Engineering human JMJD2A tudor domains for an improved understanding of histone peptide recognition

Abstract

JMJD2A is a histone lysine demethylase which recognizes and demethylates H3K9me3 and H3K36me3 residues and is overexpressed in various cancers. It utilizes a tandem tudor domain to facilitate its own recruitment to histone sites, recognizing various di- and tri-methyl lysine residues with moderate affinity. In this study, we successfully engineered the tudor domain of JMJD2A to specifically bind to H4K20me3 with a 20-fold increase of affinity and improved selectivity. To reveal the molecular basis, we performed molecular dynamics and free energy decomposition analysis on the human JMJD2A tandem tudor domains bound to H4K20me2, H4K20me3, and H3K23me3 peptides to uncover the residues and conformational changes important for the enhanced binding affinity and selectivity toward H4K20me2/3. These analyses revealed new insights into understanding chromatin reader domains recognizing histone modifications and improving binding affinity and selectivity of these domains. Furthermore, we showed that the tight binding of JMJD2A to H4K20me2/3 is not sufficient to improve the efficiency of CRISPR-CAS9 mediated homology directed repair (HDR), suggesting a complicated relationship between JMJD2A and the DNA damage response beyond binding affinity toward the H4K20me2/3 mark.

Keywords: CRISPR; JMJD2A; epigenetics; histone; molecular dynamics.

FIGURE 1

Predicted delta delta G (from simulation) vs experimentally measured binding affinity

FIGURE 2

(Left image is to H3K23me3, right to H4K20me3). No comparable figure is shown for H4K20me2 since an experimental crystal structure is not available for the H4K20me2 complex

FIGURE 3

Snapshots from the final stage of the trajectory for the H4K20me2 complex. The mutant is shown in blue, the wild type in green

FIGURE 4

A comparison of the interactions formed by 17R for the H4K20me2 complex in the wild type (in green) and in the mutant (in blue). The 17R residue directly forms a strong electrostatic interaction with 931D in the mutant, replacing its weak H-bond interaction with 931N in the wild type and causing a large −8.6 kcal/mol change in the contribution of 17R to total binding energy

FIGURE 5

Important interactions between peptide and protein for the H3K23me3 complex. The mutant is shown in blue, the wild type in green and the residues from Table 2 that are adjacent to the binding pocket are labeled for clarity

FIGURE 6

The simulation results suggest a conformational shift in the backbone of the complex with the H4K20me2 versus the H4K20me3 peptide, although the conformation of the “head” region that binds the peptide remains largely unchanged

FIGURE 7

mClover assay with overexpression of NLS-tagged Y973A, WT, and J1 tudor domains (% mClover in U2OS cells) (A), non-NLS tagged Y973A, WT, and J1 tudor in U20S cells (B), NLS-tagged tudors in HEK293A cells (C), and full-length H188A/A482E JMJD2A (with or without NLS tags) in HEK293A cells (D). “Empty” means empty vector control. Each assay was performed in triplicate in panel D

FIGURE 8

mAG1 assay in U2OS cells with NLS or non-NLS tagged WT, J1, Y973A tudor vectors (A), and H188A/A482E full-length WT JMJD2A (with or without NLS tags, each assay in duplicate) (B)