Insights Into the Allosteric Inhibition of the SUMO E2 Enzyme Ubc9

Abstract

Conjugation of the small ubiquitin-like modifier (SUMO) to protein substrates is an important disease-associated posttranslational modification, though few inhibitors of this process are known. Here, we report the discovery of an allosteric small molecule binding site on Ubc9, the sole SUMO E2 enzyme. An X-ray crystallographic screen was used to identify two distinct small molecule fragments that bind to Ubc9 at a site distal from its catalytic cysteine. These fragments and related compounds inhibit SUMO conjugation in biochemical assays with potencies of 1.9-5.8 mM. Mechanistic and biophysical analyses, coupled with molecular dynamics simulations, point toward ligand-induced rigidification of Ubc9 as a mechanism of inhibition.

A Small-Molecule Microarray Approach for the Identification of E2 Enzyme Inhibitors in Ubiquitin-Like Conjugation Pathways

“…In addition to wildtype enzyme, two Ubc9 mutants (K59A and E42A) that disrupt a recently identified allosteric site were evaluated. 6 Compound 2 was able to inhibit SUMO conjugation to the fluorescent peptide substrate for all three enzymes without any apparent change in potency (Suppl. Fig.…”

“…The in vitro sumoylation assay was performed in 20 μL reaction buffer (50 mM Tris [pH 9], 5 mM MgCl 2 , 1 mM DTT) containing SUMO E1 (0.1 μM), Ubc9 (0.15 μM), SUMO-1 (His-tag, 1.4 μM), and a fluorescent peptide (FL-AR) substrate 5 (1 μM) incubated with various concentrations of small molecule in DMSO or DMSO alone (4% final concentration). Experiments with mutant Ubc9, obtained as previously described, 6 were conducted according to the same procedure using K59A or E42A Ubc9 in place of the wild-type E2. The reaction was then initiated by the addition of ATP (2 mM final concentration) and incubated at room temperature for 90 min.…”

“…The in vitro sumoylation assay was executed as previously described. 5,6 The assay was performed in a 384-well plate format in 20 μL Tris buffer (50 mM Tris [pH 9], 5 mM MgCl 2 , and 1 mM DTT) with SUMO E1 (0.1 μM), SUMO-1 (His-tag, 1.4 μM), Ubc9 (0.15 μM), fluorescent peptide FL-AR (1 μM), and small molecule (4% DMSO final). For experiments with detergent, either Triton X-100 or Tween 20 (0.01%, v/v) was incorporated into the assay buffer as well.…”

“…5 A more recent fragment–based approach revealed an allosteric small-molecule binding site on Ubc9 that inhibits thioester formation, with fragments having IC 50 values in the low millimolar range. 6 Inhibitors that target the SUMO E1 enzyme, including ginkgolic acid and tannic acid, have also been reported. To date, these existing inhibitors of sumoylation suffer from weak potency in biochemical or cell–based assays, polyphenolic structures, redox activity, pleiotropic mechanisms of action, or a combination of these issues.…”

Allosteric network in Ube2T drives specificity for RING E3 catalysed ubiquitin signals

“…Biochemical, structural and computational studies have revealed how RING/U-box E3s stimulate internal dynamics in different E2s that are linked to their ubiquitination activity (Benirschke et al, 2010; Chakrabarti et al, 2017; Das et al, 2013; Ozkan et al, 2005). Furthermore, recent efforts in fragment-based inhibitor discovery have revealed promising lead compounds that bind Ube2T (Morreale et al, 2017) as well as the unrelated Ube2I (Hewitt et al, 2016) at regions distal from their active/E3 binding sites, nevertheless can allosterically regulate the respective E2 activities. These observations collectively suggest that allosteric networks could operate across the E2 family and that future investigations into such networks could prove instrumental for basic and translational research in ubiquitin biology.…”