From molecular understanding to organismal biology of N-terminal acetyltransferases

Summary

In this issue of Structure, Deng et al. (2019) determine the structure of the yeast N-terminal acetyltransferases Naa10 and Naa50 in complex with the Naa15 and demonstrate that Naa50 has negligible catalytic activity on its own but modulates Naa10/Naa15. This study provides insights into mechanisms involving amino-terminal acetylation of proteins.

N-terminal acetylation (NTA) is among the most widespread co-translational modifications found in eukaryotic proteins. NTA is estimated to affect up to 90% of human proteins and influences their folding, localization, complex formation, and degradation, along with a variety of cellular functions ranging from apoptosis to gene regulation. From the ubiquity of the process alone we would expect its dysregulation to carry serious consequences, and this is indeed the case: disruption of NTA is associated with a number of cancers and developmental disorders (Aksnes et al., 2019).

NTA is carried out by N-terminal acetyltransferases (NATs), which catalyze the transfer of an acetyl moiety from acetyl coenzyme A to the N-terminal amino group of the nascent polypeptides as they emerge from the ribosome. The process is reminiscent of histone acetylation, in which histone acetyltransferases (HATs) transfer an acetyl moiety to a lysine side chain (Friedmann and Marmorstein, 2013). The difference is that, whereas over a dozen histone deactylases (HDACs) counterbalance the activity of twice that many HATs, there are no known Nt-deacetylases, which suggests that NTA is an irreversible protein modification.

There are seven known NAT types (NatA through NatG), each composed of one or more specific subunits and having specific substrates defined by the very first amino acid residue (serine, alanine, etc.) (Aksnes et al., 2019). The major acetyltransferase in humans, for which 40–50% of all human proteins are potential substrates, is the NatA complex. It comprises the catalytic subunit NAA10, the auxiliary subunit NAA15, and HYPK. Notably, mutations in the X-linked gene NAA10 cause Ogden Syndrome, which affects numerous aspects of development (Wu and Lyon, 2018).

In the present study, Marmorstein and colleagues build upon their prior work characterizing the human NatA complex, with and without HYPK, using hNatA prepared from baculovirus-infected Sf9 insect cells (Gottlieb and Marmorstein, 2018). They had previously revealed the X-ray crystal structure of the 100-kDa holo-NatA complex from Schizosaccharomyces pombe, in the absence and presence of a bisubstrate peptide-CoA–conjugate inhibitor, as well as the structure of the uncomplexed Naa10p catalytic subunit (Liszczak et al., 2013). Seeking to obtain a crystal structure of a NatA/Naa50 complex (also referred to as NatE), they were foiled by the poor diffraction of the S. pombe NatE, so they turned to Saccharomyces cerevisiae and found they could obtain crystals using recombinant proteins overexpressed and purified from E. coli cells—full-length ScNaa15 (residues 1–854), C-terminally truncated ScNaa10 (1–226 out of 238 total residues), and full-length ScNaa50 (residues 1–176)—in the presence of inositol hexaphosphate (IP₆) and bi-substrate analogs for both Naa10 and Naa50. As expected, ScNaa15 has a high degree of structural conservation with the previously published SpNaa15 and hNaa15 structures, and ScNaa10 is similarly and completely locked into a cradle by the surrounding Naa15 helices. The new information is that ScNaa50 sits adjacent to ScNaa10, and though it interacts with several ScNaa15 helices, the extent of the contact between ScNaa50 and ScNaa15 is much more modest. This allows more flexibility for ScNaa15 in the crystal, which likely explains why: 1) some residues away from the ScNaa50-ScNaa15 interface had poor side chain density; 2) some α-helices and β strands were not resolved and thus were built as loops; 3) the NatA bisubstrate analog that was used in the co-crystallization could not be resolved; and 4) only the CoA portion of the Naa15 bisubstrate inhibitor could be resolved. Given the prior identification of a bound IP₆ molecule in the human NatA structure, they added IP₆ to the crystallization of the ternary ScNatA/Naa50 complex, and this confirmed its presence there as well (with a dissociation constant of ~130 nM and 1:1 stoichiometry, measured using isothermal titration calorimetry (ITC)).

These structural findings are supported by a series of biochemical analyses using the S. pombe and human NatA complexes, which revealed that SpNaa50 has a robust interaction with SpNatA that is maintained even in high salt concentrations (1 M NaCl), which is also very similar to ScNatA. Consistent with the failure to obtain crystals of the human NatE complex, hNatA and hNaa50 failed to form a stoichiometric complex in the high salt buffer. Experiments with a fluorescence polarization (FP) assay, however, did reveal tight binding of Naa50 to NatA in both the S. pombe and human systems. This was confirmed by differential scanning fluorimetry experiments in which thermal melting temperature increases were consistent with complex formation, and Deng et al. identified hydrophobic residues that mediate the interaction. The authors also explored various residues that are conserved among the yeast and human complexes, and they find that a single SpNaa15-T412 mutation to either tyrosine or lysine completely disrupts complex formation with SpNaa50.

