Site-Specific Acetylation of the Transcription Factor Protein Max Modulates Its DNA Binding Activity

Chemical protein synthesis provides a powerful means to prepare novel modified proteins with precision down to the atomic level, enabling an unprecedented opportunity to understand fundamental biological processes. Of particular interest is the process of gene expression, orchestrated through the interactions between transcription factors (TFs) and DNA. Here, we combined chemical protein synthesis and high-throughput screening technology to decipher the role of post-translational modifications (PTMs), e.g., Lys-acetylation on the DNA binding activity of Max TF. We synthesized a focused library of singly, doubly, and triply modified Max variants including site-specifically acetylated and fluorescently tagged analogs. The resulting synthetic analogs were employed to decipher the molecular role of Lys-acetylation on the DNA binding activity and sequence specificity of Max. We provide evidence that the acetylation sites at Lys-31 and Lys-57 significantly inhibit the DNA binding activity of Max. Furthermore, by utilizing high-throughput binding measurements, we assessed the binding activities of the modified Max variants across diverse DNA sequences. Our results indicate that acetylation marks can alter the binding specificities of Max toward certain sequences flanking its consensus binding sites. Our work provides insight into the hidden molecular code of PTM-TFs and DNA interactions, paving the way to interpret gene expression regulation programs.


Analytical HPLC-MS analysis
Analytical HPLC were acquired using Thermo Scientific Vanquish HPLC, Mobile phases used are solvent A (0.05% formic acid in water) and solvent B (0.05% formic acid in acetonitrile) and mass spectrometry using Thermo Scientific LCQ Fleet Ion Trap Mass spectrometer.

pH measurements for ligation reactions
All pH values in aqueous 6 M Gn.HCl were determined using a VWR pH meter with a SENTEK electrode.

GGSDSSSESEPEEPQSRKKLRMEAS-K
The Met residue at positions 1, 65 and 148 were replaced with the isologous norleucine (Nle) residue to avoid Met oxidation.

Synthesis of segment 2.1 Cys-Max(53-91)-NHNH2
The synthesis was carried out according to the following scheme: The synthesis of the segment 2.1 Cys-Max(53-91)-NHNH2 was carried out using stepwise Fmoc chemistry SPPS on hydrazide resin (139 mg, loading 0.36 mmol/g, 0.05 mmol scale).The resin was pre-swelled in DMF for 30 min and then transferred to the CSBio automated peptide synthesizer and residues 91-52 added in a stepwise fashion with Fmoc-amino acid (10 equiv., 0.5 mmol, 33.3 mM), using HBTU/HOBt (10 equiv., 0.5 mmol, 33.3 mM) and 0.18 mL DIEA (20 equiv., 1 mmol, 66.7 mM).The coupling was carried out at 60 °C for 15 minutes coupling time.When the synthesis was completed, the peptide resin was washed with DMF (5 mL x 3), MeOH (5 mL x 3), and DCM (5 ml x 3) and dried under vacuum.To remove side chain protecting groups and release the peptide chains, a mixture of TFA/H2O/TIS (95:2.5:2.5, 7 mL for 0.025 mmol scale) was added to the resin which was shaken for 3 h at RT.The resin was removed by filtration and washed with TFA (2 × 1 mL).To precipitate the peptide, the combined filtrate was added dropwise to cold diethyl ether (25 mL for 0.025 mmol resin) followed by centrifugation at 4000 rpm for 7 min.Then, the diethyl ether was decanted, followed by the dissolution of the peptide in 25%
When the synthesis was completed, the peptide resin was washed with DMF (5 mL x 3), MeOH (5 mL x 3), and DCM (5 ml x 3) and dried under vacuum.To remove side chain protecting groups and release the peptide chains, a mixture of TFA/H2O/TIS (95:2.5:2.5, 7 mL for 0.025 mmol scale) was added to the resin which was shaken for 3 h at RT.The resin was removed by filtration and washed with TFA (2 × 1 mL).To precipitate the peptide, the combined filtrate was added dropwise to cold diethyl ether (25 mL for 0.025 mmol resin) followed by centrifugation at 4000 rpm for 7 min.Then, the diethyl ether was decanted, followed by the dissolution of the peptide in 25% acetonitrile/water and lyophilized to get crude Cys-MaxK57Ac(53-91)-NHNH2 (231 mg, 46.4 µmol) as white powder.The crude dry peptide powder was purified by RP-HPLC (Method C described in Section 1.3) affording the product Cys-MaxK57Ac(53-91)-NHNH2 (45 mg, 9.0 µmol, 18% yield based on 0.05 mmol resin) as a white powder.

