Structural basis for human PHF2 Jumonji domain interaction with metal ions

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• PHF2 is an α-ketoglutarate-Fe(2+)-dependent dioxygenase containing plant homeodomain (PHD) and Jumonji domains.
• PHF2's PHD domain binds trimethylated histone H3 Lys4, and it has been suggested to possess in vivo histone H3 Lys9 demethylase activity.

Purpose of the Study:

• To investigate the in vitro enzymatic activity of PHF2, specifically its potential histone demethylase function.
• To determine the crystal structure of the PHF2 Jumonji domain and elucidate its metal-binding site.

Main Methods:

• Biochemical assays to test PHF2's demethylase activity on histone peptides.
• X-ray crystallography to determine the structure of the PHF2 Jumonji domain.
• Site-directed mutagenesis (Y321H) to assess the role of the tyrosine residue in metal binding and activity.

Main Results:

• PHF2 did not exhibit detectable demethylase activity on histone peptides in vitro, even with its Jumonji domain alone or with the PHD domain.
• Crystal structures revealed that Fe(2+) or Ni(2+) binds in an octahedral coordination, with a tyrosine residue (Y321) acting as a key ligand, differing from other Jumonji domains.
• Neither the Y321H mutation nor high metal concentrations restored catalytic activity on histone peptides; wild-type and mutant PHF2 bound Ni(2+) with similar affinity.

Conclusions:

• PHF2's histone demethylase activity may require additional regulatory factors in vivo or it may act on non-histone substrates.
• The structural data provides insights into the metal-binding mechanism of PHF2, highlighting a unique tyrosine ligand.
• PHF2 shares homology with Epe1 from S. pombe, which is crucial for heterochromatin but lacks known enzymatic activity, suggesting a potential conserved regulatory role.