br Substoichiometric substrate modification E
Substoichiometric substrate modification E3 ligases catalyze the rapid transfer of the SUMOD from the E2 enzyme to the substrate. By definition, enzymes are recycled in the reaction, allowing multiple rounds of substrate modification by a single enzyme. Thus, enzymes function at substoichiometric amounts relative to the substrate (low enzyme to substrate ratio) in a concentration- and time-dependent manner. To monitor sumoylation in vitro we use purified recombinant enzymes following the protocols described in detail by the Melchior lab (Werner, Moutty, Moller, & Melchior, 2009). Ideally, all assay components are purified from Escherichia coli as bacteria lack a SUMO system, thus avoiding copurification of SUMO E3 ligases or proteases which at undetectable concentrations could affect the outcome of the in vitro reaction. Any known and putative SUMO substrate can be tested in this system if can be purified. All known SUMO E3 ligases show some substrate specificity in vitro, but they usually have broad substrate spectra; this is especially when working with a truncated E3 ligase (Cox et al., 2015; Koidl et al., 2016; Pichler et al., 2004). Here, we use GST-Sp100 as model substrate as it is a promiscuous substrate that can be sumoylated by all known classes of SUMO E3 ligases as well as by the sumoylated E2 (S*E2) without an E3 (Knipscheer et al., 2008; Koidl et al., 2016; Pichler et al., 2002, Pichler et al., 2004; Sternsdorf, Jensen, & Will, 1997). For in vitro sumoylation reactions, all components can simply be added together in a test tube with ATP and Mg and incubated for up to 30min in a multiturnover reaction. Detection of sumoylation is performed by immunoblotting using substrate-specific antibodies. We use anti-GST Hyperoside mg that detect the N-terminally fused GST-tag of Sp100, and thus we can rule out that sumoylation interferes with detection. Because the E2 enzyme directly recognizes a SCM, it can modify substrates in vitro at high enzyme concentrations as we illustrate in Fig. 1A. Hence, it is important to use a low E2 concentration in E3-dependent reactions that either show no or only marginal substrate modification by the E2 alone. At high enzyme concentrations, the mammalian E2 itself gets sumoylated at Lys 14 (S*E2). We have shown that this particular modification stabilizes the E2 interaction with GST-Sp100 due to a SIM in the substrate that is in close proximity to the SCM. The E2*S adduct therefore also enhances GST-Sp100 modification to a degree comparable to some E3 ligases (Knipscheer et al., 2008) (Fig. 1A). However, E3 ligases are usually more potent and promote higher sumoylation rates when at low (substoichiometric) levels; this can be seen both by varying E3 concentrations (Fig. 1B) and by following sumoylation over time (Fig. 1C).
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 classes 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).