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Sequence alignment of the CW domain constructs in relation to the ASHH2 protein. The numbering on the top and the bottom panel corresponds to the numbering in the entire ASHH2 protein and the current construct, correspondingly. The underscored residues represent the part of the plasmid solely. Small letters indicate the residues remaining from the linker sequence connecting the GST-expression tag to the protein sequence
Source publication
The ASHH2 CW domain is responsible for recognizing the methylation state at lysine 4 of histone 3 N-terminal tails and implicated in the recruitment of the ASHH2 methyltransferase enzyme correctly to the histones. The ASHH2 CW domain binds H3 lysine motifs that can be either mono-, di-, or tri-methylated [ARTK(meX)QTAR, where X denotes the number o...
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Citations
... The 1 H-15 N HSQC NMR spectra of CW in complex with mono-, di-, and trimethylated ligands were acquired and compared to the unbound form. Using the backbone chemical shift assignments available for the four situations (Dobrovolska et al., 2018), the chemical shift perturbation (CSP) was calculated, and the residues involved in the corresponding complex formation were determined ( Figure 2A). The NMR data in the case of all the three histone-mimicking peptides suggest that binding causes fairly extensive structural changes that were not limited to W865 and W874 of the binding pocket. ...
... Protein expression and purification was performed as described in the work of Dobrovolska et al. (2018). Mutant versions of the ASHH2 CW domain were generated using site-directed mutagenesis and PCR. ...
The CW domain binds to histone tail modifications found in different protein families involved in epigenetic regulation and chromatin remodeling. CW domains recognize the methylation state of the fourth lysine on histone 3 and could, therefore, be viewed as a reader of epigenetic information. The specificity toward different methylation states such as me1, me2, or me3 depends on the particular CW subtype. For example, the CW domain of ASHH2 methyltransferase binds preferentially to H3K4me1, and MORC3 binds to both H3K4me2 and me3 modifications, while ZCWPW1 is more specific to H3K4me3. The structural basis for these preferential bindings is not well understood, and recent research suggests that a more complete picture will emerge if dynamical and energetic assessments are included in the analysis of interactions. This study uses fold assessment by NMR in combination with mutagenesis, ITC affinity measurements, and thermal denaturation studies to investigate possible couplings between ASHH2 CW selectivity toward H3K4me1 and the stabilization of the domain and loops implicated in binding. The key elements of the binding site—the two tryptophans and the α1-helix form and maintain the binding pocket— were perturbed by mutagenesis and investigated. Results show that the α1-helix maintains the overall stability of the fold via the I915 and L919 residues and that the correct binding consolidates the loops designated as η1 and η3, as well as the C-terminal. This consolidation is incomplete for H3K4me3 binding to CW, which experiences a decrease in overall thermal stability on binding. Loop mutations not directly involved in the binding site, nonetheless, affect the equilibrium positions of the key residues.
... Using the backbone chemical shift assignments available for the four situations [27], the chemical shift perturbation (CSP) was calculated, and the residues involved in the corresponding complex formation were determined ( Fig. 2A). The NMR data in the case of all the three histone-mimicking peptides suggests that binding causes fairly extensive structural changes that are not limited to W865 and W874 of the binding pocket. ...
... It is The copyright holder for this preprint this version posted August 13, 2021. ; https://doi.org/10.1101/2021.08.12.456084 doi: bioRxiv preprint Protein expression and purification: Protein expression and purification was performed as described in [27]. Mutant versions of ASHH2 CW-domain were generated using site-directed mutagenesis and PCR. ...
The CW domain binds to histone-tail modifications found in different protein families involved in epigenetic regulation and chromatin remodelling. CW domains recognize the methylation state of the fourth lysine on histone 3, and as such could be viewed as a reader of epigentic information. The specificity towards different methylation states such as me1, me2 or me3 depend on the particular subtype. For example, the CW domain of ASHH2-methyltransferase binds preferentially to H3K4me1, MORC3 binds to both H3K4me2 and me3 modifications, while ZCWPW1 is more specific to H3K4me3. The structural basis for these preferential bindings are not understood well, and recent research suggests that a more complete picture will emerge if dynamical and energetic assessments are included in analysis of interactions. This study uses fold assessment by NMR in combination with mutagenesis, ITC affinity measurements and thermal denaturation studies to investigate possible couplings between ASHH2 CW selectivity towards H3K4me1, and the stabilization of the domain. Key elements of the binding site are the two tryptophans and the a1-helix form and maintain the binding pocket were perturbed by mutagenesis and investigated. Results show that a1-helix maintains the overall stability of the fold via the I915 and L919 residues, and that correct binding consolidates the coils designated n1, n3, as well a the C-terminal. This consolidation is incomplete for H3K4me3 binding to CW, which experiences a decrease in overall thermal stability upon binding. Moreover, loop-mutations not directly involved in the binding site nonetheless affect the equilibrium positions of key residues.
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. β‐augmentation and rearrangement of coils coincide with changes in the flexibility of the complex, in particular the η1, η3 and C‐terminal coils, but also in the β1 and β2 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.
Databases
Structural data are available in the PDB database under the accession code 6QXZ. Resonance assignments for CW42 in its apo‐ and holo‐forms are available in the BMRB database under the accession code 27251.
