UbFluor: a mechanism-based probe for HECT E3 ligases

UbFluor is a mechanism-based probe that undergoes a direct transthiolation reaction with the catalytic cysteine of the model HECT E3 ligase Rsp5. We show that UbFluor can be utilized to conduct high-throughput screens (HTS) of small molecules against HECT ligases.


Supporting Data
. Domain map of Rsp5. We used the ∆WW Rsp5 construct and Rsp5 HECT construct in this study. The ∆WW Rsp5 construct used in this work and in our previous study was found to harbour the mutations N463D and F512S (numbered according to full length Rsp5). 1 To the best of our knowledge, these mutations do not affect the catalytic mechanism. These mutations were corrected during cloning of the HECT domain construct. (C) The reactions of (B) were analysed with -ubiquitin western blot to show that the Rsp5 catalytic cysteine Cys 777 is required to process UbFluor. Mixtures of UbFluor (0 -5 μM), Ub (0 -5 μM), and Fluor-SH (0 -5 μM) were incubated in 150 mM NaCl, 50 mM HEPES pH 7.5, 6 μM Tween-20, and 0.5 mM TCEP. The solutions were loaded to wells in a 384 well low volume, low binding plate (Corning 3820). Fluorescence polarization for each solution was read on the Synergy4 plate reader. The solutions were loaded to wells in a 384 well low volume, low binding plate (Corning 3820).
Fluorescence polarization was read on the Synergy4 plate reader over 2 minutes and averaged.
Sensitivity was set to 46. (C) ∆WW Rsp5 (       Cys 777 is highlighted in yellow, and the conserved Rsp5 Asn 779 is highlighted in green. N779 is shown in an Rsp5 crystal structure. PDB ID: 3OLM. 3 The involvement of N779 in the E2~Ub transthiolation reaction was not known before our study.  (D) With UBE2K, chase performed at room temperature. ** indicates a fluorescent Ub adduct that was built independent of the Rsp5 HECT catalytic cysteine. * indicates UBE2W~Ub-Ub.
Cognate and non-cognate E2 enzymes were selected according to Sheng,et al. 4 Graphs in (A-D) have plotted mean  SEM from 3 separate reaction trials run on separate gels. Note: the fluorescent ubiquitin used in these assays is not UbFluor.

General Information
UbE2W and UBE2K were purchased from R&D Systems. Sodium 2-mercaptoethanesulfonate (MESNa) and wild-type ubiquitin from bovine erythrocytes were purchased from Sigma-Aldrich. These purchased proteins and chemicals were used without further purification. In-gel fluorescence scanning was performed using the Typhoon 9400 (GE Healthcare  4, 129.8, 128.6, 127.4, 67.7, 36.9, 29.4.

Ubiquitin-Fluor-SH conjugation
Lyophilized Fluor-SH was dissolved in a few milliliters of methanol and transferred to a tared 20 mL glass scintillation vial. The majority of methanol was removed with rotovap, and then the

Fluorescence polarization measurement
Fluorescence polarization (FP) was measured using a Synergy4 (BioTek) fluorescence plate reader running Gen5 software (BioTek). All components for a given reaction were added to a 1.5 mL microcentrifuge tube, the appropriate volume of Rsp5 was placed on the wall of the tube, and then the reaction was initiated by centrifuging the tube at 10,000 g for seven seconds. The reaction solution was mixed by pipetting five times and then loaded (20 μL) to a 384-well plate (Corning 3820). Once all the samples were loaded to the plate, the plate was spun at 1,500 g for eight seconds, and then added to the plate reader to record FP. All the reactions contained 150 mM NaCl, 6 μM Tween-20, 0

UbcH7
GST-UbcH7 cloned into the pGEX-6P vector was transformed into E. Coli  UbcH5B was purified from uncleaved enzyme, GST, and thrombin via size exclusion chromatography using a Superdex 75 column (GE Healthcare).