Divergent architecture of the heterotrimeric NatC complex explains N-terminal acetylation of cognate substrates

Abstract

The heterotrimeric NatC complex, comprising the catalytic Naa30 and the two auxiliary subunits Naa35 and Naa38, co-translationally acetylates the N-termini of numerous eukaryotic target proteins. Despite its unique subunit composition, its essential role for many aspects of cellular function and its suggested involvement in disease, structure and mechanism of NatC have remained unknown. Here, we present the crystal structure of the Saccharomyces cerevisiae NatC complex, which exhibits a strikingly different architecture compared to previously described N-terminal acetyltransferase (NAT) complexes. Cofactor and ligand-bound structures reveal how the first four amino acids of cognate substrates are recognized at the Naa30–Naa35 interface. A sequence-specific, ligand-induced conformational change in Naa30 enables efficient acetylation. Based on detailed structure–function studies, we suggest a catalytic mechanism and identify a ribosome-binding patch in an elongated tip region of NatC. Our study reveals how NAT machineries have divergently evolved to N-terminally acetylate specific subsets of target proteins.

Molecular mechanism of N-terminal acetylation by the ternary NatC complex

“…While Glu109 is positioned to function as a general base to deprotonate the α-amino group of the peptide substrate, Tyr121 is positioned to function as a general acid to re-protonate the CoA leaving group. This is consistent with the proposed roles of the analogous Glu118 and Tyr130 residues in Sc NatC (Grunwald et al, 2020) , as well as the conserved nature of these residues ( Figure 4a ). In addition, Naa30-Asn114 is highly conserved among all NATs (data not shown) and it was recently suggested to play a structural role in orienting the catalytic tyrosine residues ( Sp Naa30-Tyr135 and Tyr136) by ensuring the proper position of the active site helix (α4) for interaction with Ac-CoA (Deng et al, 2020b).…”

“…To investigate the catalytic roles of Naa38, we could readily purify both binary ( Sc Naa30 1-161 and Sc Naa35 19-733 ) and ternary Sc NatC complexes ( Sc Naa30 1-161 and Sc Naa35 19-733 , and Sc Naa38 1-70 ) from Saccharomyces cerevisiae to compare their activities. Consistent with the studies by Grunwald et al (Grunwald et al, 2020 ), evaluation of the activities of the binary and ternary Sc NatC complexes toward the canonical NatC substrate (MLRF peptide) revealed that the ternary complex showed robust activity, while the binary complex showed compromised activity ( Figure 1b ). Thus, it appears that Naa38 is required for optimal NatC activity in yeast.…”

“…These two Naa30 residues likely play a role in recognizing positions 3 and 4 of high-affinity NatC peptide substrates, as supported by the recent report by Grunwald et al . (Grunwald et al, 2020)…”

“…We also report the Cryo-EM structure of ~100 kDa S. pombe NatC ( Sp NatC) bound to a NatE/C-type bisubstrate analogue and IP 6 , with related biochemistry, to reveal the molecular basis for IP 6 -mediated stabilization of the complex and the similar substrate profiles of NatE and NatC. Comparison of these studies with the recently published structural and functional studies of the Sc NatC complex (Grunwald et al, 2020) reveal evolutionarily conserved and divergent features of NatC.…”

“…Although the recently reported crystal structures and associated biochemical studies of S. cerevisiae NatC ( Sc NatC) by Grunwald et al . have provided some important insights into NatC substrate specificity and its catalytic mechanism (Grunwald et al, 2020), several questions remain unanswered. In particular, those regarding the mechanisms dictating the overlapping yet distinct substrate profiles of NatC and NatE and the influence of IP 6 on NatC.…”