Perhaps the most surprising finding from the current study is the diminished catalytic activity of the yeast Naa50 enzymes, including SpNaa50 and ScNaa50, at least when measured in vitro with one of the canonical Naa50 substrates with the first four amino acids being MLGP. By contrast, hNaa50 activity is significantly promoted within the ternary hNatA/hNaa50 complex. The authors note that SpNaa50 and ScNaa50 do not contain an optimal Q/RxxGxG/A consensus acetyl-CoA binding motif, as suggested in a prior paper (Van Damme et al., 2015), and they could not detect any acetyl-CoA binding to SpNaa50, although they do detect binding to ScNaa50 and hNaa50. They then go on to show that the substrate peptide binding grooves are much narrower in SpNaa50 and ScNaa50 than in hNaa50, and they lack the characteristic YY motif in the β6-β7 loop that facilitates substrate binding in most NATs.

It thus remains puzzling that Naa50 is conserved and retained in yeast, given the apparent absence of any phenotype in S. cerevisiae lacking Naa50 (Gautschi et al., 2003). Deng et al. do show that when SpNaa50 binds to SpNatA, it increases its affinity for a canonical NatA substrate (a peptide with the first four amino acids being SASE). In addition, the SpNatA/Naa50 complex binds acetyl-CoA about 4.5 more strongly than SpNatA alone, and the authors reveal that ScNaa50 sits relatively close to the Q/RxxGxG/A consensus acetyl-CoA binding motif of ScNaa10, which suggests a structurally plausible explanation for these observations. It is therefore possible that these somewhat indirect effects on NatA activity explain the retention of Naa50 in yeast throughout evolution, although it seems worthwhile to explore whether yeast mutants involving Naa50 possess phenotypes that just have not yet been discovered. That said, the current results are consistent with what was previously reported in S. cerevisiae with N-terminome analyses comparing wild-type and scNaa50 deletion strains (Van Damme et al., 2015). In that study, only six N-termini displayed an absolute reduction in Nt-acetylation of 10% or more in the ynaa50Δ strain, and these few affected N-termini were not predicted Naa50 substrates but rather typical NatA (Naa10) substrates, all starting with small amino acids (alanine, serine, threonine, or valine) after excision of methionine (Van Damme et al., 2015)—a stark contrast with other yNat deletion strains (e.g., yNatA and yNatB) in which Nt-acetylation levels decrease dramatically. The crystal structure provides support for the lack of catalytic activity, and this study provides convincing evidence that the yeast Naa50 enzymes are likely not catalytically active but rather serve a supporting and/or regulatory role for the NatA complex—with the caveat, as pointed out by the authors, that their activity measurements were performed in vitro on single canonical peptide substrates.

More broadly, although the present work focuses on these enzymes and single canonical peptide substrates, it presents an important step forward for a field trying to interpret large-scale proteomic measurements to determine whether NTA of particular substrates is necessary and sufficient for the expression of any particular phenotypic trait. Perhaps the best evidence to date comes from studies of yeast prion propagation, which show that loss of NTA promotes general protein misfolding, a redeployment of chaperones to these substrates, and a corresponding stress response that subsequently reduces the size of prion aggregates and reverses their phenotypic consequences (Holmes et al., 2014). It is possible to make proteins no longer susceptible to NTA by switching the second amino acid (after methionine) into a proline, and it was shown that the expression of such mutants in the proteins Ssa1, Ssa2, Ssb1, Ssb2 or Sup35 on genetic backgrounds null for these proteins only partially mimicked the effects of ΔNatA. Mutation of certain of these proteins on genetic backgrounds that were also disrupted for the corresponding Hsp70 paralogues [ssa1(S2P)Δssa2, ssb1(S2P)Δssb2, sup35(S2P)], however, did mimic the effects of disruption of NatA in the [PSI+] strain, including induction of smaller Sup35 aggregates and reversion of stop-codon readthrough in colony growth ade1–14 (UGA) assays (Holmes et al., 2014). This indicated that the increased chaperones and loss of Sup35 NTA combinatorially contributed to the effect of NatA disruption on prion propagation, and it seems likely to be the case that the role of NTA will be even more complex in the expression and modulation of various phenotypes in mammalian organisms. A better understanding of NTA will be necessary if we are to unravel the wide-ranging developmental defects observed in humans with mutations in NAA10 and NAA15 (Cheng et al., 2019; Wu and Lyon, 2018).