NAA40 and NAC cooperate in co-translational histone acetylation in humans

Domain: biology
Published: 2026-03-12
Editorial outcome: minor_revision

Abstract
N-terminal acetylation is an abundant and predominantly co-translational modification in eukaryotes that profoundly affects folding, compartmentalization fidelity and turnover of target proteins. Unlike other N-acetyltransferases, human NatD is composed solely of the catalytic subunit NAA40 and exclusively modifies histone proteins H2A and H4. However, the molecular details of co-translational NAA40 activity have remained elusive. Here, we show biochemically and by cryo-EM how NAA40 activity is coordinated at the ribosomal peptide tunnel exit involving the NAC complex. We demonstrate that the NAA40-NAC interaction is required for efficient ribosome binding and histone acetylation. Furthermore, we provide insights on the potential coordination of methionine removal and subsequent NAA40-mediated acetylation by formation of a multienzyme complex on the ribosome involving METAP1. Therefore, our results illustrate the details of N-terminal histone acetylation by NAA40 and highlight the role of NAC as a general coordinator of nascent protein modification.

Introduction
N-terminally acetylated (Nt-acetylation) 1, a modification which in most cases is added co-translationally, when the nascent polypeptide chain emerges from the ribosome. For this modification, N-terminal α-acetyltransferases (NATs) catalyze the transfer of an acetyl group from acetyl-CoA to the α-amino group of the nascent peptide chain, often requiring the removal of the N-terminal methionine. Of the eight distinct NATs that have been identified in eukaryotes (NatA-NatH), five act co-translationally on the ribosome (NatA to NatE) 2. At the ribosome, NATs compete with each other and an array of other enzymes, which also bind to the ribosomal tunnel exit to ensure the proper maturation of the newly made polypeptide 3. Protein Nt-acetylation has been shown to be decisive for a diversity of general processes in the cell, such as protein-protein interactions, subcellular localization, protein targeting and protein half-life. Yet, also more specific activities such as sister chromatid cohesion and chromosome condensation in the nucleus are dependent on accurate Nt-acetylation 2. Of the five NATs acting co-translationally, four (NatA, NatB, NatC, NatE) are responsible for the vast majority of protein Nt-acetylation and typically consist of a catalytic subunit and one or two accessory subunits that mediate the interaction with the ribosome 4–7. NAA40 (or NatD), on the other hand, is one of the most specialized NATs since in humans it Nt-acetylates exclusively histone proteins H2A, H4 and histone variant H2A.X 8. Due to the distinct structural features of its catalytic domain, NAA40 provides specificity for the N-terminal four amino acids of its cognate substrates 9,10. In contrast, NATs with a broad substrate range, like NatA and NatE, mainly recognize the first two target residues 11,12. Furthermore, NAA40 binds directly to the ribosome, therefore lacking any accessory subunits 9.

NAA40 is active only after the starter methionine has been removed by the methionine aminopeptidases METAP1 or METAP2, thereby exposing the recognition sequence Ser-Gly-Arg-Gly 10. Importantly, Nt-acetylation of these histone proteins antagonizes other histone modifications, specifically H2A/H4S1Ph (Ph, phosphorylation) and H4R3me (me, methylation), thereby influencing chromatin activity and gene regulation 13,14. Most notably, NAA40 levels are altered in various cancers, and NAA40 has been shown to promote oncogenesis (summarized in ref. 13), which is why NAA40 has been recognized as a potential therapeutic drug target. However, it is not known how NAA40 interacts with the ribosome to modify nascent H2A and H4.

Here we present the structure of human NAA40 bound to the 80S ribosome. We discovered that NAA40 is positioned near the ribosomal tunnel exit and that its ribosome interaction largely depends on the nascent polypeptide-associated complex (NAC). Notably, we demonstrate that within cells, the ubiquitin-associated (UBA) domain of NAC is required for a productive interaction of NAA40 with the ribosome. Finally, we provide structural evidence for the coordination of methionine excision and NAA40-mediated Nt-acetylation by formation of a multienzyme complex on the ribosome.

