1H, 13C, and 15N resonance assignments of CW domain of the N-methyltransferase ASHH2 free and bound to the mono-, di- and tri-methylated histone H3 tail peptides

Abstract

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 of methylations], but binds strongest to monomethylated instances (K values reported in the range of 1 µm to 500 nM). Hoppmann et al. published the uncomplexed NMR structure of an ASHH2 CW domain in 2011. Here we document the assignment of a shortened ASHH2 CW construct, CW42, with similar binding affinity and better expression yields than the one used to solve the uncomplexed structure. We also perform H-N HSQC-monitored titrations that document at which protein-peptide ratios the complex is saturated. Backbone resonance assignments are presented for this shortened ASHH2 CW domain alone and bound to an H3 histone tail mimicking peptide monomethylated on lysine 4 (ARTK(me1)QTAR). Likewise, the assignment was also performed for the protein in complex with the dimethylated (ARTK(me2)QTAR) and trimethylated (ARTK(me3)QTAR) peptide. Overall, these two latter situations displayed a similar perturbation of shifts as the mono-methylated instance. In the case of the monomethylated histone tail mimic, side-chain assignment of CW42 in this complex was performed and reported in addition to backbone assignment, in preparation of a future solution structure determination and dynamics characterization of the CW42-ARTK(me1)QTAR complex.

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

“…Several crystallization attempts were unsuccessful, and consequently, the decision was made to characterize the complex using NMR. For subsequent work, CW42 construct was selected as it expressed well and had an affinity indistinguishable from that of the longer CWs [24].…”

“…Buffer components were acquired from Sigma-Aldrich. CW constructs were prepared using ligation-independent cloning in the KpnI/SacI restriction sites of pET-49b vector (Novagen/Merck, Darmstadt, Germany), and the protein and all mutants were expressed and purified as described [24]. For more details, see Supporting Information: Cloning of CW constructs, site-directed mutagenesis, protein expression and purification.…”

“…Structural calculation, refinement and validation of the CW42-H3K4me1 complex Assignment of ARTKme1QTARY in complex with CW42 was carried out using 2D-filtered 1 H-1 H TOCSY and 2D-filtered 1 H-1 H NOESY spectra. The peptide assignment was used to establish the intermolecular NOE connectivities with CW42 (BMRB ID: 27251, [24]). Intra-and intermolecular NOE cross-peaks were assigned manually using the CARA program v 1.9.1.2 [52].…”

“…Data were collected at 25°C on an 850 MHz Bruker Avance III HD Spectrometer fitted with a 1 H/ 13 C/ 15 N TCI CryoProbe and a SampleJet with temperature control for storing samples in between runs (set to 4°C). Samples were prepared in NMR buffer consisting of 20 mM potassium phosphate, 50 mM NaCl and 1 mM DTT adjusted to pH 6.4, and all NMR experiments related to the backbone and side-chain assignment performed for this study (summarized in Table S2) were collected, processed and analysed as described previously [24]. Protein diffusion measurements were performed on protons at 25°C using stimulated echo, bipolar gradients and 3-9-19 pulse train for solvent suppression.…”

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

“…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).…”