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  • br E mediated E discharge E ligases simultaneously interact

    2020-09-16


    E3-mediated E2 discharge E3 ligases simultaneously interact with the substrate and the SUMOD charged E2 enzyme to catalyze the discharge of the thioester-bound SUMOD from the E2 to the substrate. E3 interaction with SUMOD via a SIM results in a closed conformation which is highly reactive and leads to rapid discharge of the thioester bond as it was shown for all three Celastrol synthesis of bona fide SUMO E3 ligases (Cappadocia et al., 2015; Eisenhardt et al., 2015; Reverter & Lima, 2005; Streich & Lima, 2016). By using E1 and E2 enzymes together with ATP, Mg, and SUMO, a SUMOD~E2 thioester is formed. To monitor the discharge of SUMOD from the E2, ATP needs to be hydrolyzed by apyrase (an ATP diphosphohydrolase) to prevent recharging of the E2. As this assay allows only a single round of E2 discharge (called a “single-turnover” assay), it requires much higher enzyme concentrations compared to the “multiturnover” reactions described in Fig. 1 that cycle through multiple rounds of charging and discharging. The major challenge of single-turnover reactions is the ability of the E2 to also be discharged in the absence of an E3, and it discharges within minutes at 30°C. This is likely due to the high E2 enzyme concentration that allows E3-independent modification of the E1, the E2, SUMO, and the substrate. Hence, E3-dependent discharge has to be executed at high E3 concentrations and in a short time frame to visualize SUMOD~E2 discharge and substrate modification (Fig. 2).
    E3–E2 backside interaction The E2 possesses an important regulatory interface which is termed its backside as it is opposite to the catalytic cleft that bears the active-site cysteine forming the thioester with SUMOD. This backside site interacts noncovalently with a scaffold SUMOB and was originally shown to be important for E2-mediated SUMO chain formation in vitro (Capili & Lima, 2007; Knipscheer, van Dijk, Olsen, Mann, & Sixma, 2007). Moreover, it partially overlaps with the E2–E1 interface (Duda et al., 2007) and is required for direct or indirect E2–E3 interactions. SUMOB is essential for the E3 activity of ZNF451 family members (Cappadocia et al., 2015; Eisenhardt et al., 2015) and strongly enhances the activity of Siz/Pias family members (Mascle et al., 2013; Streich & Lima, 2016), whereas RanBP2 directly interacts with the backside of the E2 independent of a SUMO (Pichler et al., 2004; Reverter & Lima, 2005). The requirement of the E2-SUMOB interaction for E3 activity can be tested in vitro by employing a SUMO or E2 mutant which are specifically impaired in this interaction: SUMO2 D62R abolishes, and E2 F22A weakens this particular interaction (Capili & Lima, 2007; Knipscheer et al., 2007). Here we show RanBP2 as an example of an E3 that displays no major defects with either mutant (Fig. 3A, upper panel) as SUMOB is dispensable for its catalytic activity. However, E2 F22A has mild effects on RanBP2’s activity as E2-Phe22 contributes to the larger RanBP2–E2 interface (Pichler et al., 2004; Reverter & Lima, 2005). By contrast, PIAS1 clearly requires the SUMOB-E2 interaction for efficient Sp100 sumoylation (Fig. 3A, lower panel) as does ZNF451 (Eisenhardt et al., 2015; Koidl et al., 2016). At low PIAS1 concentrations, SUMO2 D62R results in minimal GST-Sp100 modification. The E2 F22A mutation only partially interrupts the E2-SUMOB interaction and hence causes a more mild reduction in PIAS1-dependent substrate sumoylation. Of note, Sp100 modification in the absence of an E3 (E2 or S*E2 dependent) remains unaffected (Fig. 3B). In vitro sumoylation assays used here are similar to substrate modification described in Section 2 and also set up in a multiturnover reaction.
    Donor SUMO positioning Initially, E3 ligases were thought to interact simultaneously with the charged E2 enzyme and the substrate to bring them in close distance for an efficient SUMOD transfer. As this definition may better describe a cofactor than an enzymatic activity, the question was raised as to whether E3 ligases also involve a catalytic component. The first evidence in this direction came from the crystallographic analysis of RanBP2 interacting with a donor SUMOD-charged E2 mimic; this revealed that the E3 ligase also binds the modifier SUMOD (Reverter & Lima, 2005). Subsequent biochemical analysis demonstrated that this SIM-dependent interaction is indeed essential for RanBP2’s catalytic activity (Reverter & Lima, 2005). Meanwhile, all three classes of SUMO E3 ligases were shown to depend on this feature that is also called the “closed conformation” as it positions the SUMOD modifier optimally for the nucleophilic attack of the incoming substrate lysine (Cappadocia et al., 2015; Eisenhardt et al., 2015; Reverter & Lima, 2005; Streich & Lima, 2016; Yunus & Lima, 2009).