Results
NAA40 and NAC co-associate with ribosomes in vivo
The N-terminal histone acetylation activity of NAA40 occurs both post- and co-translationally, however, the significance of the co-translational activity has been poorly characterized 9,15,16. To better understand this co-translational activity of NAA40, we first aimed at investigating the association of NAA40 with ribosomes in vivo. Therefore, we expressed NAA40 with a C-terminal 3C protease cleavage site and 3xFLAG tag in HEK293T cells. We isolated the tagged NAA40 under native conditions from cell lysate and indeed enriched 80S ribosomes (Fig. 1a). In addition to ribosomes, we noticed enrichment of histones as well as XRCC5/6 and PRKDC (Fig. 1a), which are essential actors in DNA damage repair via nonhomologous end joining 17,18. Association with histones was expected, as H2A, H4, and H2A.X are established NAA40 substrates 8 and the enzyme localizes to both cytosol and nucleus 9. Also, the enrichment of DNA repair factors is in line with the recently reported involvement of the NAA40 yeast homolog in DNA damage response 16.

To elucidate the molecular details of the NAA40-ribosome interaction, we subjected the isolated NAA40 complexes to cryo-EM single particle analysis (SPA). The majority of 2D classes obtained were representative of 80S ribosomes, and only a few classes corresponded to co-purified nucleosomes (Supplementary Fig. 1). After 3D classification of ribosomal particles, we obtained classes representing translating ribosomes but mainly inactive/hibernating ribosomes, likely as a result of NAA40 overexpression. Yet, all these classes shared a distinct density on the large ribosomal subunit at the peptide exit site and thus were combined to gain an improved overall resolution (2.7 Å) (Supplementary Fig. 2a–d upper panels, Supplementary Table 1). The resulting combined reconstruction mainly resembled a hibernating state with SERBP1, eEF2 and E-site tRNA bound (Fig. 1b) 19,20 and allowed us to identify NAA40 and, unexpectedly, NAC binding together in the vicinity of the peptide exit. NAC is a heterodimeric complex consisting of NACA and NACB that has been recognized as a constituent component of the translation machinery, coordinating co-translational modifications and protein translocation as well as acting as an early chaperon on the ribosome 3,21,22. To assess potential interactions between NAC and NAA40, we performed an AF2 (AlphaFold2) multimer analysis 23,24, which confidently predicted an interaction of the NACA UBA-domain and the catalytic domain of NAA40 (Supplementary Fig. 3a–c). The interaction was predicted to be mediated via both negatively charged (NACA: E177, D180, D205; NAA40: R50, K153, R216) and hydrophobic residues (NACA: L183, I210; NAA40: F45, L161) of the UBA domain (Supplementary Fig. 3d), and was in agreement with additional density observed on NAA40 in our reconstruction (Fig. 1b).

To gain further insights into the co-translational substrate engagement of NAA40 we isolated a smaller but defined class of particles representing actively translating ribosomes with both P- and E-site tRNAs (Fig. 1c, Supplementary Figs. 1 and 3e, f). Here, NAA40 and NAC associate with the ribosome in essentially the same manner as for inactive ribosomes. Despite lower overall resolution (2.9 Å) (Supplementary Fig. 2a–d lower panels, Supplementary Table 1) the nascent polypeptide chain could still be traced from the P-site tRNA to the peptide tunnel exit site but density is lost after exiting the ribosome and could not be unambiguously assigned within the catalytic domain of NAA40 (Fig. 1c, d left panel and Supplementary Fig. 3e, f). However, using a crystal structure of substrate analogue-bound NAA40 25 (Supplementary Fig. 3g) as a reference point, one can estimate that nascent chains need to span a distance of about 4.5 nm or at least 12 amino acids after leaving the ribosome to reach the active site of NAA40 (Fig. 1d right panel). Compared to recent structures of the ribosome, NAA40/NatD is similarly positioned to NatA/E acetyltransferase complexes 6,7 (Supplementary Fig. 3h, i), but nascent polypeptides theoretically require at least 16 or 20 amino acids outside the peptide exit to reach their respective catalytic subunits NAA10 and NAA50 (Supplementary Fig. 3j).

