Catalysis by KDM6 Histone Demethylases - A Synergy between the Non-Heme Iron(II) Center, Second Coordination Sphere, and Long-Range Interactions

Abstract

KDM6A (UTX) and KDM6B (JMJD3) are human non-heme Fe(II) and 2-oxoglutarate (2OG) dependent JmjC oxygenases that catalyze the demethylation of trimethylated lysine 27 in the N-terminal tail of histone H3, a post-translational modification that regulates transcription. A Combined Quantum Mechanics/ Molecular Mechanics (QM/MM) and Molecular Dynamics (MD) study on the catalytic mechanism of KDM6A/B reveals that the transition state for the rate-limiting hydrogen atom transfer (HAT) reaction in KDM6A catalysis is stabilized by polar (Asn217) and aromatic (Trp369)/non-polar (Pro274) residues in contrast to KDM4, KDM6B and KDM7 demethylases where charged residues (Glu, Arg, Asp) are involved. KDM6A employs both σ- and π-electron transfer pathways for HAT, whereas KDM6B employs the σ-electron pathway. Differences in hydrogen bonding of the Fe-chelating Glu252(KDM6B) contribute to the lower energy barriers in KDM6B vs. KDM6A. The study reveals a dependence of the activation barrier of the rebound hydroxylation on the Fe-O-C angle in the transition state of KDM6A. Anti-correlation of the Zn-binding domain with the active site residues is a key factor distinguishing KDM6A/B from KDM7/4s. The results reveal the importance of communication between the Fe center, second coordination sphere, and long-range interactions in catalysis by KDMs and, by implication, other 2OG oxygenases.

Keywords: KDM6; QM/MM; histone demethylases; long-range; molecular dynamics; second coordination sphere.

Figure 1.

Folds and active sites of KDM6A and KDM6B. (a) Views derived from crystal structures of KDM6A (PDB ID:3avr) (left) and KDM6B (PDB ID:5oy3) (right). The JmjC domain is in green, the Zn(II)-binding domain is in yellow, the linker is in blue, and the helical domain is in magenta. (b) View of the active site of KDM6A at the Fe(IV)=O intermediate stage generated using VMD visualization software.

Figure 2.

Residues stabilizing the H3 histone substrate in KDM6A-Fe(III)-OO^•−.H3_(17–33)K27me3. Substrate residues are labeled in black and enzyme residues are labeled in magenta.

Figure 3.

PCA and DCCA of Dynamics in KDM6A for the a) KDM6A-Fe(II)-OO^•− b) KDM6A-Fe(IV)=O c) KDM6A-Fe(III)-OH intermediates. The boxed regions in (a) show the flexible motion of the residues (177–208), and in (b) show flexible motion on PCA and anti-correlation of the Zn(II)-binding domain (residues 445–508) with the JmjC domain on DCCA. More flexible parts are shown tighter, and the direction of their motion is represented by a change of color from blue to yellow.

Figure 4.

Reaction profiles of a) KDM6A and b) KDM6B, c) Energies of the reaction and characteristics of the TS states are tabulated.

Figure 5

Frontier Molecular Orbitals of Fe(IV)=O intermediate.

Figure 6.

Hydrogen Bonding Scheme for the Fe(IV)=O.H3_(17–33)K27me3 low activation energy RC-HAT states of KDM6A(left) and KDM6B(right). Hydrogen bonds are depicted by dashed blue lines. Torsion angles are depicted by labels in red (O-Fe-O1-O2)

Figure 7.

Rebound mechanism for conversion of H3_(17–33)K27me3 to H3_(17–33)K27me2-OH. Energies are in kcal/mol as calculated at the QM(B2)/MM (black) and QM(B2+ZPE)/MM (red) levels of theory.

Figure 8.

Spin Natural Orbitals of KDM6A Rebound TS State corroborates the electron transfer between σ_CH and σ*_z2. Occupancies are in parentheses.

Figure 9

Summary of the insights obtained from the study. All SCS and LR residues, which are important for the subsequent reaction steps, are shown. Unique residues for respective systems are depicted in a single color. The KDM6A residues are colored blue, and the KDM6B residues are colored pink. Boltzmann averaged activation energies are given for each reaction.

Scheme 1.

Outline mechanism for the JmjC N^ε-Methyl lysine demethylases (KDMs). The boxed intermediates were studied by MD and QM/MM in this study.