Generation and Characterization of Site‐Specifically Mono‐Ubiquitylated p53

Abstract The tumor suppressor p53 is regulated by various posttranslational modifications including different types of ubiquitylation, which exert distinct effects on p53. While modification by ubiquitin chains targets p53 for degradation, attachment of single ubiquitin moieties (mono‐ubiquitylation) affects the intracellular location of p53 and/or its interaction with chromatin. However, how this is achieved at the molecular level remains largely unknown. Similarly, since p53 can be ubiquitylated at different lysine residues, it remains unclear if the eventual effect depends on the position of the lysine modified. Here, we combined genetic code expansion with oxime ligation to generate p53 site‐specifically mono‐ubiquitylated at position 120. We found that mono‐ubiquitylation at this position neither interferes with p53 ubiquitylation by the E3 ligases HDM2 and E6AP in complex with the viral E6 oncoprotein nor affects p53 binding to a cognate DNA sequence. Thus, ubiquitylation per se does not affect physiologically relevant properties of p53.


Expression and purification of p53-120KeK
To test whether oxime ligation is a valid strategy for the generation of mono-ubiquitylated p53 variants, we switched to position 120 of p53. Besides the notion that K120 is known to be ubiquitylated and to contribute to the DNA binding capabilities of p53, we chose this position, because in most cases ACS is not quantitative resulting in a mixture of full-length and truncated versions of the protein of interest. The more N-terminal the truncation occurs, the easier is the separation of the full-length product from the truncated product, e.g. by size exclusion chromatography or, as in this case, by heparin affinity chromatography (as binding of p53 to heparin depends on the presence of its DNA binding domain which comprises approximately residues 100-300). A codon-optimized cDNA encoding human full-length p53 was cloned into a pGEX backbone containing the Pyl-tRNA cassette and an N-terminal His 9 -lipoyl domain (HLD) tag [6] instead of the GST tag. The lysine codon at position 120 was replaced by the amber codon TAG by site-directed mutagenesis. E. coli BL21 DE3 were co-transformed with pGEX-HLD p53-120TAG and pRSF-duet1 containing the AcK-RS tRNA mutant of M. barkeri. [7] A single clone was inoculated in LB medium supplemented with the appropriate antibiotics and cultivated to OD 600 = 1. The pre-culture was diluted in LB medium supplemented with the appropriate antibiotics to OD 600 = 0.1 and cultivated at 37 °C. At OD 600 = 0.3, Ketolysine (KeK) was added to a final concentration of 10 mM, and at OD 600 = 0.6-0.8 expression was induced by adding 1 mM IPTG. Cells were cultivated at 25 °C for 20 h; afterwards cells were harvested by centrifugation. Cells were lysed in 50 mM phosphate buffer pH 8.0, 300 mM NaCl, 0.01 % (v/v) NP-40, 1 μg/mL aprotinin and leupeptin, 100 μM Pefabloc, 10 mM DTT. The lysate was cleared by centrifugation (15,000x g, 4 °C, 20 min) and the supernatant was loaded onto a Ni-NTA column. HLDtagged p53 was eluted with a gradient of 25 column volumes from His-Trap p53 buffer A (50 mM phosphate buffer pH 8.0, 150 mM NaCl, 0.01 % (v/v) NP-40, 2 mM DTT, 20 mM imidazole) to 100 % His-Trap p53 buffer B (50 mM phosphate buffer pH 8.0, 150 mM NaCl, 0.01 % (v/v) NP-40, 2 mM DTT, 1 M imidazole) and dialyzed overnight in p53 dialysis buffer (50 mM phosphate buffer pH 7.2, 150 mM NaCl, 0.01 % (v/v) NP-40, 2mM DTT) at 4 °C in the presence of thrombin (Sigma-Aldrich), as thrombin cleaves between the HLD tag and p53. p53 was further purified by affinity chromatography on a heparin column. p53 was eluted from the heparin column with a gradient over 25 column volumes from 15 % heparin p53 buffer A (50 mM phosphate buffer pH 7.2, 0.01 % (v/v) NP-40, 2 mM DTT) to 100% heparin p53 buffer B (50 mM phosphate buffer pH 7.2, 1 M NaCl, 0.01 % (v/v) NP-40, 2 mM DTT). Fractions containing purified p53 were pooled followed by dialysis against p53 storage buffer (50 mM phosphate buffer pH 7.2, 150 mM NaCl, 0.01 % (v/v) NP-40, 2 mM DTT, 10 % glycerol), flash-frozen with liquid nitrogen and stored at -80 °C.

