Research ArticleBIOCHEMISTRY

Deciphering and engineering chromodomain-methyllysine peptide recognition

See allHide authors and affiliations

Science Advances  07 Nov 2018:
Vol. 4, no. 11, eaau1447
DOI: 10.1126/sciadv.aau1447
  • Fig. 1 Pipeline of the chromodomain-peptide interaction screening experiments.

    (A) Workflow of comprehensive screening for chromodomain recognition profiling. The screened peptides (combinatorial modification library) were composed of 153 possible single trimethylated histone peptides in the human proteome and 72 histone peptides that include up to three modifications at sites K4, K9, K27, K36, K56, and K79 of the H3 histone protein and 232 non-histone peptides in the human proteome (selected by various filters) that are likely bound by the chromodomains [proteome-wide methylation filters; see the Supplementary Methods and Materials and details in (19, 20)]. The peptides were printed on a microarray to test their binding to 29 human chromodomains (peptide microarray screening of chromodomain). The chromodomain-peptide recognition specificity can be predicted using a quantitative model (chromo-methyl recognition model) that captures the structural and energetic features at the binding interface (complex structure construction/MD, MIEC-SVM model, and feature selection). (B) Chromodomain-peptide binding intensities on the microarray (shown as z scores; red, binding; green, nonbinding). The z scores were calculated by standard normalization using all the signal intensities for each chromodomain. For any data point x, z score = (x − mean)/SD. Three categories of chromodomains and six clusters of peptides with different chromodomain binding preferences (the size and the composition of peptide sequences for each peptide cluster are shown on the right) were identified by hierarchical clustering. (C) Literature comparison of H3K9me3 and H3K27me3 z scores to reported Kd values (in μM) of interactions of the CBX chromodomains. Green color indicates consistent with previous study, and yellow is inconsistent.

  • Fig. 2 Chromodomain binding sequence motif analysis from the peptide array screens.

    (A) No significant sequence motif was observed in the sequences for each peptide cluster. Peptides were aligned such that methylated lysine was set as the eighth amino acid position. (B) Alignment of sequence motifs for each individual chromodomain shows a lack of any strong binding motif, consistent with (A).

  • Fig. 3 Construction and validation of the chromodomain MIEC-SVM model.

    (A) Flowchart of MIEC-SVM that predicts binding specificity between chromodomains and methyllysine peptides. Complex structures between 13 chromodomains and 457 peptides were constructed by computationally mutating peptide sequence from a template complex for each chromodomain (virtual mutagenesis). From the modeled complex structures, MIEC terms between peptide-protein residues at the binding interface were computed. The MIECs and the binding/nonbinding label (obtained from microarray experiments) for each domain-peptide pair were input to a LASSO logistic regression model to select most predictive MIECs (LASSO feature selection). These selected MIEC features were then used to train an SVM model to discriminate binding from nonbinding events. VDW, Van der Waals forces; ELE, electrostatic forces; GB, polar contribution to the desolvation energy; SA, nonpolar contribution to the desolvation energy. (B) Performance of MIEC-SVM model on three different peptide groups (all peptides, singly modified peptides, and multiply modified peptides). The MIEC-SVM model showed consistent performance regardless of the number of modifications on the peptides, indicating that chromodomain-peptide recognition share the same MIEC features for singly and multiply modified peptides. (C) SVM decision value distribution of the four classes of peptides (binders/nonbinders with single or multiple modifications). Binders and nonbinders are well separated regardless of the modification number. (D) Pairwise Jensen-Shannon (JS) divergences between the SVM decision value distributions of the four classes. The differences between any binder class and nonbinder class (regardless of the PTM number) are large (larger JS divergence value) singly modified binder–singly modified nonbinder, JS = 0.468 (P < 1.0 × 10−20); singly modified binder–multiply modified nonbinder, JS = 0.396 (P < 1.0 × 10−19); multiply modified binder–single modified nonbinder, JS = 0.704 (P < 1.0 × 10−20); and multiply modified binder–multiply modified nonbinder, JS = 0.603 (P < 1.0 × 10−20). In contrast, binder (or nonbinder) peptides are similar to each other regardless of the PTM numbers: JS values of 0.113 for binders (P = 7.0 × 10−15 for statistical similarity) and 0.027 for nonbinders (P = 6.1 × 10−10). All P values were calculated on the basis of the background distributions of JS divergence of randomly selected decision values for the same number of binders or nonbinders as the foreground.

  • Fig. 4 Application of the chromodomain MIEC-SVM model to engineering the CBX1 chromodomain.

