Histone acetylation is a versatile post-translational modification essential for diverse biological processes. While nearly all histone acetyltransferases (HATs) act promiscuously on multiple lysine residues, the human male-specific lethal (MSL) acetyltransferase complex mediates strictly site-specific acetylation of histone H4 at lysine 16 (H4K16ac), with its underlying mechanism remaining enigmatic. Here, we leveraged chemical protein synthesis to engineer a semisynthetic nucleosome probe that traps the MSL complex in a catalytically engaged state and determined its 3.12 Å cryo-electron microscopy (cryo-EM) structure, which reveals a hierarchical anchoring–gating mechanism responsible for stringent H4K16 selectivity. The MSL complex adopts a dual-anchoring mode to bind nucleosomal DNA at superhelix location (SHL) 1.5 and the H2A–H2B acidic patch. Three spatially coordinated gating elements (the MSL1 anchor helix, KAT8 gating helix, and KAT8 gating hairpin) then conformationally constrain the H4 N-terminal tail, funneling H4K16 side chain exclusively into the active site while sterically occluding flanking lysines. Targeted mutations disrupting these interfaces impair H4K16ac in vitro and in cells, with concomitant defects in chromatin accessibility and transcription. Beyond the monomeric 1:1 complex, cryo-EM analysis identifies a 2:2 MSL–nucleosome assembly, wherein MSL3 dimerization mediates inter-nucleosomal contacts linked to genome-wide H4K16ac patterning. Collectively, our work delineates a residue-to-domain framework in which a chromatin acetylation writer hardwires specific catalysis into nucleosome engagement and scales this precision to higher-order chromatin architecture, providing a mechanistic foothold for understanding aberrant MSL-encoded chromatin programs in diseases.