Expression, purification and functionalization of Ub75C-Keto
The cDNA encoding Ub75C (replacement of glycine codon 75 by cysteine codon) was cloned into pET3a. Ub75C was expressed in E. coli BL21 DE3. A single clone was inoculated in LB medium supplemented with the appropriate antibiotics and cultivated to OD 600 = 1. The pre-culture was diluted in LB supplemented with the appropriate antibiotics to OD 600 = 0.1 and cultivated at 37 °C to OD 600 = 0.5-0.8. Gene expression was induced with 1 mM IPTG, and upon incubation overnight at 25 °C cells were harvested by centrifugation. Cells were resuspended in 20 mM sodium acetate buffer pH 4.5, lysed by sonication, and centrifuged (27,000x g, 4 °C, 20 min). The extract was heated to 70 °C for 20 min. After centrifugation, the supernatant was purified using 1 mL HiTrap SP Sepharose High Performance column (GE Healthcare). Ub75C was eluted using a linear gradient of NaCl from 25 mM to 1000 mM in 25 mM NaOAc, pH 4.0, 1 mm DTT. Ub75C containing fractions were pooled and chromatographed on a HiLoad Superdex 75 pg 26/600 (GE Healthcare) equilibrated with 1x PBS using the ÄKTA purifier FPLC system. Protein was eluted with 1x PBS using an isocratic gradient. Ub75C containing fractions were pooled and concentrated. A 100 µM solution of Ub75C in PBS pH 7.2 was treated with 1 mM TCEP for 30 min at 37 °C. Afterwards, reduced Ub75C was diluted 5-fold with 1x PBS pH 7.2 supplemented with 1000 eq. chloroacetone (Keto) and incubated by shaking for 90 min at 25 °C. To monitor the reaction, 20 µL of the reaction were incubated with 25 eq. fluorescein maleimide in the dark (10 min at room temperature) and analyzed by SDS-PAGE followed by measuring fluorescence intensity on an FLA Imager at 473 nm and by Coomassie blue staining. The reaction mixture was dialyzed against 25 % MeOH/1x PBS pH 7.2, then 2.5 % MeOH/1x PBS pH7.2 and finally against 0.1x PBS pH 7.2. The protein solution was concentrated and stored at -20°C. The molecular mass of Ub75C-Keto was determined by HR-MS.

Oxime Ligation
Incorporation of U1 into ubiquitin did not proceed to completion ( Figure S4 C), resulting in a mixture of truncated Ub (Ub75) and full-length Ub76ONH-Boc. Thus, for oxime ligation, a mixture consisting of Ub75 and Ub76ONH 2 (1 nmol) was mixed with p53-120KeK (20 pmol) in p53 buffer (50 mM phosphate buffer pH 7.2, 150 mM NaCl, 0.01 % (v/v) NP-40, 2 mM DTT) containing 0.5 mM SDS followed by incubation at 25 °C for 20 h (Fig. S4 E) or by 2 freeze-thaw cycles with -20 °C as freezing temperature (Fig. 1). The reaction products were analyzed by SDS-PAGE followed by Coomassie brilliant blue staining or Western blotting against p53 using the anti-p53 antibody DO-1. While the efficiency of oxime ligation was similar for both procedures (incubation at 25 °C, freeze-thaw cycles), the freezethaw cycle protocol was generally used for reasons of time and to exclude the possibility that the extended incubation time at 25 °C does not negatively affect p53 properties. For generation of oxime-linked H1.2-206Ub, 50 µM Ub75C-Keto were mixed with 10 µM H1.2-206U1 and 2 mM SDS at pH 7.0 followed by 3 freeze-thaw cycles. The reaction mixture was analyzed by SDS-PAGE followed by Coomassie brilliant blue staining.

Purification of oxime-linked p53-120-Ub
For purification of p53-120-Ub, the reaction mixture was added to a heparin column (200 µl, Repligen) equilibrated in buffer A (50 mM phosphate buffer pH 7.2, 150 mM NaCl, 0.01 % (v/v) NP-40, 2 mM DTT). The flow-through of the heparin column was loaded onto a Q Sepharose column (50 µL). Upon loading, the column was washed with 5 column volumes of buffer A. Protein was eluted in a step gradient from 200-1000 mM NaCl. Fractions were examined by SDS-PAGE followed by Coomassie brilliant blue staining. As p53 mainly exists in a tetrameric form, the advantage of this procedure is that tetramers consisting mainly of p53-120-Ub subunits can be separated from tetramers mainly S7 consisting of non-modified p53-120KeK, as the latter almost quantitatively bind to heparin (see Fig.  S5).

