Structural and mechanistic basis of selective inhibition of the fungal‐specific histone deacetylase Hos3 from the pathogenic fungus Aspergillus fumigatus

Histone deacetylases (HDACs) are critical epigenetic regulators of fungal growth and pathogenicity and constitute promising targets for antifungal drug development. Hos3 defines a fungal-specific HDAC subclass that lacks a clear mammalian ortholog; however, the structural determinants underlying its selective inhibition remain poorly characterized. Here, we integrated x-ray crystallography with biochemical and biophysical analyses to comprehensively characterize the catalytic properties of Hos3 from the pathogenic fungus Aspergillus fumigatus (AfHos3) and its inhibition by the hydroxamate-based compound T2383. Despite high conservation of catalytic residues, AfHos3 displayed significantly higher intrinsic catalytic activity and markedly enhanced sensitivity to T2383 compared with its yeast homolog ScHos3. The crystal structure of the AfHos3-T2383 complex revealed a canonical zinc-dependent inhibitor-binding mode, coupled with a distinct, fungal-specific architecture of the substrate and inhibitor binding pocket. Structural and functional studies identified an aromatic clamp, comprising residues F371 and F443, that is indispensable for both catalytic activity and ligand stabilization. Furthermore, fungal-specific residues M538 and V205, together with structural elements loopA and α12, shape the catalytic-pocket environment and differentially influence enzymatic activity and T2383 responsiveness. Notably, the dimeric assembly of AfHos3 was associated with optimal enzymatic activity but did not influence inhibitor recognition. Collectively, these findings elucidate how fungal-specific structural elements synergize with the evolutionarily conserved catalytic core to govern selective inhibitor recognition, providing a robust structural and mechanistic framework for the rational design of fungal-selective HDAC inhibitors.