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
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 (Kd 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 1H–15N 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.
KeywordsCW Methylation Histone-tail modification Assignment
The authors would like to acknowledge the Norwegian NMR Platform (NNP) for support in the form of instruments access and experimental time (Grant 226244/F50). The authors wish to thank the NNP Bergen Node staff for facilitating the research. The authors also wish to thank Drs. V. De Marco, E. Christodoulou, and S. J. Gamblin, NIMR/MRC, London, for their advice and generous support for construction of CW42.
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