In vitro ubiquitylation
For in vitro ubiquitylation, 20-50 ng of recombinant p53 variants, 150 ng of baculovirus-expressed E1, 150 ng of UbcH5b, 5 µg of ubiquitin were incubated in the presence or absence of 200 ng Hdm2 or 200 ng of bacterially expressed E6AP and 200 ng GST-E6 in 25 mM Tris-HCl pH 7.5, 1 mM DTT, 2 mM ATP, 4 mM MgCl 2 in a total reaction volume of 30 µL. After incubation at 30 °C for 0 to 90 min, total reaction mixtures were stopped by addition of 5x Laemmli buffer and boiling at 95 °C for 5 min. Samples were analyzed by SDS-PAGE followed by Western blot analysis using an anti-p53 antibody (DO-1). Expression and purification of E1 (UBA1), UbcH5b, Hdm2, E6AP, and a GST fusion protein of the E6 protein of human papillomavirus type 16 were performed as described. [8] EMSA For electrophoretic mobility shift assay (EMSA), a 4 % (v/v) native gel was casted using 10x TBE buffer    Figure S4. Generation of mono-functionalized Ub76ONH-Boc using ACS. A) Ub76ONH-Boc was generated and purified by size exclusion chromatography as described in the Experimental Section. An aliquot of the respective fractions was analyzed by SDS-PAGE followed by Coomassie blue staining. Note that incorporation of U1 did not proceed to completion (see panel C), resulting in a mixture of truncated ubiquitin (Ub75) and full-length Ub76ONH-Boc (Ub75/ONHBoc). B) Schematic of the deprotection of Nε-aminooxy-(tert-butoxycarbonyl)-L-lysine (U1) to Nε-aminooxy-L-lysine using 60 % TFA. C) Deconvoluted ESI-MS spectra of purified protected Ub75/Ub76ONH-Boc (left panel) and unprotected Ub75/Ub76ONH 2 (right panel). Calculated Mass: Ub75 [M+] = 8507.8 Da, Ub76ONHBoc [M+] = 8738.9 Da, Ub76-ONH 2 [M+] = 8637.8 Da. D) Efficiency of oxime ligation is slightly enhanced by increasing amounts of Ub76ONH 2 . p53-120KeK (20 pmol) was mixed with 0.5 mM SDS and different molar excess (1:6.25-1:100) of the Ub76ONH 2 /Ub75 mixture (see right panel in C). Samples were subjected to two freeze-thaw cycles, centrifuged, and subjected to SDS-PAGE followed by Coomassie blue staining. E) Generation of p53-120-Ub by oxime ligation at room temperature. p53-120KeK (20 pmol) was mixed with a 50-molar excess of Ub76ONH 2 in presence of increasing concentrations of SDS as indicated and incubated at 25 °C for 20 h. Reaction products were analyzed by SDS-PAGE followed by Coomassie blue staining. Running positions of p53-120KeK and p53-120-Ub are indicated by an arrow and an asterisk, respectively.

Fig. S5.
Purification of p53-120-Ub via heparin affinity chromatography followed by anion exchange chromatography. A) Upon oxime ligation, the resulting mixture of p53-120-Ub / p53-120KeK was subjected to a heparin column and bound proteins were eluted by a salt gradient from 150 mM to 1 M NaCl. The p53 variants eluted between 280-350 mM NaCl. However, compared to the input (20 %), the ratio between p53-120-Ub to p53-120KeK was reversed. This indicates that p53-120-Ub has a lower affinity to heparin than p53-120KeK and that approximately 50 % of p53-120-Ub did not bind to heparin under the conditions used. B) Upon oxime ligation, the resulting mixture of p53-120-Ub / p53-120KeK was subjected to a Q sepharose column and bound proteins were eluted by a salt gradient from 150 mM to 1 M NaCl. The majority of the p53 variants eluted between 350-400 mM NaCl, and there was no difference in the elution behavior between p53-120-Ub and p53-120KeK. Input represents 2 % of the mixture applied. C) Because of the different binding behavior of p53-120-Ub and p53-120-KeK, upon oxime ligation the mixture of p53 variants was loaded first onto a heparin column. The flow-through (FT) was collected and applied to Q sepharose. Proteins bound to Q sepharose were eluted with 400 mM NaCl ("flow-thr. → Q"). As control, p53-120KeK was subjected to the same purification procedure, showing that in contrast to p53-120-Ub, p53-120KeK bound to heparin almost quantitatively and eluted between 280-350 mM NaCl. 5 % of the respective elution fractions were subjected to SDS-PAGE followed by Coomassie blue staining. Running positions of p53-120KeK and p53-120-Ub are indicated by an arrow and an asterisk, respectively.