consistently shown to abolish Parkin’s ubiquitination of substrates^25–27 and genetically predisposes for Parkinson’s disease²⁸. The results presented above are not affected by the presence of a GST-domain, as several results were reproduced with non-GST versions of HHARI (Supplementary Fig. 13).

HHARI_R1-IBR-R2-C357A effectively binds E2 as GST-pulldowns with purified UbcH7 and constructs of GST-HHARI demonstrate that HHARI_R1-IBR-R2-C357A interacts with UbcH7 as efficiently as HHARI_R1-IBR-R2-WT (Fig. 4d). In contrast, a HHARI-RING1 mutant (I188A²³, analogous to BRCA1-I26A) does not interact detectably with UbcH7 in this assay. Furthermore, C357 is surface accessible and reactive, as HHARI_R2-WT but not HHARI_R2-C357A is readily derivitized by cysteine-modifying reagents (Supplementary Fig. 14).

In the absence of a bona fide substrate, anf in vitro product of HHARI-catalyzed Ub transfer is the non-reducible ubiquitination of UbcH7 (Fig. 4b). Ubiquitination of UbcH7 is E3-dependent as mutation of HHARI_R1-IBR-R2-C357 to serine or alanine abolishes the formation of this product (Fig. 4b). Our finding that HHARI_R1-IBR-R2-C357S forms an oxyester-linked Ub without subsequent transfer of Ub to UbcH7 suggests an ordered mechanism that involves formation of an E3~Ub prior to modification of UbcH7 (or substrates). Thus, HHARI, unlike other RING E3s, does not facilitate direct transfer from an E2~Ub to a target. We note that the HHARI oxyester-linked Ub conjugate accumulates in low yield compared to the available number of active sites. This is consistent with our failure to observe intrinsic serine reactivity for UbcH7, even at serine concentrations as high as 0.5 M (Fig. 1a and data not shown) and suggests that the unique chemical environment surrounding a target residue (in this case an enzyme active site) contributes to catalysis—a contribution that is absent in the nucleophile assay. Our combined results are consistent with HHARI and Parkin functioning via a HECT-like mechanism whereby RING1 harbors the E2-binding site and RING2 harbors the active-site cysteine.

Our characterization of the reactivity of UbcH7 resulted in two unexpected discoveries. First, while E2s known to work with RING-type E3s have E3-independent reactivity toward lysine, UbcH7’s intrinsic reactivity is restricted to cysteine and consequently, its Ub transfer activity is restricted to HECT-type ligases. Second, while confirming reports that UbcH7 is active with E3s in the RBR family, we discovered that these RING-containing E3s function like HECTs in that they require an obligate trans-thiolation step during Ub transfer. Both findings have important implications for guiding our understanding of ubiquitination pathways. RING1 of HHARI does not harbor catalytic activity, and neither HHARI_R1-IBR, nor HHARI_R2 enhance lysine reactivity of UbcH5c (Supplementary Fig. 15). Our results underscore the diversity of structures that facilitate thiol-based Ub transfer, enzymes that include bacterial HECT-like E3s that bear no homology to eukaryotic HECT counterparts²⁹. Knockdown and over-expression studies indicate that UbcH7 regulates S-phase progression into G2, but neither the E3(s) nor targets responsible have been identified¹⁰. Our results suggest that the relevant E3(s) will be found among HECT or RBRs. Although it is possible that UbcH7 cooperates with RINGs such as BRCA1 to modify substrate cysteines, we have not observed such species.

Among human E2s, only five residue types are found at the position analogous to UbcH5c-D87: aspartate, serine, asparagine, glutamate, and proline. The tolerance for asparagine and serine at position 87 suggests that the negative charge of D87 may not be critical for its role in catalysis, but instead a hydrogen-bonding function seems likely, possibly interacting with the conjugated Ub. Of human E2s, only UbcH7 and UbcH8 have a proline at position 87. Although we did not test UbcH8 for lysine reactivity, it functions primarily with the HECT-type ligase HERC5 to transfer the Ub-like protein ISG15 to substrates³⁰. Like UbcH7, we anticipate UbcH8 activity will be limited to HECT or RBR E3s. In conclusion, our effort to understand the mechanism of UbcH7-mediated Ub transfer highlights the predictive power of elucidating E2 mechanisms to understand the E3s with which they function.

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