Molecular details of the NAA40-NAC ribosome interaction
Next, we analyzed the structural basis of the interaction of NAA40 and NAC with the ribosome. When looking at the overall positioning of NAA40 and NAC on our best resolved ribosome (2.7 Å, Supplementary Fig. 2a–d), both proteins are situated in close proximity to the peptide exit site for immediate engagement with emerging substrate polypeptides (Fig. 2a). Notably, NAA40 consists of two parts: the catalytic GCN5-related N-acetyltransferase (GNAT) domain and an N-terminal helical extension (α0) that is unique to NatD compared to other NATs 10. The catalytic domain resides on top of ribosomal protein uL24 and the 5.8S rRNA helix H7 with its substrate binding cavity facing towards the peptide tunnel exit (Fig. 2a, b left, Supplementary Fig. 4a). However, the lower local resolution of the GNAT domain (Supplementary Fig. 4a) as well as the minimal contact points with uL24 and 5.8S H7 indicate a certain degree of flexibility for the catalytic domain which might hint at a dynamic conformational adaptability for its continuously elongating substrates. The N-terminal α0 helix on the other hand is well resolved (Supplementary Fig 4c), anchoring NAA40 to the ribosome via interactions with uL24 as well as rRNA helices H19, H24 and H46 (Fig. 2b right, Supplementary Fig. 4b). Its highly positively charged portion engages with the negatively charged surface of rRNA elements H19 and H24 (Fig. 2c left and middle, Supplementary Fig. 4c and d). The unstructured very N-terminus of NAA40 further protrudes towards H46, held in place by interaction of lysine K4 with H19 and π-charge interaction of arginine R3 with guanine G409 of H24 (Fig. 2c right), and is essentially buried at the convergence site of the three rRNA segments. In essence, the α0 helix of NAA40 appears to act as a substitute for additional auxiliary subunits, which have been shown to serve as ribosome tethers for other NATs 4–7. Furthermore, NAA40 is highly conserved in vertebrates for both its GNAT domain and N-terminus (Supplementary Fig. 5a, b), supporting the notion of functional importance of the α0 helical element, including its ribosome interaction capabilities.

The NAC complex interacts with the ribosome as observed before 26. It is anchored to the ribosome via the NACB N-terminus, which is attached to ribosomal protein eL22 (Fig. 2d, Supplementary Fig. 4e, f). The NAC globular domain is positioned near the peptide exit site and close to an unstructured loop of the NAA40 catalytic domain (191–213 aa) (Fig. 2e). However, similar to the NAA40 GNAT domain, the lower local resolution (Supplementary Fig. 4e, f) suggests a level of positional flexibility for the NAC globular domain possibly allowing it to adapt to the nascent polypeptide. Consistent with this assumption, we observed the NAC beta sandwich positioned closer to the peptide exit in our reconstruction of actively translating ribosomes than for hibernating 80S (Fig. 2f). Lastly, the NACA UBA-domain was found interacting with the ribosome distal side of the NAA40 catalytic domain, as predicted by AF2 (Fig. 2g, Supplementary Fig. 3a–d). This suggests that, similar to the N-terminal acetyltransferase complexes NatA/E, which require the NACA UBA interaction for stable ribosome association 6, NAA40 ribosome binding might also depend on NAC. Further, when comparing NAA40 with NatA/E, it becomes evident that they share a common binding site as the NatA/E auxiliary subunit NAA15 also binds to rRNA helices H19 and H24 6,7 (Supplementary Fig. 4g and h). Taken together, this architecture implies a similar mode of interaction of NAA40 and NatA/E with NAC. Yet, it also highlights the competitive environment of the peptide exit site for co-translational modification factors that requires appropriate coordination for each target peptide.