Synthesis of segment 3.2 MaxK31Ac(1-51)-NHNH2
The synthesis was carried out according to the following scheme: The synthesis of segment 3.2 MaxK31Ac(1-51)-NHNH2 was carried out using stepwise Fmoc chemistry SPPS on hydrazide resin (278 mg, loading 0.36 mmol/g, 0.1 mmol scale).The resin was pre-swelled in DMF for 30 min and then transferred to the CSBio automated peptide synthesizer and residues 51-32 added in a stepwise fashion with Fmoc-amino acid (10 equiv., 1 mmol, 66.7 mM), using HBTU/HOBt (10 equiv., 1 mmol, 66.7 mM) and 0.37 mL DIEA (2 mmol, 0.13 M).The coupling was carried out at 30 °C for 45 S12 minutes coupling time.To the resin was manually coupled Fmoc-Lys(Ac)-OH (5 equiv., 0.5 mmol, 0.23 M) using HATU (5 equiv., 0.5 mmol, 0.23 M) and 0.18 mL DIEA (10 equiv., 1 mmol, 0.46 M) in 2 mL DMF for 1 h.The resin was again transferred to the CSBio automated peptide synthesizer and continued step wise addition of the remaining amino acids in a similar manner till residue 12. Residues 11-1 was added with Fmoc-amino acid (10 equiv., 1 mmol, 66.7 mM) using DIC (10 equiv., 1 mmol, 66.7 mM) and HOBt (10 equiv., 1 mmol, 66.7 mM) in the same way using CSBio automated peptide synthesizer.When the synthesis was completed, the peptide resin was washed with DMF (5 mL x 3), MeOH (5 mL x 3), and DCM (5 ml x 3) and dried under vacuum.To remove side chain protecting groups and release the peptide chains, a mixture of TFA/H2O/TIS (95:2.5:2.5, 7 mL for 0.025 mmol scale) was added to the resin which was shaken for 3 h at RT.The resin was removed by filtration and washed with TFA (2 × 1 mL).To precipitate the peptide, the combined filtrate was added dropwise to cold diethyl ether (25 mL for 0.025     equiv., 0.3 µmol, 2 mM) was then dissolved in the reaction mixture and the pH was adjusted to 6.8 using 1 N NaOH at 0 °C.The mixture was then incubated for 1.5 h at 25 °C and then 75 µL of TCEP (40 equiv., 16.8 µmol, 0.22 M; based on 4.2) in 6 M Gun.HCl, 0.2 M Na2HPO4 buffer at pH 6.8 was added and continued incubating for 30 min at 25 °C.The reaction was monitored using analytical HPLC-MS (Method A described in Section 1.2).The ligation was completed in 2 h.After completion of the reaction, the crude HPLC-MS (Method A described in Section 1.2).The ligation was completed in 2 h.After completion of the reaction, the crude reaction was desalted by pipetting the reaction mixture into a 10 kDa molecular weight cutoff spin filter (Amicon® Ultra-2mL, 10K).The reaction mixture was diluted with a 6 M Gun.HCl, 0.2 M Na2HPO4 buffer (pH 7.2) to 2.0 mL and concentrated to 1.0 mL by Centrifuging the spin filter at 5000 rpm for 15 min.This process was repeated four more times.In the final process, the reaction mixture was concentrated into 400 µL.After that, the reaction mixture was collected by reverse centrifuge and then treated with VA044 (80 µmol, 200 mM), TCEP (0.1 mmol, 250 mM), and L-Glutathione (GSH, 24 µmol, 60 mM) for 12 h.Progress of reaction was monitored by analytical HPLC-MS using Method A (Section 1.2).After the completion of the reaction, purification was carried out using RP-HPLC (Method E described in Section 1.3) affording 1.9 mg (0.11 µmol) of final product MaxK31AcK57Ac as a white powder (37% yield, based on the limiting segment 1).(Method A described in Section 1.2).The ligation was completed in 2 h.After completion of the reaction, the crude reaction was desalted by pipetting the reaction mixture into a 10 kDa molecular weight cutoff spin filter (Amicon® Ultra-2mL, 10K).The reaction mixture was diluted with a 6 M Gun.HCl, 0.2 M Na2HPO4 buffer (pH 7.2) to 2.0 mL and concentrated to 1.0 mL by Centrifuging the spin filter at 5000 rpm for 15 min.This process was repeated four more times.In the final process, the reaction mixture was concentrated into 400 µL.After that, the reaction mixture was collected by reverse centrifuge and then  at pH 6.8 was added and continued incubating for 30 min at 25 °C.The reaction was monitored using analytical HPLC-MS (Method A described in Section 1.2).The ligation was completed in 2 h.After completion of the reaction, the crude reaction was desalted by pipetting the reaction mixture into a 10 kDa molecular weight cutoff spin filter (Amicon® Ultra-2mL, 10K).The reaction mixture was diluted with a 6 M Gun.HCl, 0.2 M Na2HPO4 buffer (pH 7.2) to 2.0 mL and concentrated to 1.0 mL by Centrifuging the spin filter at 5000 rpm for 15 min.This process was repeated four more times.In the final process, the reaction mixture was concentrated into 400 µL.After that, the reaction mixture was collected by reverse

