Highlights d LSD2 demethylase binds nucleosomes by discrete promiscuity at multiple sites d Nucleosomal DNA represents an intrinsic barrier to histone demethylation d NPAC has a rigid and catalytically inert oligomerization core and flexible arms d The NPAC/LSD2 multifunctional enzyme complex modifies transcribed chromatin

Genetic diseases often lead to rare and severe syndromes and the identification of the genetic and protein alterations responsible for the pathogenesis is essential to understand both the physiological and pathological role of the gene product. Recently, de novo variants have been mapped on the gene encoding for the lysine-specific histone demethylase 1 (LSD1)/lysine(K)-specific histone demethylase 1A in three patients characterized by a new genetic disorder. We have analyzed the effects of these pathological mutations on the structure, stability and activity of LSD1 using both in vitro and cellular approaches. The three mutations (Glu403Lys, Asp580Gly and Tyr785His) affect active-site residues and lead to a partial impairment of catalytic activity. They also differentially perturb the ability of LSD1 to engage transcription factors that orchestrate key developmental programs. Moreover, cellular data indicate a decrease in the protein cellular half-life. Taken together, these results demonstrate the relevance of LSD1 in gene regulation and how even moderate alterations in its stability, catalytic activity and binding properties can strongly affect organism development. This depicts a perturbed interplay of catalytic and non-catalytic processes at the origin of the pathology.

No abstract

NPAC is a cytokine-like nuclear factor involved in chromatin modification and regulation of gene expression. In humans, the C-terminal domain of NPAC has the conserved structure of the β-hydroxyacid dehydrogenases (β-HAD) protein superfamily, which forms a stable tetrameric core scaffold for demethylase enzymes and organizes multiple sites for chromatin interactions. In spite of the close structural resemblance to other β-HAD family members, the human NPAC dehydrogenase domain lacks a highly conserved catalytic lysine, substituted by a methionine. The reintroduction of the catalytic lysine by M437 K mutation results in a significant decrease of stability of the tetramer. Here, we have computationally investigated the molecular determinants of the functional differences between methionine and lysine-containing NPAC proteins. We find that the single mutation can determine strong consequences in terms of dynamics, stability, and ultimately ability to assemble in supramolecular complexes: the higher stability and lower flexibility of the methionine variant structurally preorganizes the monomer for tetramerization, whereas lysine increases flexibility and favors conformations that, while catalytically active, are not optimal for tetrameric assembly. We combine structure-dynamics analysis to an evolutionary study of NPAC sequences, showing that the methionine mutation occurs in a specifically flexible region of the lysine-containing protein, flanked by two domains that concentrate most of the stabilizing interactions. In our model, such separation of stability nuclei and flexible regions appears to favor the functional innovability of the protein.

No abstract