NAC coordinates the co-translational activity of NAA40
Recently, NAC has been identified as a master regulator for the recruitment of co-translational protein modification enzymes such as METAP1 26, NatA/E 6,7 and for protein translocation complexes like SRP to the ribosome 27. The consistent coappearance of NAC and NAA40 in our ex vivo cryo-EM data, as well as a similar interaction with the NACA UBA-domain for NatA/E suggest comparable recruitment of NAA40 to translating 80S by NAC. To test this hypothesis, we performed in vitro binding assays with purified recombinant NAA40-FLAG together with NAC and NAC-ΔUBA complexes to assess their association with purified 80S ribosomes (Supplementary Fig. 6a–c). The respective components were incubated together with anti-FLAG resin, immobilized via tagged NAA40 and bound complexes were eluted with FLAG peptide. NAA40 alone bound only weakly to ribosomes, but in the presence of NAC, enrichment of ribosomes was strongly increased (Fig. 3a, Supplementary Fig. 6d). Conversely, when incubated with NAC-ΔUBA, the ribosome association of NAA40 was lost entirely (Fig. 3a). To further examine the negative effect of NAC-ΔUBA on NAA40 ribosome association, we performed another binding assay but this time titrated NAC against NAC-ΔUBA (Supplementary Fig. 6e, f). Here, increasing the proportion of NAC-ΔUBA led to a decrease in associated ribosomes until binding is again essentially lost when only NAC-ΔUBA is added. We speculate that the high affinity of NAC for the ribosome, in combination with its flexible positioning around the peptide exit site, might lead to a steric block for NAA40 binding in the case of NAC-ΔUBA. Therefore, it seems that the UBA-domain acts in an almost allosteric manner, both assisting and allowing proper NAA40 ribosome binding. Taken together, NAA40 binding to ribosomes appeared indeed largely dependent on NAC in vitro and, similar to NatA/E and in agreement with our structure, the NAC interaction with NAA40 is mediated by the NACA UBA-domain.

Based on our structural data, we identified the N-terminal α0 helix of NAA40 as its main anchor for ribosome association (Fig. 2b, c, Supplementary Fig. 4c, d). Recently, a second isoform of NAA40 has been reported that lacks the first 21 amino acids (NAA40S) generated either by alternative translation initiation at M22 of the canonical NAA40 transcript or expression of a shorter transcript variant 28,29. Interestingly, this part of the α0 helix contains the entirety of highly conserved, positively charged residues responsible for interaction with rRNA elements (Fig. 3b, compare Fig. 2c). In order to assess the contribution of the positively charged N-terminus to ribosome association, we performed our binding assay also with recombinantly purified short form, NAA40S-FLAG. As expected, we could not observe any binding to ribosomes of NAA40S, whether on its own or in the presence of NAC (Fig. 3a last two lanes, Supplementary Fig. 6d). This demonstrates that the positively charged N-terminus is essential for NAA40 ribosome binding and also suggests that the NAA40S isoform is unlikely to participate in co-translational Nt-acetylation, but possibly active in post-translational modification in the cytosol. Furthermore, we conclude that the interaction of NAC with NAA40 via the NACA UBA-domain is necessary (Fig. 3c) but not sufficient to mediate a stable ribosome association.

Next, we aimed to determine whether NAC had an effect on NAA40-mediated histone Nt-acetylation within cells. Therefore, we raised an antibody recognizing Nt-Ac on an SGRG sequence shared by H2A and H4, but with the antibody detecting primarily histone H4 Nt-acetylation (Supplementary Fig. 6g, h). Using this antibody, we probed the acetylation as well as the antagonistic H4/H2AS1Ph phosphorylation levels in NACA knockdown (Supplementary Fig. 6i) and NAA40 knockout (NAA40-KO) cell lysates (Fig. 3d). As expected, in NAA40-KO cells the signal for H4 Nt-acetylation was significantly reduced but not completely abolished, probably due to weak H4 backbone recognition by the antibody (Supplementary Fig. 6h). At the same time, the H4/H2AS1Ph signal was strongly increased compared to wild type lysate. More interestingly, knockdown of NACA also led to a decrease in H4 Nt-acetylation and an increase in H4/H2AS1Ph levels (Fig. 3d). As expected, the effect was not as profound as for NAA40-KO cells, likely due to the incomplete dissociation of NAA40 from ribosomes in the absence of NAC and/or due to the remaining post-translational modification activity of NAA40. Additionally, while the almost complete reduction in NACA mRNA levels confirm the efficient NACA knockdown (Supplementary Fig. 6i), we cannot exclude that residual H4 Nt-acetylation may be present due to remaining NACA protein. Importantly, NACA depletion did not affect NAA40 expression levels or its localization (Supplementary Fig. 6i and j, respectively), suggesting that its effect on histone Nt-acetylation is primarily due to the weaker association of NAA40 with the ribosome. To further determine whether the observed decrease in histone Nt-acetylation is directly dependent on the NACA UBA-domain interaction we complemented cells that were depleted of NACA with siRNA resistant constructs expressing either N-terminally FLAG tagged NACA or NACA-ΔUBA (Supplementary Fig. 6k) and monitored the antagonistic H4/H2AS1Ph levels as a proxy for Nt-acetylation (Fig. 3e). While the phosphorylation levels were increased three-fold when depleting NACA compared to non-targeting siRNA, as observed above (Fig. 3e, f), the levels of H4/H2AS1Ph were equivalent between control cells and cells depleted for NACA but complemented with exogenous full-length NACA (Fig. 3f). However, when NACA-depleted cells were complemented with NACA-ΔUBA the H4/H2AS1Ph levels remained higher again by more than two-fold compared to the control cells, indicative of compromised Nt-acetylation activity (Fig. 3f). Taken together, our in vitro and in vivo data indicate a substantial role of the NACA UBA-domain interaction with NAA40 for ribosome binding/recruitment (Fig. 3c) and thereby for overall histone Nt-acetylation in cells.