Folding and analysis of Max variants
Synthetic Max variants were dissolved in DMSO (2 mM) and then diluted into 10 mM MES, 150 mM KCl, 1 mM MgCl2, 10% glycerol buffer (pH 6) using an Amicon® Ultra-0.5 mL 3K MWCO spin filtration unit to provide the desired Max variants in 50 μM final concentration in 10 mM MES, 150 mM KCl, 1 mM MgCl2, 10% glycerol buffer (pH 6).The concentrations of Max analogs were determined using a NanoDrop ND-1000 spectrophotometer.
The folded Max analogs were characterized via Size Exclusion Chromatography (SEC) using ÄKTA Pure on a Superdex 75 column.The column was equilibrated with a buffer consisting of 10 mM MES, 500 mM KCl, 100 mM NaCl and 1 mM MgCl2.All Max variants were eluted as a single peak at around 9 ml, which corresponds to the mass of a Max homodimer (~35 kDa).

Figure S2 .
Figure S2.Analytical HPLC-MS analysis of segment 2.1 Cys-Max(53-91)-NHNH2 with the observed mass 4936.7 ± 0.1 Da, calculated mass 4937.6 Da (average isotopes).The UV absorbance was monitored at 214 nm and the m/z data acquired over the marked region in the chromatogram.HPLC-MS analysis was carried out with Method A depicted in section 1.2 (5-50% acetonitrile gradient; 4.5% per min).

Figure S3 .
Figure S3.Analytical HPLC-MS analysis of segment 2.2 Cys-MaxK57Ac(53-91)-NHNH2 with the observed mass 4979.0 ± 0.1 Da, calculated mass 4979.6 Da (average isotopes).The UV absorbance was monitored at 214 nm and the m/z data acquired over the marked region in the chromatogram.HPLC-MS analysis was carried out with Method A depicted in section 1.2 (5-50% acetonitrile gradient; 4.5% per min).

Figure S4 .
Figure S4.Analytical HPLC-MS analysis of segment 3.1 Max(1-51)-NHNH2 with the observed mass 5896.7 ± 0.2 Da, calculated mass 5896.4Da (average isotopes).The UV absorbance was monitored at 214 nm and the m/z data acquired over the marked region in the chromatogram.HPLC-MS analysis was carried out with Method A depicted in section 1.2 (5-50% acetonitrile gradient; 4.5% per min).

Figure S5 .
Figure S5.Analytical HPLC-MS analysis of segment 3.2 MaxK31Ac(1-51)-NHNH2 with the observed mass 5938.1 ± 0.1 Da, calculated mass 5938.4Da (average isotopes).The UV absorbance was monitored at 214 nm and the m/z data acquired over the marked region in the chromatogram.HPLC-MS analysis was carried out with Method A depicted in section 1.2 (5-50% acetonitrile gradient; 4.5% per min).

