Molecular role of NAA38 in thermostability and catalytic activity of the human NatC N-terminal acetyltransferase

N-terminal acetylation occurs on over 80% of human proteins and is catalyzed by a family of N-terminal acetyltransferases (NATs). All NATs contain a small catalytic subunit, while some also contain a large auxiliary subunit that facilitates catalysis and ribosome targeting for co-translational acetylation. NatC is one of the major NATs containing an NAA30 catalytic subunit, but uniquely contains two auxiliary subunits, large NAA35 and small NAA38. Here, we report the cryo-EM structures of human NatC (hNatC) complexes with and without NAA38, together with biochemical studies, to reveal that NAA38 increases the thermostability and broadens the substrate-specificity profile of NatC by ordering an N-terminal segment of NAA35 and reorienting an NAA30 N-terminal peptide binding loop for optimal catalysis, respectively. We also note important differences in engagement with a stabilizing inositol hexaphosphate molecule between human and yeast NatC. These studies provide new insights for the function and evolution of the NatC complex. [Display omitted] •NAA38 broadens the substrate-specificity profile of NatC•NAA38 reorients an NAA30 N-terminal peptide binding loop for optimal catalysis•NAA38 orders an N-terminal segment of NAA35 and increases NatC thermostability•Unlike in yeast, Inositol hexaphosphate (IP6) does not contribute to NatC stability The N-terminal acetyltransferases NatC uniquely requires a third small NAA38 subunit for activity. Deng et al. find that NAA38 increases the thermostability and broadens the substrate-specificity profile of NatC by ordering an N-terminal segment of NAA35 and reorienting an NAA30 N-terminal peptide binding loop for optimal catalysis, respectively.