NatA co-translationally acetylates the N termini of over 40% of eukaryotic proteins and can associate with another catalytic subunit, Naa50, to form a ternary NatA/Naa50 dual enzyme complex (also called NatE). The molecular basis of association between Naa50 and NatA and the mechanism for how their association affects their catalytic activities in yeast and human are poorly understood. Here, we determined the X-ray crystal structure of yeast NatA/Naa50 as a scaffold to understand coregulation of NatA/Naa50 activity in both yeast and human. We find that Naa50 makes evolutionarily conserved contacts to both the Naa10 and Naa15 subunits of NatA. These interactions promote catalytic crosstalk within the human complex, but do so to a lesser extent in the yeast complex, where Naa50 activity is compromised. These studies have implications for understanding the role of the NatA/Naa50 complex in modulating the majority of the N-terminal acetylome in diverse species.

No abstract

The human N-terminal acetyltransferase E (NatE) contains NAA10 and NAA50 catalytic, and NAA15 auxiliary subunits and associates with HYPK, a protein with intrinsic NAA10 inhibitory activity. NatE co-translationally acetylates the N-terminus of half the proteome to mediate diverse biological processes, including protein half-life, localization, and interaction. The molecular basis for how NatE and HYPK cooperate is unknown. Here, we report the cryo-EM structures of human NatE and NatE/HYPK complexes and associated biochemistry. We reveal that NAA50 and HYPK exhibit negative cooperative binding to NAA15 in vitro and in human cells by inducing NAA15 shifts in opposing directions. NAA50 and HYPK each contribute to NAA10 activity inhibition through structural alteration of the NAA10 substratebinding site. NAA50 activity is increased through NAA15 tethering, but is inhibited by HYPK through structural alteration of the NatE substrate-binding site. These studies reveal the molecular basis for coordinated N-terminal acetylation by NatE and HYPK.

NatB is one of three major N-terminal acetyltransferase (NAT) complexes (NatA-NatC), which co-translationally acetylate the N-termini of eukaryotic proteins. Its substrates account for about 21% of the human proteome, including well known proteins such as actin, tropomyosin, CDK2, and α-synuclein (aSyn). Human NatB (hNatB) mediated N-terminal acetylation of αSyn has been demonstrated to play key roles in Parkinson's disease pathogenesis and as a potential therapeutic target for hepatocellular carcinoma. Here we report the cryo-EM structure of hNatB bound to a CoA-aSyn conjugate, together with structure-guided analysis of mutational effects on catalysis. This analysis reveals functionally important differences with human NatA and Candida albicans NatB, resolves key hNatB protein determinants for aSyn N-terminal acetylation, and identifies important residues for substrate-specific recognition and acetylation by NatB enzymes. These studies have implications for developing small molecule NatB probes and for understanding the mode of substrate selection by NAT enzymes.