Facile Preparation of UFMylation Activity-Based Probes by Chemoselective Installation of Electrophiles at the C-Terminus of Recombinant UFM1

Aberrations in protein modification with ubiquitin-fold modifier (UFM1) are associated with a range of diseases, but the biological function and regulation of this post-translational modification, known as UFMylation, remain enigmatic. To provide activity-based probes for UFMylation, we have developed a new method for the installation of electrophilic warheads at the C-terminus of recombinant UFM1. A C-terminal UFM1 acyl hydrazide was readily produced by selective intein cleavage and chemoselectively acylated by a variety of carboxylic acid anhydrides at pH 3, without detriment to the folded protein or reactions at unprotected amino acid side chains. The resulting UFM1 activity-based probes show a range of tunable reactivity and high selectivity for proteins involved in UFMylation processes; structurally related E1s, E2s, and proteases associated with Ub or other Ubls were unreactive. The UFM1 probes were active both in cell lysates and in living cells. A previously inaccessible α-chloroacetyl probe was remarkably selective for covalent modification of the active-site cysteine of de-UFMylase UFSP2 in cellulo.


Safety statement
No unexpected or unusually high safety hazards were encountered.

General reagent information
Chemical reagents were purchased from Sigma Aldrich (Buchs, Switzerland), Acros Organics (Geel, Belgium), TCI Europe (Zwijndrecht, Belgium) and were used without further purification.
CH2Cl2, DMF, EtOH and MeOH were purchased from Fisher Scientific (Geel, Belgium) and Sigma Aldrich (Buchs, Switzerland) and used without further purification (reagent or HPLC grade). Milli-Q water was obtained from a Millipore purification system. Q5 High-Fidelity DNA

Gel electrophoresis
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on a Mini-PROTEAN Tetra Cell system (Bio-Rad) connected to the PowerPac Basic (Bio-Rad power supply. Reducing samples were treated with sample buffer (Laemmli 2x or 4x Concentrate, Sigma Aldrich), heated to 95 °C for 5 min and used for separation. A 10-180 kDa pre-stained protein ladder (Thermo Fisher) was applied to at least one well of each gel (3 μL).

Reverse-phase high-performance liquid chromatography (RP-HPLC)
RP-HPLC was carried out on Jasco analytical instrument with simultaneous monitoring of the eluent at 220 nm, 254 nm, and 301 nm at room temperature (rt). The mobile phase was Milli-Q water with 0.1% TFA (solvent A) and HPLC-grade MeCN with 0.1% TFA (solvent B).
Shiseido C18 (5 μL, 4.6 mm I.D. x 250 mm) column or Agilent Eclipse XDB-C8 (5 μm, 4.6 mm x 150 mm) at a flow rate of 1 mL/min, was used with the following method: 20% solvent B for 3 min, 20 to 95% solvent B over 14 min, and 95% solvent B for 3 min. acetonitrile with 0.1% formic acid. The gradient started at 2% of B and was linearly increased to 35% in 120 min at flowrate of 300 μl min −1 . A second gradient profile, started at 35% of B and was linearly increased to 95% in 2 min at flowrate of 300 μl min −1 . Followed by isocratic conditions of 95% B at flowrate of 300 μl min −1 for 8 min. Total run time, including the conditioning of the column to the initial conditions, was 163 min. Further data processing was performed with Data Analysis 5.3 software (Bruker Daltonics, Germany) using a processing script to generate export files and reports. Advanced raw data processing for bioanalytical interpretations were performed using PEAKS Studio software (Bioinformatics Solutions Inc., Canada).

CD-measurements
Retention of UFM1 structural integrity was confirmed by circular dichroism spectroscopy (CD). UFM1-hydrazide 1 and UFM1-chloroacetyl 2 were buffer exchanged to PBS and diluted to approximately 20 μM final concentration. Circular dichroism was measured using a JASCO J-1500 Spectrometer at 20 °C. CD spectra were recorded ranging from 300 to 180 nm at a scan rate of 20 nm per minute and a scan width of 0.5 nm using a quartz cuvette with a 1 mm path length. Measurements were plotted using Graphpad PRISM.

General procedure for synthesis of anhydrides 10, 11, 12
To a solution of carboxylic acid (0.075 mmol, 1.0 equiv.) in CH2Cl2 at 0°C was added dicyclohexylcarbodiimide (0.037 mmol, 0.49 equiv) and the solution was stirred for 30 min. The precipitate was filtered off and resulting solution was concentrated under reduced pressure to afford symmetrical anhydrides 10, 11, 12. Anhydrides were further used for protein modification without additional purification.  Supplementary Figures 1 -6

Electroporation experiments
FLAG-UFM1 variants were delivered into HEK293T-dCas9-KRAB-sgUFM1 cells using a Lonza 4D-Nucleofector according to manufacturer's protocol with slight modifications. Briefly, 15 M cells were mixed in 120 μl of Lonza SF buffer and 7.5 nmol of each FLAG-UFM1 variant.
The mixture was then equally split into two nucleofection cuvettes and nucleofected using preprogrammed HEK293T code on the Lonza 4D-nucleofector. Cells were then transferred into a 15 cm plate for 24 h recovery. The next day, cells were harvested and lysed in 500 μl of RIPA buffer on ice for 10 min. Cells were spun at 20 x g for 5 min at 4 °C to remove cell debris and nuclei. 450 μl of lysates were transferred to a screw cap tube and SDS was added to a final concentration of 2% and boiled at 95 °C for 5 min for denaturation. The denatured lysates were then transferred to a 15 ml falcon tube and 9.5 ml of RIPA buffer was added to dilute the SDS to a final 0.1% before FLAG-immunoprecipitation.

Immunoprecipitation on anti-FLAG beads
For each FLAG-IP, 100 μl Pierce Anti-DYKDDDDK Magnetic Agarose Beads were preequilibrated with RIPA lysis buffer. Cell lysates with nucleofected probes 2 and 6 were incubated with anti-FLAG magnetic beads for 1 h at room temperature. Samples were washed twice with 1 ml of high-salt RIPA lysis buffer, followed by a final wash with 1 ml regular RIPA lysis buffer. The samples were then prepared for on-bead digestion and tandem mass spectrometry analysis. Samples were visualized with Western Blot against UFM1 and against UFSP2.

On-bead digestion
Beads were washed twice with 100 μl of digestion buffer (10 mM Tris, 2 mM CaCl2, pH 8.2) and the wash was discarded. The samples were resuspended in 45 μl of 10 mM Tris, 2 mM Cysteine residues were searched with a variable modification for carboxyamidomethylation (+57.02 C), methionine oxidation was searched as a variable modification (+15.99 M).

SDS-PAGE analysis of UFM1 probes in vitro crosslinking with UBA1 and UBA5
Supplementary Figure 8. Coomassie-stained SDS-PAGE analysis for in vitro crosslinking of UFM1 C-terminal probes 2 -6 with E1s: a, UBA1 (Ub) and b, UBA5 (UFM1). Cropped gel is shown in Figure 4A in the main text.

SDS-PAGE analysis of UFM1 probes in vitro crosslinking with UFC1
Supplementary Figure 10. Coomassie-stained SDS-PAGE analysis for in vitro crosslinking of UFM1 C-terminal probes 2 -4 with UFC1 C116A and UFC1 wt. Cropped gel is shown in Figure 4B in the main text.