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. 2016 Sep 6;24(9):1606-12.
doi: 10.1016/j.str.2016.05.023. Epub 2016 Aug 18.

Structural Insights into Histone Crotonyl-Lysine Recognition by the AF9 YEATS Domain

Qiang Zhang  1 , Lei Zeng  2 , Chengcheng Zhao  3 , Ying Ju  3 , Tsuyoshi Konuma  4 , Ming-Ming Zhou  5
Affiliations

Structural Insights into Histone Crotonyl-Lysine Recognition by the AF9 YEATS Domain

Qiang Zhang et al. Structure. .

Abstract

Histone lysine acylations play an important role in the regulation of gene transcription in chromatin. Unlike histone acetyl-lysine, molecular recognition of a recently identified crotonyl-lysine mark is much less understood. Here, we report that the YEATS domain of AF9 preferentially binds crotonyl-lysine over acetyl-lysine in histone H3. Nuclear magnetic resonance structural analysis reveals that crotonyl-lysine of histone H3 lysine 18 is engulfed deep in an aromatic cage of the YEATS domain where the carbonyl oxygen of crotonyl-lysine forms a hydrogen bond with the backbone amide of protein residue Tyr78. The crotonyl-lysine, through its unique electron-rich double-bond side chain, engages π-π aromatic stacking and extended hydrophobic/aromatic interactions with the YEATS domain compared with acetyl-lysine. Our mutational analysis confirmed key protein residues Phe59 and Tyr78 for crotonyl-lysine recognition. Importantly, our findings present a new structural mechanism of protein-protein interactions mediated by histone lysine crotonylation, and show how the cells interpret acyl-lysine marks in different biological contexts.

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Conflict of interest statement

STATEMENT The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. AF9 YEATS domain binds crotonyl-lysine better than acetyl-lysine
(A) Chemical structures of acetyl-lysine (Kac), crotonyl-lysine (Kcr), and butyryl-lysine (Kbu). (B) 2D 1H-15N-HSQC NMR spectra of the AF9 YEATS domain depicting changes of the protein backbone amide resonances upon the addition of histone H3 peptides containing acetylation (blue), crotonylation (red) or butyrylation (green) at lysine 9 or lysine 18 (residues 3–15, TKQTAR-Kcr, Kac or Kbu-STGGKA; residues 12–24, GGKAPR-Kcr or Kac-QLATKA). The unmodified histone H3 (residues 3–15, TKQTAR-K-SSGGKA) shows no binding to the protein (left panel). (C) Isothermal titration calorimetry (ITC) measurements of the AF9 YEATS domain binding to histone H3 peptides containing acetyl-lysine, crotonyl-lysine, or butyryl-lysine at H3K9, H3K18 or H3K27. The H3K9 and H3K18 peptides are same as in B, and H3K27 peptides consist of ATKAAR-Kcr or Kac-SAPATG (residues 21–33).
Figure 2
Figure 2. 3D structure of the AF9 YEATS domain bound to an H3K18cr peptide
(A) Left, ribbon diagram depicting the average minimized NMR structure of the YEATS domain bound to an H3K18cr peptide (green). Right, space-filled representation of the YEATS domain structure highlights the H3K18cr binding site. (B) Electrostatic potential surface representation of the AF9 YEATS domain structure depicting its recognition of an H3K18cr peptide. Lower, depiction of the inter-molecular interactions between the protein and peptide. (C) Comparison of H3K18cr (green) versus H3K18ac (salmon) recognition by the AF9 YEATS domain, showing key residues engaged in the inter-molecular protein/peptide interactions. The key residues involved in protein/peptide interactions are color-coded by atom type, with blue for H3K18cr and yellow for H3K18ac bound forms, respectively. (D) Effects of mutations of key residues in the AF9 YEATS domain on histone lysine acylation binding, as assessed by NMR binding study using 1H-15N HSQC spectra.
Figure 3
Figure 3. Crotonyl-lysine recognition by distinct histone binding modules
(A) H3K18cr recognition by the AF9 YEATS domain, as depicted in the overall protein structure (upper) and the detailed interactions at the crotonyl-lysine binding site (lower). (B) H4K5cr recognition by the second bromodomain of TAF1 (TAF1-BD2), as shown in the crystal structure of the TAF1_BD2/H4K5cr complex (PDB: 4YYN). The side-chain carbonyl of the crotonyl-lysine H4K5cr is hydrogen-bonded to the conserved Asn1583, similar to what’s seen for acetyl-lysine recognition by the bromodomain. (C) H3K4cr recognition by Sirt3, as shown in the crystal structure of human Sirt3/H3K4cr complex (PDB: 4V1C). The side-chain amide of crotonyl-lysine is hydrogen-bonded to the backbone carbonyl of Val292 of the protein.
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