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. 2020 Apr 9:8:241.
doi: 10.3389/fcell.2020.00241. eCollection 2020.

A Degenerate Peptide Library Approach to Reveal Sequence Determinants of Methyllysine-Driven Protein Interactions

Ariana Kupai  1 , Robert M Vaughan  1 , Bradley M Dickson  1 , Scott B Rothbart  1
Affiliations

A Degenerate Peptide Library Approach to Reveal Sequence Determinants of Methyllysine-Driven Protein Interactions

Ariana Kupai et al. Front Cell Dev Biol. .

Abstract

Lysine methylation facilitates protein-protein interactions through the activity of methyllysine (Kme) "reader" proteins. Functions of Kme readers have historically been studied in the context of histone interactions, where readers aid in chromatin-templated processes such as transcription, DNA replication and repair. However, there is growing evidence that Kme readers also function through interactions with non-histone proteins. To facilitate expanded study of Kme reader activities, we developed a high-throughput binding assay to reveal the sequence determinants of Kme-driven protein interactions. The assay queries a degenerate methylated lysine-oriented peptide library (Kme-OPL) to identify the key residues that modulate reader binding. The assay recapitulated methyl order and amino acid sequence preferences associated with histone Kme readers. The assay also revealed methylated sequences that bound Kme readers with higher affinity than histones. Proteome-wide scoring was applied to assay results to help prioritize future study of Kme reader interactions. The platform was also used to design sequences that directed specificity among closely related reader domains, an application which may have utility in the development of peptidomimetic inhibitors. Furthermore, we used the platform to identify binding determinants of site-specific histone Kme antibodies and surprisingly revealed that only a few amino acids drove epitope recognition. Collectively, these studies introduce and validate a rapid, unbiased, and high-throughput binding assay for Kme readers, and we envision its use as a resource for expanding the study of Kme-driven protein interactions.

Keywords: functional proteomics; lysine methylation; lysine-orientated peptide libraries; non-histone proteins; reader domains.

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Figures

FIGURE 1
FIGURE 1
Kme-OPL assay overview. (A) Design of Kme-OPL. (B) Schematic of assay workflow. (C) Cartoon of the magnetic bead pulldown assay developed for screening the activities of Kme readers. PEG, polyethylene glycol.
FIGURE 2
FIGURE 2
Preferred methyl orders for histone Kme readers are recapitulated with Kme-OPL. (A–C) GST-subtracted fluorescent signals from Kme-OPL pools reacted with the indicated wild-type or mutant Kme reader domains. Error bars are SD from replicate binding reactions (black dots). A biotinylated H3(1–20)K9me2 peptide was included in B.
FIGURE 3
FIGURE 3
Binding determinants of MPP8 chromo and validation of Lowest Bin (LoB) scoring. (A) MPP8 chromo co-structure with an H3K9me3 peptide (PDB:3QO2). (B) MPP8 chromo Kme3-OPL profile. Each Kme3-OPL set is shown in the heatmap as an average of 4 replicate GST subtracted fluorescence measurements, and data is scaled from 0 (no signal, blue) to 1 (highest average signal, red). (C) MPP8 chromo specificity profile. Specificity is graphed as the number of Kme3-OPL sets with a given signal intensity range. (D) MPP8 chromo fluorescence polarization. Data points are plotted as an average of 4 measurements from 2 independent experiments. Error is SD (E) Scatterplot of normalized MPP8 LoB scores from data with GST subtraction vs without GST subtraction. Red, green, and blue points correspond to same colored peptides as in F. (F) Fluorescence polarization of MPP8 chromo. Data points are plotted as an average of 5 replicate measurements from 2 independent experiments. Error is SD.
FIGURE 4
FIGURE 4
Binding determinants of CDYL2 chromo. (A) CDYL2 chromo Kme3-OPL profile. Each Kme3-OPL set is shown in the heatmap as an average of 4 GST subtracted fluorescence measurements from 2 independent binding reactions. Data is scaled as in Figure 3B. (B) CDYL2 chromo specificity profile. (C) CDYL2 chromo fluorescence polarization. Data points are plotted as an average of 4 measurements from 2 independent experiments. Error is SD (D) Quantification of CDYL2 signal from western blots of biotinylated-peptide pulldown from HEK293 cell lysates (see also Supplementary Figure S5C). Quantified signal from each lane was normalized to input signal from the same blot. Error bars are SD.
FIGURE 5
FIGURE 5
Promiscuity in Kme binding is recognized by Kme-OPL. (A) L3MBTL3 3xMBT Kme2-OPL profile. Two independent binding measurements with 2–3 technical replicate measures for each Kme2-OPL set were averaged. Data was GST background subtracted and normalized as in Figure 3B. (B) L3MBTL3 3xMBT specificity profile. (C) 53BP1 TTD histone peptide microarray data. Data points were normalized to the highest peptide signal, and error is SEM (D) 53BP1 TTD Kme2-OPL profile. Two independent binding measurements with 2 technical replicate measures for each Kme2-OPL set were averaged. Data was GST subtracted and normalized as in Figure 3B. (E) 53BP1 TTD specificity profile.
FIGURE 6
FIGURE 6
Kme-OPL reveals few sequence drivers for Kme-specific antibody target recognition. (A) Histone peptide microarray data. Signals were normalized to the highest peptide average per subarray. On target is defined as a peptide that contains the intended methyllysine mark. (B) Kme-OPL pools. Bar graphs are an average of 4 replicate measurements. Error is SD (C) Kme-OPL profiles. Profiles are an average of 4 fluorescent binding measurements. ab8895 is 2 individual experiments while all others are replicates from one experiment. Data was normalized as in Figure 3B. (D) Histone Kme antibody specificity profiles. Millipore 07-449 LOT: 2455635 dilution 1:5,000, CST 9733 LOT:14 dilution 1:5,000. Active Motif #39161 LOT: 14418003 dilution 1:5,000. ab8895 LOT: GR141677-4 dilution 1:10,000. ABclonal A2355 combination of LOT: 2200170102 and 2200170202 dilution 1:5,000.
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References

    1. Alix J.-H., Hayes D., Lontie J.-F., Colson C., Glatigny A., Lederer F. (1979). Methylated amino acids in ribosomal proteins from Escherichia coli treated with ethionine and from a mutant lacking methylation of protein L11. Biochimie 61 671–679. 10.1016/s0300-9084(79)80165-0 - DOI - PubMed
    1. Ambler R. P., Rees M. W. (1959). ε-N-Methyl-lysine in Bacterial Flagellar Protein. Nature 184 56–57. 10.1038/184056b0 - DOI - PubMed
    1. Bannister A., Zegerman P., Allshire R., Kouzarides T. (2001). Selective recognition of methylated lysine 9 on histone H3 by the HP1chromo domain. Nature 410 120–124. 10.1038/35065138 - DOI - PubMed
    1. Barnash K. D., Lamb K. N., Stuckey J. I., Norris J. L., Cholensky S. H., Kireev D. B., et al. (2016). Chromodomain Ligand Optimization via Target-Class Directed Combinatorial Repurposing. ACS Chem. Biol. 11 2475–2483. 10.1021/acschembio.6b00415 - DOI - PMC - PubMed
    1. Botuyan M. V., Lee J., Ward I. M., Kim J.-E., Thompson J. R., Chen J., et al. (2006). Structural basis for the methylation state-specific recognition of histone H4-K20 by 53BP1 and Crb2 in DNA Repair. Cell 127 1361–1373. 10.1016/j.cell.2006.10.043 - DOI - PMC - PubMed

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