    (A) Selection of key sites to randomize for the yeast display experiment with the CBX1 chromodomain. Sites “A” and “B” are just two representative residues being analyzed for site selection for demonstration purposes. The other strategy of selecting residues to randomize on CBX1 (comparing H3K27me3 to CBX1 binders) used the same procedure. (B) Kd values obtained from fluorescence polarization binding studies between the WT CBX1 and MPP8 chromodomains, along with the V22E mutants isolated from yeast surface display selections. The Kd values were derived from a nonlinear regression equation after performing experiments in triplicate against 1 nM H3K9me3 [NH2-ARTKQTARK(me3)STGG-mini-PEG-K(5-fam)-NH2] and 1 nM H3K27me3 [Ac-QLATKAARK(me3)SAPA-mini-PEG-K(5-fam)-NH2] peptides. Kd values toward H3K27me3 are based on visual approximation from unsaturated binding curves; for the V22E mutants, up to 180 μM of each protein was used in an attempt to get a binding curve to H3K27me3. (C) Peptide array screening of the V22E/K25E/D59S CBX1 chromodomain mutant. An Active Motif histone peptide array, containing 384 peptide spots printed in duplicate, was screened against 1 nM GST-CBX1 mutant. Spots were visualized by chemiluminescence, and the spot intensities were analyzed by Active Motif array analysis software. The height of the y axis (specificity factor) represents the ratio of the average intensity of all array spots containing the mark (listed on the x axis) over the average intensity of spots not containing the mark. (D) Left: Representative reconstructed PALM image of HeLa cells (top row) and MEF cells (bottom row) transiently transfected with CBX1 (V22E/K25E/D59S)–PAmCherry (left) WT CBX1-PAmCherry (center), or immunostained with Alexa 647–labeled anti-H3K9me3 antibody (right). Scale bars, 10 μm. Right: Quantification of fraction of PALM localizations located within the nuclear region for HeLa cells (top) and MEF cells (bottom) (unpaired two-tailed t test). Mut: V22E/K25E/D59S triple mutant.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/11/eaau1447/DC1

    Supplementary Methods and Materials

    Fig. S1. Hierarchical clustering of chromodomains based upon z scores.

    Fig. S2. Two-dimensional hierarchical clusters of peptides binding to chromodomains based on z scores (as separate PDF file).

    Fig. S3. Multiple sequence alignment of chromodomains.

    Fig. S4. Getis-Franklin single-molecule coclustering analysis (35) of H3K9me3 and the CBX1 (V22E/K25E/D59S) chromodomain.

    Table S1. List of chromodomains screened by microarray.

    Table S2. Four hundred sixty-seven peptides printed on the histone microarray (as separate Excel file).

    Table S3. Sequence and averaged signal intensities of identified binders from the peptide microarray for all 29 chromodomains (as separate Excel file).

    Table S4. Receptor-ligand residue pairs after LASSO feature selection (as separate Excel file).

    Table S5. Nested cross-validation performed to evaluate overfitting in the training process (as separate Excel file).

    Table S6. List of ranked sites for the CBX1 chromodomain that were considered for randomization (as separate Excel file).

    Movie S1. WT CBX1-PAmCherry in MEF cells.

    Movie S2. V22E/K25E/D59S CBX1-PAmCherry in MEF cells.

    Movie S3. WT CBX1-PAmCherry in HeLa cells.

    Movie S4. V22E/K25E/D59S CBX1-PAmCherry in HeLa cells.

    References (3645)

  • Supplementary Materials

    The PDF file includes:

    • Supplementary Methods and Materials
    • Fig. S1. Hierarchical clustering of chromodomains based upon z scores.
    • Legend for fig. S2
    • Fig. S3. Multiple sequence alignment of chromodomains.
    • Fig. S4. Getis-Franklin single-molecule coclustering analysis (35) of H3K9me3 and the CBX1 (V22E/K25E/D59S) chromodomain.
    • Table S1. List of chromodomains screened by microarray.
    • Legends for tables S2 to S6
    • Legends for movies S1 to S4
    • References (3645)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Fig. S2. Two-dimensional hierarchical clusters of peptides binding to chromodomains based on z scores (as separate PDF file).
    • Table S2. Four hundred sixty-seven peptides printed on the histone microarray (as separate Excel file).
    • Table S3. Sequence and averaged signal intensities of identified binders from the peptide microarray for all 29 chromodomains (as separate Excel file).
    • Table S4. Receptor-ligand residue pairs after LASSO feature selection (as separate Excel file).
    • Table S5. Nested cross-validation performed to evaluate overfitting in the training process (as separate Excel file).
    • Table S6. List of ranked sites for the CBX1 chromodomain that were considered for randomization (as separate Excel file).
    • Movie S1 (.avi format). WT CBX1-PAmCherry in MEF cells.
    • Movie S2 (.avi format). V22E/K25E/D59S CBX1-PAmCherry in MEF cells.
    • Movie S3 (.avi format). WT CBX1-PAmCherry in HeLa cells.
    • Movie S4 (.avi format). V22E/K25E/D59S CBX1-PAmCherry in HeLa cells.

    Files in this Data Supplement:

Navigate This Article