Molecular mimicry and ligand recognition in binding and catalysis by the histone demethylase LSD1-CoREST complex

Abstract

Histone demethylases LSD1 and LSD2 (KDM1A/B) catalyze the oxidative demethylation of Lys4 of histone H3. We used molecular dynamics simulations to probe the diffusion of the oxygen substrate. Oxygen can reach the catalytic center independently from the presence of a bound histone peptide, implying that LSD1 can complete subsequent demethylation cycles without detaching from the nucleosomal particle. The simulations highlight the role of a strictly conserved active-site Lys residue providing general insight into the enzymatic mechanism of oxygen-reacting flavoenzymes. The crystal structure of LSD1-CoREST bound to a peptide of the transcription factor SNAIL1 unravels a fascinating example of molecular mimicry. The SNAIL1 N-terminal residues bind to the enzyme active-site cleft, effectively mimicking the H3 tail. This finding predicts that other members of the SNAIL/Scratch transcription factor family might associate to LSD1/2. The combination of selective histone-modifying activity with the distinct recognition mechanisms underlies the biological complexity of LSD1/2.

Figure 1

Histone demethylation by LSD1. (A) Scheme of the LSD1-catalyzed amine oxidation reaction. This enzyme acts on mono- and di-methylated Lys4 of histone H3 and is able to subsequently remove two methyl groups from a dimethylated substrate. The reactants involved in the simulated system are highlighted in red and correspond to the protein-bound FADH⁻ (Fig. 2), the histone H3 N-terminal peptide (Fig. 1B), and O₂. (B) Sequence alignment of the N-terminal residues of histone H3 and the N-terminal sequences of an evolutionary-related family of C₂H₂ zinc-finger transcription factors which includes SNAIL1 (Barrallo-Gimeno and Nieto, 2009). OVO-like1 is an epidermal proliferation/differentiation factor homologous to a protein originally identified in D. melanogaster ovary cells (Nair et al., 2006). Scratch proteins are expressed in the nervous system in both developing and adult cells (Marín and Nieto, 2006). Growth factor independence 1 (gfi1) is a gene repressor involved in hematopoiesis whose expression was already demonstrated to be regulated by LSD1-containing complexes (Saleque et al., 2007). Insulinoma-associated 1 (insm1) was originally isolated from neuroendocrine tumour cells and data suggest a role in differentiation of both neural and pancreatic precursors (Lukowski et al., 2006). Conserved residues are highlighted in green (identity) or magenta (similarity). Lys4 of LSD1 is indicated in blue.

Figure 2

Chemical representation of the reduced flavin adenine dinucleotide (FADH⁻) molecule. The C4a and C5a atoms relevant for the present study are labeled. The flavin is facing the viewer with its re-side (the si-side is on the opposite side). See also Figure S5.

Figure 3

X-ray structure of LSD1-CoREST in complex with the SNAIL1 peptide. (A) Overall ribbon representation of the ternary complex of LSD1 (cyan), CoREST (blue) and the SNAIL1 peptide (orange). The FAD cofactor is in yellow sticks. (B) Fitting of the SNAIL1 peptide (carbons in orange) into the unbiased electron density map contoured at 1.2 σ calculated with weighted 2Fo-Fc coefficients. Color-coding and orientation are as in panel A. (C) Binding of the SNAIL1 peptide in the LSD1 active site. Color-coding and orientation are as in panel A with H-bonds shown as dashed lines. Red labels are used to highlight residues in direct contact with oxygen pathways in O₂-bound simulations. (D) Superposition between SNAIL1 (orange) and histone H3 (gray) peptides. Conserved peptide side chains (Fig. 1B) are drawn in stick representation. The FAD cofactor and the Lys661 side chain are shown as reference. The water bridging Lys661 and flavin N5 (as observed in the unbound enzyme structure that has been solved at higher resolution) are also displayed (Yang et al., 2006). See also Figure S1.

Figure 4

Backbone C^α atom-positional RMSD of LSD1-CoREST MD trajectory snapshots. Time series (left panels) and normalized probability distributions (right panels) of the RMSD values were calculated either for all C^α atoms of the complex (top panels) or for the individual LSD1 or CoREST subunits (middle and bottom panels) from both unbound and bound 50 ns simulations using the X-ray structures as reference (Yang et al., 2006; Forneris et al., 2007). Structures were superimposed using all backbone C^α atoms for which RMSD values are reported. Corresponding time series and distributions of the scaled size-independent similarity parameter (Maiorov and Crippen, 1995) are reported as Supplemental Information, Fig. S2.

Figure 5

Successful oxygen diffusion paths in the LSD1-CoREST complex. Time series of the O₂ – C4a distance are displayed with different colors (green, yellow, red, blue, and black) for independent MD simulations in O₂-saturated conditions (O₂-unbound and O₂-bound). The dashed horizontal line defines the flavin as a sphere of 7 Å radius centered on C4a. See Fig. 2 for the atom numbering of FADH⁻. See also Figure S3.

Figure 6

O₂ spontaneous diffusion into the bound LSD1-CoREST complex. (A) Overall location, (B) top view, and (C) side view of the O2 diffusion pathways displayed with color-coding corresponding that of the right panel of Fig. 5. For graphical purposes the blue pathway is displayed in Supplemental Information, Fig. S4. The bound histone H3 tail (gray coil) and LSD1 (cyan coil) are also shown. (D) Side view of the time-dependent representations along the simulation time: O₂ molecules are colored from red (entrance into the protein matrix) to blue (exit from the active site). All displayed paths conduct O₂ molecules to the C4a-N5 locus of the FADH⁻ reduced flavin cofactor (yellow sticks). Residues Lys661 (orange sticks) and Tyr761 (purple sticks) are also highlighted.