Catalytic mechanism investigation of lysine-specific demethylase 1 (LSD1): a computational study

Abstract

Lysine-specific demethylase 1 (LSD1), the first identified histone demethylase, is a flavin-dependent amine oxidase which specifically demethylates mono- or dimethylated H3K4 and H3K9 via a redox process. It participates in a broad spectrum of biological processes and is of high importance in cell proliferation, adipogenesis, spermatogenesis, chromosome segregation and embryonic development. To date, as a potential drug target for discovering anti-tumor drugs, the medical significance of LSD1 has been greatly appreciated. However, the catalytic mechanism for the rate-limiting reductive half-reaction in demethylation remains controversial. By employing a combined computational approach including molecular modeling, molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations, the catalytic mechanism of dimethylated H3K4 demethylation by LSD1 was characterized in details. The three-dimensional (3D) model of the complex was composed of LSD1, CoREST, and histone substrate. A 30-ns MD simulation of the model highlights the pivotal role of the conserved Tyr761 and lysine-water-flavin motif in properly orienting flavin adenine dinucleotide (FAD) with respect to substrate. The synergy of the two factors effectively stabilizes the catalytic environment and facilitated the demethylation reaction. On the basis of the reasonable consistence between simulation results and available mutagenesis data, QM/MM strategy was further employed to probe the catalytic mechanism of the reductive half-reaction in demethylation. The characteristics of the demethylation pathway determined by the potential energy surface and charge distribution analysis indicates that this reaction belongs to the direct hydride transfer mechanism. Our study provides insights into the LSD1 mechanism of reductive half-reaction in demethylation and has important implications for the discovery of regulators against LSD1 enzymes.

Figure 1. The proposed catalytic mechanism for the overall demethylation reaction of LSD1.

Figure 2. Three major suggested catalytic mechanism proposals for the reductive half-reaction of the flavin-dependent amine oxidases.

Figure 3. The overall structure of LSD1-CoREST-substrate complex and key interactions in the catalytic chamber.

Cartoon diagram of the LSD1-CoREST-substrate complex highlights the SWIRM domain (green), AOL domain (wheat), Tower domain (golden yellow), CoREST (light blue) and substrate peptide (magenta). (A) H-bond interactions in the conserved lysine-water-flavin motif. The bridging water molecular is shown in red sphere and the residues and FAD are shown in green and yellow sticks, respectively. H-bonds are indicated with purple dashed lines. (B) Hydrophobic interactions of dimethylated H3K4 with its surrounding residues in the catalytic chamber. Dimethylated H3K4 is shown in cyan for the sake of clarity. (C) H-bond interactions of FAD with its surrounding residues. (D) Hydrophobic interactions of FAD with its surrounding residues.

Figure 4. Time dependencies of the weighted root-mean-square deviations for the backbone atoms of the three domains of LSD1 and CoREST from their initial positions during the 30-ns simulation.

Figure 5. Distance evolution along simulation time for the two hydrogen bonds in the lysine-water-flavin motif.

The red curve and blue curve were obtained by 5 ps averaged.

Figure 6. The residues involved in hydrophobic interactions with dimethylated H3K4 versus simulation time.

Different colors are used for the sake of clarity.

Figure 7. The potential energy surface of the reductive half-reaction in the demethylation of LSD1-CoREST-substrate complex.

(A) The QM/MM optimized structures of the reactant (R), transition state (TS), and immediate product (P). For clarity, only the structures in the QM region have been shown. (B) The potential energy surface (PES) of the reductive half-reaction along the defined reaction coordinates. (C) Contour plot of the PES corresponding to the central part in (B). The pink triangle line represents the lowest energy pathway on the calculated PES and the position of the TS is marked by a red triangle.

Figure 8. ESP charge distributions of the four groups in the QM region along the hydride transfer reaction.