Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)

Graduate Group


First Advisor

Ronen Marmorstein


N-terminal acetylation (NTA) is one of the most widespread protein modifications, which occurs on most eukaryotic proteins, but is significantly less common on bacterial and archaea proteins. This modification is carried out by a family of enzymes called N-terminal acetyltransferases (NATs). To date, 12 NATs have been identified, harboring different composition, substrate specificity, and in some cases, modes of regulation. In the first chapter, we review the molecular features of NATs.NatA/E, NatB and NatC, are multi-subunit enzymes, responsible for the majority of eukaryotic protein NTA. Their mechanisms of action and regulation remain poorly understood before this dissertation. In the second chapter, 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 found 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. Thirdly, we reported the Cryo-EM structures of human NatE and NatE/HYPK complexes and associated biochemistry. We revealed that NAA50 and HYPK exhibit negative impacts on their 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 substrate binding site. Fourthly, we reported the Cryo-EM structure of hNatB bound to a CoA-αSyn conjugate, together with structure-guided analysis of mutational effects on catalysis. This analysis revealed functionally important differences with human NatA and Candida albicans NatB, resolved key hNatB protein determinants for αSyn N-terminal acetylation, and identified important residues for substrate-specific recognition and acetylation by NatB enzymes. Lastly, we report the Cryo-EM structure of S. pombe NatC with a NatE/C-type bi-substrate analogue and inositol hexaphosphate (IP6), and associated biochemistry. We find that all three subunits are prerequisite for normal NatC acetylation activity, IP6 binds tightly to NatC to stabilize the complex, and we determine the molecular basis for IP6-mediated stability of the complex and the overlapping yet distinct substrate profiles of NatC and NatE.

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Biochemistry Commons