Structure and Mechanism of Acetylation by the N-Terminal Dual Enzyme NatA/Naa50 Complex

Abstract

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.

Molecular Basis for N-terminal Alpha-Synuclein Acetylation by Human NatB

“…Acetyltransferase Activity Assays. All acetyltransferase assays were carried out at room temperature in a reaction buffer containing 75 mM HEPES, pH 7.5, 120 mM NaCl, 1 mM DTT as described [33,34] Cryo-EM data collection. For initial sample screening, 0.6 mg/ml fresh hNatB sample with three-molar excess bisubstrate was used.…”

“…NatC/E/F have overlapping substrates, acting on N-terminal methionine when it is followed by several residues excluding D, E, N and Q [23,[26][27][28][29][30][31]. When another catalytic subunit (NAA50) binds to NatA, a dual enzyme complex NatE is formed [32,33], with catalytic crosstalk between NAA10 and NAA50 [33,34]. We recently demonstrated that both NAA50, and a protein with intrinsic NatA inhibitory activity -Huntingtin-interacting protein K (HYPK) [35][36][37], can bind to NatA simultaneously to form a larger tetrameric complex [33].…”

“…Compared to the comprehensive structural and biochemical characterization of NatA [20][21][22][34][35][36], the study of NatB has been limited, particularly in humans. Recently, the crystal structure of Candida albicans (Ca) NatB bound to a bisubstrate CoA-peptide conjugate was determined, providing important insights into substrate specificity and NTA by caNatB [57].…”

Molecular basis for N-terminal acetylation by human NatE and its modulation by HYPK

“…HYPK and hNAA50 display negative cooperative binding to hNatA. Previous studies demonstrated that both human HYPK and hNAA50 bind tightly to hNatA, with dissociation constants in the nanomolar range 16,46 . We set out to determine if the HYPK-and hNAA50 binding properties to hNatA are altered in the context of the tetrameric complex.…”

“…The trimeric hNAA10/hNAA15/hNAA50 complex is referred to as hNatE, and we refer to the tetrameric hNAA10/hNAA15/hNAA50/HYPK complex as hNatE/HYPK. Previous studies have demonstrated that HYPK may be important for cellular NatA activity and that NAA50 and NatA can affect each other's function 16,41,[44][45][46] . Previous studies also demonstrated that hNatA can physically associate with hNAA50 (ref.…”