Lysine-Specific Demethylase 1A (LSD1/KDM1A): Identification, Characterization, and Biological Implications of an Extended Recognition Interface for Product and Substrate Binding

The posttranslation modification of histone proteins within the nucleosomes of chromatin expands the epigenetic code. Among these modifications is methylation; playing a dual role in both gene activation and repression. Lysine-specific demethylase 1A (LSD1/KDM1A), an FAD-dependent enzyme that catalyzes the oxidative demethylation of histone H3K4me1/2 and H3K9me1/2, is an enzymatic target with therapeutic potential. Using steady-state kinetic analyses, we show that unmodified histone H3 is a tight-binding, competitive inhibitor of KDM1A and the KDM1A/H3 binary complex reaches rapid equilibrium. Rapid dilution experiments confirmed the increased binding affinity was caused by a slow off-rate and SPR experiments independently confirmed these observations. In order to identify region, we turned to the use of cysteine labeling, cross-linking coupled to LC-MS/MS, HDX-MS, and the design of a chimeric, tower domain deletion KDM1A mutant. We have determined the KDM1A tower domain is dispensable for demethylation activity and contributes to the extended binding interface of the KDM1A/H3 interaction. Our discovery and characterization of this secondary binding site within the tower domain provides a foundational understanding of this binding interaction. It also further uncovers how pivotal this region is to the control and localization of KDM1A enzymatic activity as this unprecedented domain also serves as a protein-protein interaction motif for the nucleation of heteromeric complexes.