PMC

Structural basis for recognition of crotonyllysine by Taf14-YEATS G82A. a The ribbon diagram of the Taf14-YEATS G82A:H3K9cr complex. Dashed lines and red sphere represent hydrogen bonds and a water molecule, respectively. b Close up view of the H3K9cr-binding site of Taf14-YEATS G82A. Red dashed lines represent short distances indicative of the aromatic-aliphatic-aromatic π stacking interaction. c A representative HOMO-8 (see also Supplementary Figure )

Engineering the Taf14-YEATS-based reader of H3K9cr. a Surface view of the Taf14-YEATS G82A mutant structure in the apo-state. The side chains of W81, A82, and F62 are shown as green sticks. b Structural overlay of the acyllysine-binding site in the Taf14-YEATS:H3K9bu (lavender/orange) complex and the apo state of Taf14-YEATS G82A (green). c Superimposed ¹H,¹⁵N HSQC spectra of uniformly ¹⁵N-labeled G82A mutant of Taf14-YEATS recorded while the indicated peptides were added stepwise. The spectra are color coded according to the protein:peptide molar ratio. d Binding curves used to determine K_d of taf14-YEATS G82A by tryptophan fluorescence. e Real-time qPCR analysis of various transcripts in the wild-type strain, TAF14 delete strain, and taf14 mutant strains. The mean ± SD are calculated from three biological replicates

AF9-YEATS binds DNA. a Binding affinities of AF9-YEATS and Taf14-YEATS to indicated histone peptides. Values represent the average of three separate experiments with error calculated as the SD between the runs. b Electrostatic potential surface representation of the AF9-YEATS in complex with H3K9cr generated in PyMol (PDB ID 5hjb). Basic residues are labeled. c EMSA with 147-bp 601 DNA (1.88 pmol/lane) incubated with increasing amounts of AF9-YEATS. DNA to protein molar ratio is shown below the gel image. d Superimposed ¹H,¹⁵N HSQC spectra of uniformly ¹⁵N-labeled AF9-YEATS collected upon titration with 601 DNA. The spectra are color coded according to the protein:DNA molar ratio. e–g EMSA assays with 1 pmol/lane H3K9cr-NCP (e, g) or unmodified NCP (f) incubated with increasing amounts of WT AF9-YEATS or F59A/Y78A mutant. Band intensities in (c, e–g) were quantified by densitometry using ImageJ

Bivalent binding of AF9-YEATS is not conserved in Taf14-YEATS. a EMSA with 5 pmol/lane 20 bp dsDNA incubated with increasing amounts of AF9-YEATS. b Binding curves used to determine K_d for the DNA:AF9-YEATS complex by EMSA. The band of free DNA was used for quantification of the complex formation. Binding constants are obtained from duplicate measurements as mean ± standard error. c A schematic of the bivalent interaction of AF9-YEATS with histone H3K9cr and DNA. d Alignment of the YEATS domain sequences: absolutely, moderately, and weakly conserved residues are colored green, orange, and pink, respectively. Positively charged residues in three patches are indicated by blue boxes. The residues of AF9-YEATS mutated in this study are labeled. e Electrostatic surface potential of Taf14-YEATS in complex with the H3K9cr peptide. f EMSA with 1.88 pmol/lane 601 DNA and increasing concentration of Taf14-YEATS

DNA- and H3K9cr-binding sites in AF9-YEATS do not overlap. a, b Analysis of chemical shift perturbations in ¹H,¹⁵N HSQC spectra of AF9-YEATS caused by (a) H3K9cr peptide (1:3 molar ratio) or (b) 601 DNA (1:0.5 molar ratio). P indicates a proline residue. ‘*’ indicates that the residue resonances were unassigned in the apo-state. ‘-’ indicates that the residue resonances were unassigned in the H3K9cr-bound state (BMRB 26060). Dashed line indicates selection cut-off (mean + 3 SD). Residues with CSP above the cut-off value are labeled. c Identification of the H3K9cr peptide- and 601 DNA-binding sites. Residues with CSP above the cut-off value in (a, b) are mapped onto the surface of AF9-YEATS (PDB 5hjb), colored yellow and orange, respectively, and labeled. Residues with no assignments or prolines are colored light gray. d The 60th patch (R61K/K63E/K67E, light pink), 90th patch (K92E/R96E/K97E, light blue), and 130th patch (R133E/K134E/K137E, wheat) mutations are mapped onto the surface of AF9-YEATS. e–g EMSA assays with 1 pmol/lane 601 DNA (e, f) or 1 pmol/lane H3K9cr-NCP (g) incubated with increasing amounts of WT AF9-YEATS or RKK mutant. Band intensities were quantified by densitometry using ImageJ

Structural insight into the selectivity of AF9-YEATS. a Alignment of the YEATS domain sequences: absolutely, moderately, and weakly conserved residues are colored green, orange, and pink, respectively. b Binding affinities of AF9-YEATS to indicated histone peptides. Values represent the average of three separate experiments with error calculated as the SD between the runs. c Structural overlay of the acyllysine-binding sites in the AF9-YEATS:H3K9bu (pink) and AF9-YEATS:H3K9cr (light gray) complexes. Yellow dashed lines represent short distances indicative of the aromatic-amide-aromatic π stacking interaction in AF9-YEATS:H3K9cr. Red dashed lines represent short, <4 Å, distances between the crotonyl alkene group and the aromatic residues. d Superimposed ¹H,¹⁵N HSQC spectra of AF9-YEATS, wt and Y78W mutant, recorded in the presence of increasing concentration of H3K9cr peptide. Spectra are color coded according to the protein:peptide molar ratio. e Representative binding curves used to determine K_d of AF9-YEATS Y78W by tryptophan fluorescence. f The ribbon diagram of the AF9-YEATS Y78W:H3K9cr complex. Red dashed lines represent short, <4 Å, distances between the crotonyl alkene group of H3K9cr and the aromatic residues of the protein

Structural insight into the selectivity of Taf14-YEATS. a Crotonylated lysine (yellow) is sandwiched between W81 and F62 in the complex of Taf14-YEATS with H3K9cr. W81 adopts two conformations, rotamer 1 (light gray) and rotamer 2 (green). b Binding affinities of Taf14-YEATS to the indicated histone peptides, as measured by fluorescence (cr, bu, ac) or NMR (su, hib). Values represent the average of three separate experiments (two for H3K9ac) with error calculated as the SD between the runs. The enhancement in selectivity of Taf14-YEATS to crotonyllysine is comparable to the enhancement in selectivity of other well-recognized epigenetic readers, such as DPFs^,. c Representative binding curves used to determine K_d by tryptophan fluorescence. d Peptide pulldown assays for Taf14-YEATS using indicated histone H3 peptides. e The ribbon diagram of the Taf14-YEATS:H3K9bu complex. Dashed lines and red sphere represent hydrogen bonds and a water molecule, respectively. The YEATS domain is colored lavender and H3K9bu peptide is colored orange. Residues of the YEATS domain involved in the binding of K9bu are labeled. f Structural overlay of the acyllysine binding sites in the Taf14-YEATS:H3K9bu (lavender/orange), Taf14-YEATS:H3K9pr (pink), and Taf14-YEATS:H3K9ac (gray) complexes