TY - JOUR
T1 - Structural basis for human PHF2 Jumonji domain interaction with metal ions
AU - Horton, John R.
AU - Upadhyay, Anup K.
AU - Hashimoto, Hideharu
AU - Zhang, Xing
AU - Cheng, Xiaodong
N1 - Funding Information:
The Department of Biochemistry at the Emory University School of Medicine supported the use of the SER-CAT synchrotron beamline at the Advanced Photon Source of Argonne National Laboratory, local X-ray facility, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This work was supported by grant GM068680 to X.C. from the US National Institutes of Health. X.C. is a Georgia Research Alliance Eminent Scholar. J.R.H. performed crystallographic work; A.K.U performed mass spectrometry-based demethylation assays and ITC binding assays; H.H. performed cloning, mutagenesis, and fluorescence polarization assay; X.Z. and X.C. organized and designed the scope of the study; and X.C. wrote the manuscript; all were involved in the analysis of data and helped in revising the manuscript.
PY - 2011/2/11
Y1 - 2011/2/11
N2 - PHF2 belongs to a class of α-ketoglutarate-Fe2 +-dependent dioxygenases. PHF2 harbors a plant homeodomain (PHD) and a Jumonji domain. PHF2, via its PHD, binds Lys4-trimethylated histone 3 in submicromolar affinity and has been reported to have the demethylase activity of monomethylated lysine 9 of histone 3 in vivo. However, we did not detect demethylase activity for PHF2 Jumonji domain (with and without its linked PHD) in the context of histone peptides. We determined the crystal structures of PHF2 Jumonji domain in the absence and presence of additional exogenous metal ions. When Fe2+ or Ni2+ was added at a high concentration (50 mM) and allowed to soak in the preformed crystals, Fe2+ or Ni 2+ was bound by six ligands in an octahedral coordination. The side chains of H249 and D251 and the two oxygen atoms of N-oxalylglycine (an analog of α-ketoglutarate) provide four coordinations in the equatorial plane, while the hydroxyl oxygen atom of Y321 and one water molecule provide the two axial coordinations as the fifth and sixth ligands, respectively. The metal binding site in PHF2 closely resembles the Fe2+ sites in other Jumonji domains examined, with one important difference-a tyrosine (Y321 of PHF2) replaces histidine as the fifth ligand. However, neither Y321H mutation nor high metal concentration renders PHF2 an active demethylase on histone peptides. Wild type and Y321H mutant bind Ni2+ with an approximately equal affinity of 50 μM. We propose that there must be other regulatory factors required for the enzymatic activity of PHF2 in vivo or that perhaps PHF2 acts on non-histone substrates. Furthermore, PHF2 shares significant sequence homology throughout the entire region, including the above-mentioned tyrosine at the corresponding iron-binding position, with that of Schizosaccharomyces pombe Epe1, which plays an essential role in heterochromatin function but has no known enzymatic activity.
AB - PHF2 belongs to a class of α-ketoglutarate-Fe2 +-dependent dioxygenases. PHF2 harbors a plant homeodomain (PHD) and a Jumonji domain. PHF2, via its PHD, binds Lys4-trimethylated histone 3 in submicromolar affinity and has been reported to have the demethylase activity of monomethylated lysine 9 of histone 3 in vivo. However, we did not detect demethylase activity for PHF2 Jumonji domain (with and without its linked PHD) in the context of histone peptides. We determined the crystal structures of PHF2 Jumonji domain in the absence and presence of additional exogenous metal ions. When Fe2+ or Ni2+ was added at a high concentration (50 mM) and allowed to soak in the preformed crystals, Fe2+ or Ni 2+ was bound by six ligands in an octahedral coordination. The side chains of H249 and D251 and the two oxygen atoms of N-oxalylglycine (an analog of α-ketoglutarate) provide four coordinations in the equatorial plane, while the hydroxyl oxygen atom of Y321 and one water molecule provide the two axial coordinations as the fifth and sixth ligands, respectively. The metal binding site in PHF2 closely resembles the Fe2+ sites in other Jumonji domains examined, with one important difference-a tyrosine (Y321 of PHF2) replaces histidine as the fifth ligand. However, neither Y321H mutation nor high metal concentration renders PHF2 an active demethylase on histone peptides. Wild type and Y321H mutant bind Ni2+ with an approximately equal affinity of 50 μM. We propose that there must be other regulatory factors required for the enzymatic activity of PHF2 in vivo or that perhaps PHF2 acts on non-histone substrates. Furthermore, PHF2 shares significant sequence homology throughout the entire region, including the above-mentioned tyrosine at the corresponding iron-binding position, with that of Schizosaccharomyces pombe Epe1, which plays an essential role in heterochromatin function but has no known enzymatic activity.
KW - Epe1
KW - PHF2
KW - epigenetics
KW - histone lysine demethylation
KW - methyl-lysine binding
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U2 - 10.1016/j.jmb.2010.12.013
DO - 10.1016/j.jmb.2010.12.013
M3 - Article
C2 - 21167174
AN - SCOPUS:79151477106
SN - 0022-2836
VL - 406
SP - 1
EP - 8
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 1
ER -