Another important aspect of H2A and H4 Nt-acetylation is the preceding removal of the initiator methionine by methionine aminopeptidases (MetAPs) to expose the target serine and how this MetAP activity is coordinated with respect to NAA40 ribosome binding and activity. As recently shown in vitro, METAP1, NAC, and the acetyltransferase complex NatA/E can assemble at the peptide exit site simultaneously to presumably facilitate a seamless substrate handover between METAP1 and NatA/E 6,7. Since NatA/E rely on NAC in a similar fashion as NAA40 26 we next examined whether NAC could also coordinate the concomitant recruitment of METAP1 and NAA40. When superimposing our ex vivo NAC-NAA40 structure with ribosome-bound METAP1, no steric clashes were observed (Fig. 3g). Therefore, we performed another in vitro binding assay with NAA40-FLAG, 80S and METAP1 with or without NAC. As shown above, omission of NAC alone showed weak association of NAA40 with ribosomes (compare Fig. 3a), and notably also resulted in no co-enrichment of METAP1 26 (Fig. 3h and Supplementary Fig. 6l). However, inclusion of NAC resulted in enhanced NAA40 ribosome association and also binding of METAP1 (Fig. 3h), confirming the potential for a multienzyme assembly on the ribosome coordinated in the presence of NAC.

NAA40 and METAP1 can form a multienzyme assembly on the ribosome in vitro
To gain detailed molecular insights into the formation of a ternary complex consisting of NAA40, NAC and METAP1 on the ribosome, we in vitro reconstituted these components with purified 80S ribosomes and performed cryo-EM SPA. After extensive 3D classification (Supplementary Figs. 7 and 8a–d), we observed a class of particles that contained only NAA40 together with NAC, which essentially resembled our ex vivo cryo-EM data with respect to positioning of the factors and binding of the NACA UBA-domain to NAA40 (Supplementary Fig. 9a–d).

More interestingly, however, we isolated two classes of complexes with NAA40-NAC bound with additional density at the peptide exit tunnel site representing two different conformational states of METAP1 (State 1 and State 2) (Fig. 4a, b, Supplementary Figs. 7, 8, 10a, b). For both states, METAP1 was binding to the groove between ribosomal protein uL23 and rRNA helix H59 with the active site pointing towards the peptide exit site, as reported before 6,7,26 (Fig. 4c, Supplementary Fig. 10c, d). While the NACA UBA-domain on NAA40 was again clearly visible, density for the flexible zinc finger domain of METAP1 and the unstructured NACB C-terminus that contain the hydrophobic METAP1 interaction motif (NACB isoform 1: 190-VPDLV-194, isoform 2: 146–150) (Fig. 4d, e, Supplementary Figs. 10a, b and 10f–h) was not observed, as described previously 26.