Figure S13 .
Figure S13.Analytical HPLC of purified T-MaxK31Ac and m/z spectrum with the observed mass 17724.6 ± 1.6 Da, calculated mass 17729.2Da (average isotopes).The UV absorbance was monitored at 214 nm and the m/z data acquired over the marked region in the chromatogram.HPLC-MS analysis was carried out with Method A depicted in section 1.2 (5-50% acetonitrile gradient; 4.5% per min) and mass spectrometer.
centrifuge and then treated with VA044 (80 µmol, 200 mM), TCEP (0.1 mmol, 250 mM), and L-Glutathione (GSH, 24 µmol, 60 mM) for 12 h.Progress of the reaction was monitored by analytical HPLC-MS using Method A (Section 1.2).After the completion of the reaction, purification was carried out using RP-HPLC (Method E described in Section 1.3) affording 1.6 mg (0.09 µmol) of final product T-MaxK31AcK57Ac as a red powder (31% yield, based on the limiting segment 1.1).

Figure S14 .
Figure S14.Analytical HPLC of purified T-MaxK31AcK57Ac and m/z spectrum with the observed mass 17767.8± 0.5 Da, calculated mass 17771.2Da (average isotopes).The UV absorbance was monitored at 214 nm and the m/z data acquired over the marked region in the chromatogram.HPLC-MS analysis was carried out with Method A depicted in section 1.2 (5-50% acetonitrile gradient; 4.5% per min) and mass spectrometer.

Figure S17 .
Figure S17.The size exclusion chromatography (SEC) experiments show all Max variants are homogeneous species with a molecular weight corresponding to Max homodimers (35 kDa).The UV absorbance was monitored at 220 nm.

Figure S19 .
Figure S19.BLI analysis of Max variants with the E-box-1 DNA probe; the association and dissociation curves of proteins wt-Max (a), MaxK31Ac (b), MaxK57Ac (c), and MaxK31AcK57Ac (d) with E-box-1 DNA probe along with the triplicates.

Figure S20 .
Figure S20.BLI analysis of MaxK31AcK57Ac with the E-box-2 DNA probe; the association and dissociation curves of protein MaxK31AcK57Ac with E-box-2 DNA probe along with the triplicates.

Figure S22 .
Figure S22.Consistent trends in 8-mer sequences across all variants.The correlation between the 8mer median intensity values of acetylated Max variants (T-MaxK31Ac, T-MaxK57Ac and T-MaxK31AcK57Ac) and T-wt-Max is similar to the correlation between two independent experiments performed for T-wt-Max.Demonstrating similar binding preferences toward 8-mer cores for all variants.

Figure S23 .
Figure S23.Schematic representation of libraries designed to explore binding differences beyond the core 8-mer motif.(A) We selected three 6-bp-long cores and varied the three neighboring bases on each side to every possible combination, resulting in 4,096 combinations for each of the three cores.(B) To further explore the bases 3 and 4 bases away from the E-box, we chose an extended 10-bp core, and varied positions ±3,4 to all possible options.For each of these options, we varied each possible base in the extended core region.(C) To explore the distal positions 5 and 6, we selected five different 14-mer constant regions, and varied positions +5,6 to all possible combinations.(D) In addition to these sequences, we also included genomic sequences previously shown to be bound by Max,10,11 as well as additional sites not expected to bind Max as a negative control, and additional sequences containing the core motif in various flanking environments.

Figure S24 .
Figure S24.The signal distribution of T-wt-Max and T-MaxK31AcK57Ac for all possible nucleotide combinations at positions +3 and +4 is depicted.In the case of T-MaxK31AcK57Ac (far right), a subgroup with the GG combination (highlighted by a red rectangle) exhibits significantly higher binding signals compared to all other combinations (ttest p-value<0.0001).This observation highlights a distinct preference of T-MaxK31AcK57Ac for certain sequences with GG at these positions.

Figure S25 .
Figure S25.Comparison of the top 5% strongest binders within the genomic binding sites group for (A) T-wt-Max and (B) T-MaxK31AcK57Ac.The positions +3 and +4 are highlighted in red.Notably, while no GG motif is present in these positions for WT-Max (A), the top binders of MaxK31AcK57Ac (B) are enriched with GG, constituting approximately 27% of the sequences.