The irreversible and predominately co-translational N-terminal acetylation of proteins is an essential process used in the regulation of protein functions including folding, targeting, protein-protein interactions, and degradation. The seven conserved metazoan N-terminal acetyltransferase (NAT) enzymes (NatA-NatF and NatH) modify ~80% of human proteins. As an essential protein mark involved in human development and health, aberrant NAT activity contributes to several developmental disorders and is linked to human disease, including cancer and some neurodegenerative disorders. NAT enzymes minimally contain a catalytic subunit and up to two auxiliary subunits for enzymatic activity, selectivity, and ribosomal targeting. Current NAT biochemical and structural information primarily describes the basis for monomeric NAT activity. Initial insights into regulatory subunit influence on NAT activity comes from the NatA crystal structure where regulatory subunit (Naa15p) reconfigures the active site of the catalytic subunit (Naa10p) for substrate-specific N-terminal acetylation. This, however, fails to describe the influence of Naa38p and HYPK in NatC and metazoan NatA-mediated activity, respectively. In order to understand the basis for NAT regulatory mechanisms and the implications of aberrant NAT activity in human disease, we focused on the human NatA/HYPK and S. cerevisiae NatC complexes. We determined X-ray crystal structures of NatA and NatA/HYPK complexes and carried out associated biochemical, enzymatic, and structural studies. We demonstrate that human HYPK harbors intrinsic NatA inhibitory activity through a bipartite structure, presumably to more effectively regulate cognate NatA activity. We also demonstrate that the NatA–HYPK interaction reduces Naa50 targeting to NatA, likely limiting ribosomal localization of Naa50 in vivo. We evaluated the effects of human NatA disease-associated missense mutations on its function and evaluated the contribution to either NatA activity or thermal stability. We showed that binding of the small Naa38p subunit is critical for NatC complex (Naa30p/Naa35p/Naa38p) activity. Finally, we report preliminary results on the interplay between HYPK and a putative interacting partner and NatA substrate, Huntingtin (Htt). These studies provide novel mechanistic insights into the activity of NAT proteins and lay the groundwork for development of therapeutics for the exploitation of NAT regulatory mechanisms and targeting of these specialized acetyltransferases for therapy