When comparing the two conformations of METAP1, the enzyme was tilted closer toward the NAC globular domain and the peptide exit in State 1 than in State 2 (Fig. 4f). This slight conformational variability entails discrete differences for nascent chain length requirements for the enzymatic activity of METAP1. Therefore, the emerging peptide needs to span a distance of about 4.4 or 5.1 nm for State 1 or State 2, respectively (Fig. 4f). This translates to at least 12 or 14 amino acids that must have protruded from the ribosome exit and is in the same distance range as for NAA40 (12+ amino acids, compare Fig. 1d). The almost identical distances required to reach the active sites, as well as the dependency on the two different subunits of the NAC complex for NAA40 (NACA) and METAP1 (NACB), hint towards both enzymes being recruited simultaneously on the ribosome to process the nascent polypeptide chain and to potentially achieve rapid substrate exchange (Fig. 4g). This would essentially result in a similar situation as suggested for NatA/E which displayed co-association on the ribosome with NAC and METAP1 6,7. However, while METAP1 requires NAC for sufficient binding, METAP2 does not 26,30, even more so since its additional ribosome interaction domain that it shares with, e.g. EBP1, renders ribosome binding mutually exclusive with NAC 7. Yet, both MetAPs show a degree of functional redundancy in eukaryotes 31–33. Consistent with their overlapping functions but contrary to the contribution of NAC to NatA/E ribosome binding, a complex of METAP2-NatA-80S has recently been reconstituted in vitro and structurally characterized 7 (Supplementary Fig. 10i, j). For NAA40, on the other hand, simultaneous ribosome association with METAP2 is not possible as they would sterically clash (Supplementary Fig. 10k). Taken together, our structural data highlight the potential of NAC to orchestrate the coupling of methionine excision, specifically by METAP1, with Nt-acetylation by NAA40 on the ribosome. However, similar to previous reports on METAP1/2 and NatA/E, the data are largely in vitro based, and a sequential order of 80S binding and nascent chain modification events in vivo cannot be excluded for NAA40. Such a sequential order of interaction (Fig. 5) would be in agreement with our observation that we could not detect classes of ribosomes with NAA40, NAC and METAP1 bound concomitantly in our ex vivo preparations.

Methods
Factor-free 80S preparation
Empty 80S were prepared as described before 38. In short, FreeStyle 293-F cells were harvested by centrifugation at 250 x g for 5 min and washed once with 1x PBS. The cell pellet was resuspended in HS Buffer (20 mM HEPES/KOH pH 7.5, 500 mM KOAc, 2 mM MgCl2, 1 mM DTT) supplemented with 0.5% IGEPAL CA-630 (Sigma Aldrich) and incubated for 10 min. The resulting lysate was cleared by centrifugation at 2960 x g for 15 min and 36,500 x g for 25 min. Ribosomes were pelleted through a 1 M sucrose cushion in HS Buffer at 106,700 x g for 18.5 h using a Type Ti70 rotor (Beckman Coulter). The ribosome pellet was dissolved in HS Buffer, and potential aggregates were removed by centrifugation at 10,000 x g for 5 min. Ribosome subunits and nascent chains were dissociated by the addition of 1 mM puromycin and subsequent incubation for 15 min on ice and 10 min at 37 °C. Subunits were separated through a linear 10–40% (w/v) sucrose gradient in HS Buffer at 202,496 x g for 2.5 h using a SW 40 Ti rotor (Beckman Coulter). Fractions corresponding to 40S and 60S were separately collected and pelleted through a 1 M sucrose cushion in LS Buffer (20 mM HEPES/KOH pH 7.5, 100 mM KOAc, 10 mM MgCl2, 1 mM DTT) at 140,900 x g for 14 h in a Type Ti70 rotor (Beckman Coulter). Subunit pellets were resuspended in LS Buffer, and factor-free 80S were formed by incubating equimolar amounts of small and large subunits for 1 h on ice. To remove remaining subunits from the newly formed 80S, the mixture was separated through a linear 10–30% (w/v) sucrose gradient in LS Buffer at 202,496 x g for 2.5 h using a SW 40 Ti rotor (Beckman Coulter). The 80S fraction was pelleted through a 1 M sucrose cushion in LS Buffer at 494,648 x g for 1 h using a TLA-110 rotor (Beckman Coulter). Finally, the pellet was resuspended in LS Buffer, frozen in liquid nitrogen and stored at -80 °C.

