Lysine-Specific Demethylase 1A (KDM1A/LSD1): Product Recognition and Kinetic Analysis of Full-Length Histones

Abstract

Lysine-specific demethylase 1A (KDM1A/LSD1) is a FAD-dependent enzyme that catalyzes the oxidative demethylation of histone H3K4me1/2 and H3K9me1/2 repressing and activating transcription, respectively. Although the active site is expanded compared to that of members of the greater amine oxidase superfamily, it is too sterically restricted to encompass the minimal 21-mer peptide substrate footprint. The remainder of the substrate/product is therefore expected to extend along the surface of KDM1A. We show that full-length histone H3, which lacks any posttranslational modifications, is a tight-binding, competitive inhibitor of KDM1A demethylation activity with a Ki of 18.9 ± 1.2 nM, a value that is approximately 100-fold higher than that of the 21-mer peptide product. The relative H3 affinity is independent of preincubation time, suggesting that H3 rapidly reaches equilibrium with KDM1A. Jump dilution experiments confirmed the increased binding affinity of full-length H3 was at least partially due to a slow off rate (koff) of 1.2 × 10−3 s−1, corresponding to a half-life (t1/2) of 9.63 min, and a residence time (τ) of 13.9 min. Independent affinity capture surface plasmon resonance experiments confirmed the tight-binding nature of the H3/KDM1A interaction, revealing a Kd of 9.02 ± 2.3 nM, a kon of (9.3 ± 1.5) × 104 M−1 s−1, and a koff of (8.4 ± 0.3) × 10−4 s−1. Additionally, no other core histones exhibited inhibition of KDM1A demethylation activity, which is consistent with H3 being the preferred histone substrate of KDM1A versus H2A, H2B, and H4. Together, these data suggest that KDM1A likely contains a histone H3 secondary specificity element on the enzyme surface that contributes significantly to its recognition of substrates and products.

Structural analysis of lysine‐4 methylated histone H3 proteins using synchrotron radiation circular dichroism spectroscopy

“…Interestingly, the catalytic activities of LSD1 and LSD2 depend on the length of K4-methylated H3 peptides: LSD1 and LSD2 require the first 21 residues, at the least, of H3K4me1 or H3K4me2 peptides for effective demethylation, 3,4 although only the first 12 to 16 residues seem to contact the binding site of LSD1 or LSD2. 7 Considering the peptide-length dependence of the catalytic activity or binding affinity of LSD1 and LSD2, both LSD1 and LSD2 recognize not only methylated residues and their neighboring ones but also distant residues, as pointed out in previous studies. 7 Considering the peptide-length dependence of the catalytic activity or binding affinity of LSD1 and LSD2, both LSD1 and LSD2 recognize not only methylated residues and their neighboring ones but also distant residues, as pointed out in previous studies.…”

“…7 Considering the peptide-length dependence of the catalytic activity or binding affinity of LSD1 and LSD2, both LSD1 and LSD2 recognize not only methylated residues and their neighboring ones but also distant residues, as pointed out in previous studies. 3,4,6,7 Since the amino acid sequences of K4 unmethylated/methylated H3 were, understandably, the same, it is likely that the peptidelength dependence of catalytic activity or that of binding affinity between LSD1 and H3K4me0 originates from the differences in structures of H3 peptides. To understand the functions of K4-methylated H3, including binding affinities for enzymes, such as LSD1 and LSD2, more precisely, conformations of full-length K4 unmethylated/ [This article is part of the Special Issue: Proceedings 29th International Symposium on Chirality, Tokyo Japan 2017.…”

“…However, the amount of structural changes induced by K4 methylation was less than those by K9 methylation. 7 It is expected that the catalytic activities of LSD1 and LSD2 against full-length K4-methylated H3 proteins and the conformations of those complexed with LSD1 or LSD2 will be confirmed in the future. We hypothesize that the structural alterations are induced by interactions between residues distant from the methylation site, such as 65th to 86th residues in K4-methylated H3 (Figure 2), and methylated N-terminal tail.…”

“…5,6 In addition, it is also known that the binding affinity between LSD1 and full-length H3K4me0, which is a competitive inhibitor of LSD1 demethylation activity, is nearly 100-fold higher than that between LSD1 and H3K4me0 peptides (first 21 residues). 3,4,6,7 Since the amino acid sequences of K4 unmethylated/methylated H3 were, understandably, the same, it is likely that the peptidelength dependence of catalytic activity or that of binding affinity between LSD1 and H3K4me0 originates from the differences in structures of H3 peptides. 3,4,6,7 Since the amino acid sequences of K4 unmethylated/methylated H3 were, understandably, the same, it is likely that the peptidelength dependence of catalytic activity or that of binding affinity between LSD1 and H3K4me0 originates from the differences in structures of H3 peptides.…”

Conventional and unconventional interactions of the transcription factor FOXL2 uncovered by a proteome‐wide analysis

“…[53][54][55] SUPT16H is a subunit of the FACT complex, which interacts with H2A/H2B histones affecting nucleosome disassembly and transcription elongation. We were able to confirm the interaction between FOXL2 and epigenetic factors and chromatin remodelers such as KDM1A, SUPT16H and SMARCB1.…”