A previously described pET-15b vector containing 6 Г— His nО”150 KDM1A (residues 151вЂ“852) was used as a template for construction of the chimera. Joining KDM1A fragments L151вЂ“A388 and D525вЂ“M852 to KDM1B residues V494вЂ“L531 formed the chimera sequence. The chKDM1AО”Tower chimera therefore decouples tower-dependent protein interactions from catalysis and provides a tool to assess the effects of KDM1A mistargeting and orphanization. However, unlike previous tower deletion mutants, our chimera exhibits kinetic parameters nearly identical to those of unaltered KDM1A and KDM1B, suggesting that the tower domain is not required for catalytic activity. As expected, chKDM1AО”Tower failed to bind CoREST. This construct, termed chKDM1AО”Tower, copurified from Escherichia coli cellular lysates with a stoichiometric equivalent of FAD. KDM1A and KDM1B sequence and structural alignments suggested that KDM1B residues V494вЂ“L531 could replace the KDM1A tower domain (residues T389вЂ“R524) with minimal active site disturbance. Here we report the rational design and characterization of a KDM1A tower domain deletion chimera. As this paralog overcomes exclusion of the tower domain in a manner that preserves a KDM1A-like active site conformation, we sought to use it as a template for engineering a chimeric KDM1A enzyme. 1A) but conserves the AOD architecture, sharing a 2.0 Г… RMSD compared to KDM1A, despite a modest sequence similarity (<25%) ( Fig. Interestingly the recent characterization of the KDM1A paralog KDM1B provides a unique opportunity to investigate this dichotomy. These studies suggest that either the tower domain is inherently required for KDM1A catalysis or that the methods chosen to bridge the tower region were not conducive to proper active site folding. Indeed, several groups have generated such mutants, but were limited by a lack of enzymatic activity. In order to evaluate these tower-dependent interactions independent of demethylase activity, a KDM1A tower domain deletion mutant is highly desired. Therefore, one of the most significant challenges is understanding the impact of these tower-dependent interactions on KDM1A specificity, catalysis, and positioning within chromatin. KDM1A activity is further governed by other homologous proteins that bind the tower domain and perform similar functions in different molecular contexts, such as the CoREST paralogs RCOR2 and RCOR3 and the MTA proteins. Additionally, CoREST orchestrates the inclusion of enzymes such as KDM1A and histone deacetylases as catalytic subunits within modular multiprotein complexes. One of the best-studied tower domain interaction partners is the corepressor of the RE1-silencing transcription factor (CoREST/RCOR1/KIAA0071), which facilitates demethylation of nucleosomal substrates by bridging KDM1A to chromatin through its DNA-binding SANT domains. Zinc ions in KDM1B structure are shown as gray spheres.

Coloring scheme follows that of domain map above, FAD cofactor is shown in yellow, and N and C-termini are labeled. Structural overview of (B) KDM1A (PDB 2IW5) and (C) KDM1B (PDB 4HSU).

SWIRM domains are shown in green, AODs are shown in magenta, tower domain is shown in blue, C4H2C2 domain is shown in orange, Zf-CW domain is shown in cyan, and linker is shown in purple.

Дomain maps and structural overview of KDM1A and KDM1B. Unprecedented among related amine oxidases, the tower domain serves as a protein interaction motif and facilitates KDM1A incorporation into multiprotein regulatory complexes that dictate its cellular function. The вЂtowerвЂ™ domain is a nearly 100 aa AOD insert that forms an approximately 90 Г… long antiparallel coiledвЂ“coil from two О±-helices (termed TО±A and TО±B) linked by a tight turn ( Fig. The catalytic amine oxidase domain (AOD) houses a single, non-covalently bound flavin adenine dinucleotide (FAD) cofactor required for catalysis while the N-terminal SWI3p, Rsc8p, and Moira (SWIRM) domain is commonly found in chromatin-associating proteins ( Fig. This 852 amino acid (aa) polypeptide is composed of three structured domains ( Fig. KDM1A is a flavin-dependent amine oxidase that demethylates mono- and dimethylated lysine residues at positions 4 and 9 on histone H3 (H3K4me1/2 and H3K9me1/2), leading to gene activation and repression, respectively.

In 2004, lysine-specific demethylase 1A (KDM1A also known as LSD1/BHC110/AOF2/KIAA0601/p110b) became the first histone demethylase to be isolated and characterized and has subsequently been identified as a potential therapeutic target.

Chimera tool rm 917