NAA40-FLAG, NAA40S-FLAG and METAP1 protein purification
For NAA40 and NAA40S, Rosetta 2 (DE3) (Novagen) cells transformed with plasmids encoding the proteins with an N-terminal His6-SUMO and a C-terminal FLAG tag (see below). For METAP1, a plasmid encoding for His6-SUMO-METAP1 (see below) was transformed in Rosetta 2 (DE3) cells. For all proteins, cells were grown in LB medium to a density of 0.6–0.8 OD600 at 37 °C. Then, protein expression was induced with 0.1 mM IPTG at 18 °C overnight. Cells were harvested by centrifugation, washed once with 1x PBS and resuspended in Lysis Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 10 mM imidazole, 1 mM DTT, 10 μg/mL DNase I) before lysis in a continuous flow cell disruptor (Constant Systems). Cell lysates were cleared by centrifugation at 36,500 x g for 30 min and the cleared lysates were incubated with cOmplete Ni-metal affinity beads (Roche) for 1 h. Then beads were washed 3 times with HS-buffer (50 mM HEPES/NaOH pH 7.5, 500 mM NaCl, 10 mM imidazole, 5% glycerol, imidazole, 1 mM DTT) and 3 times with LS-buffer (50 mM HEPES/NaOH pH 7.5, 100 mM NaCl, 10 mM imidazole, 1 mM DTT). NAA40-FLAG and METAP1 were eluted in Elution Buffer (20 mM HEPES/NaOH, pH 7.5, 100 mM NaCl, 5% glycerol, 0.1 mg/mL SUMO-protease (homemade), 10 mM imidazole, 1 mM DTT) overnight.

NAA40S-FLAG was eluted with I-Elution Buffer (20 mM HEPES/NaOH, pH 7.5, 100 mM NaCl, 5% glycerol, 300 mM imidazole, 1 mM DTT). The tag was removed by dialysis in the presence of 0.01 mg/mL SUMO-protease (homemade) against Dialysis Buffer (20 mM HEPES/NaOH, pH 7.5, 100 mM NaCl, 10 mM imidazole, 1 mM DTT) overnight. SUMO protease was removed by running the sample over cOmplete Ni-metal affinity beads (Roche).

For both NAA40-FLAG and METAP1, elution fractions were applied to a HiTrap Q FF column (GE Healthcare). Proteins were eluted with a linear gradient from 100-500 mM NaCl in Ion Exchange Buffer (20 mM HEPES/NaOH, pH 8.0, 100 mM NaCl, 1 mM DTT). Protein-containing fractions were pooled, and buffer was exchanged using PD-10 desalting columns (Cytiva) to Storage Buffer (20 mM HEPES/KOH, pH 7.5, 100 mM KOAc, 5 mM MgCl2, 1 mM DTT.

NAA40S-FLAG was further purified exactly like NAA40-FLAG, but instead of an anion exchange column, a HiTrap SP FF column (GE Healthcare) was used.

NAC-wt and -ΔUBA purification
The genes coding for the human NAC heterodimer consisting of N-terminally His6-thrombin cleavage site-tagged NACA and NACB isoform 2 were expressed from pET28a (Novagen) (see below). Freshly transformed Escherichia coli ER2566 (NEB) were grown in LB medium at 37 °C to an OD600 of 0.8, induced by the addition of 1 mM IPTG and continued to grow for 3 h. Cells were lysed using a cell disruptor (Constant Systems Ltd.) at 30 K psi in lysis buffer (20 mM HEPES, pH 8.0, 500 mM NaCl, 0.1 mM PMSF, 20 μg/ml DNase I and 1x cComplete EDTA-free protease inhibitor cocktail (Roche). Lysates were clarified by centrifugation in a A27-8×50 rotor (Thermo Scientific) in a LYNX 6000 centrifuge (Sorvall) at 36,500 x g for 25 min. The supernatant was incubated with Protino Ni-NTA Agarose slurry (Macherey & Nagel) equilibrated in lysis buffer for 30 min at 4 °C. Beads were transferred to a gravity flow column, washed with 40 column volumes of lysis buffer and eluted with 5 column volumes of lysis buffer containing 500 mM imidazole. NAC-containing fractions were pooled and dialyzed (MWCO 12–14 kDa) overnight against buffer B (20 mM HEPES, pH 8.0, 50 mM NaCl, 2 mM CaCl2) in the presence of 20 units of thrombin (Merck) to remove the His6-tag. Dialyzed samples were loaded on a 5 ml HiTrap SP HP column (Cytiva) pre-equilibrated with 20 mM HEPES pH 7.0, 50 mM NaCl for removal of excess NACA and eluted with a 6 column volume linear gradient to 100% Buffer C (20 mM HEPES pH 7.0, 1 M NaCl). Heterodimeric NAC eluted at around 450 mM NaCl. PD10 columns (Cytivia) were used to transfer heterodimeric NAC fractions into the final Buffer D (20 mM HEPES pH 7.5, 100 mM KOAc, 5 mM MgCl2, 5% glycerol and 1 mM DTT). Protein concentration was determined using the molar extinction coefficient of human NAC (ε280 = 2980 M-1 cm−1).

