The Arabidopsis (ASHH2) CW domain binds monomethylated K4 of the histone H3 tail through conformational selection

Abstract

Chromatin post-translational modifications are thought to be important for epigenetic effects on gene expression. Methylation of histone N-terminal tail lysine residues constitutes one of many such modifications, executed by families of histone lysine methyltransferase (HKMTase). One such protein is ASHH2 from the flowering plant Arabidopsis thaliana, equipped with the interaction domain, CW, and the HKMTase domain, SET. The CW domain of ASHH2 is a selective binder of monomethylation at lysine 4 on histone H3 (H3K4me1) and likely helps the enzyme dock correctly onto chromatin sites. The study of CW and related interaction domains has so far been emphasizing lock-key models, missing important aspects of histone-tail CW interactions. We here present an analysis of the ASHH2 CW-H3K4me1 complex using NMR and molecular dynamics, as well as mutation and affinity studies of flexible coils. b-augmentation and rearrangement of coils coincide with changes in the flexibility of the complex, in particular the g1, g3 and C-terminal coils, but also in the b1 and b2 strands and the C-terminal part of the ligand. Furthermore, we show that mutating residues with outlier dynamic behaviour affect the complex binding affinity despite these not being in direct contact with the ligand. Overall, the binding process is consistent with conformational selection. We propose that this binding mechanism presents an advantage when searching for the correct post-translational modification state among the highly modified and flexible histone tails, and also that the binding shifts the catalytic SET domain towards the nucleosome.

Binding specificity of ASHH2 CW-domain towards H3K4me1 ligand is coupled to its structural stability through its α1-helix

“…This observation is in line with the flexible nature of ASHH2 CW and the proposed mechanism of conformational selection [24]. The most notable changes in chemical shifts are found in the first β-strand, perhaps related to the nascent β-sheet augmentation discussed in [24], in the conserved W891 that is not part of the binding pocket, in the ɑ1-helix and the η1-loop (Fig. 2A).…”

“…Recent X-ray (Liu et al, PDB code: 5YVX) ) and NMR (Dobrovolska et al, PDB code: 6QXZ) structural studies described the CW of ASHH2 in complex with H3K4me1. The published structures agreed on the core of the complex; however, the structural data related to the 1-helix and the following C-terminal regions differ [23,24]. The studies also concluded differently with respect to binding mechanisms and determinants.…”

“…While the ASHH2 CW domain has higher affinity towards H3K4me1 [9,23], the rest of the known CW domains bind stronger to H3K4me2 and H3K4me3 modifications [8,10,11,15]. Earlier, factors determining the ligand methylation state specificity of CW domains have been presented and discussed [9,23,24]. The C-terminal regions of the CW domain due to its variability between paralogs were suggested to be involved in ligand specificity.…”

“…The construct used by Dobrovolska et al to solve the structure was longer at the C-terminal than the one used in the Liu et al study, which allowed elucidation of the role of the I921-Q923 region in binding. Analysis of the domain's dynamics and flexibility using MD simulations and NMR further indicated that the binding mode of CW is described best by a conformational selection model [24]. A conformational selection mechanism requires that the protein has a fluctuating structural ensemble that includes the conformation(s) required for binding, even in the Maxim S. Bril'kov.…”

“…The alignment was prepared using Jalview software and UniProt entries with ClustalO default parameters, and Clustalx coloring scheme was used. MIDDLE: CW domain constructs used in earlier studies on the ASHH2 CW domain byHoppmann et al, 2011, Liu and Huang, 2018and Dobrovolska et al, 2020. The E917A mutation used by Liu and Huang, 2018 is marked by purple color.…”