The NAC-ΔUBA mutant was expressed and purified in the same way, with the exception that after the HiTrap SP HP column, NAC-containing fractions were pooled, concentrated using Amicon Ultra Centrifugal Filter (10 kDa Mw cutoff, Merck) and loaded on a HiLoad Superdex 75 pg 16 mm column (Cytiva) using Buffer E (20 mM HEPES/KOH, pH 7,5, 100 mM KOAc, 5 mM MgCl2, 1 mM DTT). NAC-containing fractions were pooled.

In vitro binding assay
Six picomoles 80S were incubated with 60 pmol of NAC, NAC-ΔUBA, NAA40-FLAG, NAA40S-FLAG or METAP1 in Binding Buffer (20 mM HEPES/KOH, pH 7,5, 100 mM KOAc, 5 mM MgCl2, 1 mM DTT) for 1.5 h. Mixtures were incubated with anti-FLAG M2 affinity gel (Sigma Aldrich) for 1 h. Beads were washed three times with Binding Buffer and eluted with Binding Buffer supplemented with 0.25 mg/mL 3x FLAG peptide (Sigma Aldrich) for 1.5 h. Input, flow-through and elution fractions were TCA precipitated and resuspended in Laemmli Buffer. Fractions were analyzed by SDS-PAGE.

Cell culture
Adherent HEK293T cells were cultured in DMEM (Gibco) supplemented with 1x GlutaMAX (Gibco), 1x Penicillin-Streptomycin (Gibco) and 10% FBS (Gibco) at 37 °C and 5% CO2. FreeStyle 293-F suspension cells were grown in HyClone HyCell TransFx-H (Cytiva) supplemented with 3× GlutaMAX (Gibco), 1x Penicillin-Streptomycin (Gibco) and 0.01% Poloxamer 188 (Sigma Aldrich) in a Multitron Cell shaker (Infors HT) at 37 °C, 80% humidity and 5% CO2. Adherent HCT116 cells were cultured in McCoy’s 5a medium (Gibco) supplemented with 10% FBS (Gibco, Invitrogen) and 1% Penicillin-Streptomycin (Gibco). Cells were grown in a humidified atmosphere at 37 °C containing 5% CO2. The HCT116 cell line was used to generate the NAA40−/− cell line (NAA40-KO) using CRISPR, as previously described 29, or for the ectopic expression of wild-type NAA40-V5, as previously described 39.

Native NAA40 ribosome complex isolation
Half confluent HEK293T cells were transiently transfected with 0.5 µg/mL pcDNA5/FRT/TO-NAA40-3C-3xFLAG plasmid for 24 h using PEI. Then, cells were harvested by scraping, pelleted at 250 x g for 5 min and washed once with 1x PBS. The cell pellet was resuspended in Lysis Buffer (20 mM HEPES pH 7.5, 150 mM KOAc, 5 mM MgCl2, 1 mM DTT, 0.1% IGEPAL CA-630 (Sigma Aldrich), 1× cOmplete EDTA-free Protease Inhibitor Cocktail (Roche)) and sonicated with four 10 s pulses and 30 s on ice in between. The lysate was cleared by two consecutive centrifugation steps at 2960 x g for 15 min and 36,500 x g for 25 min, respectively, followed by incubation for 1.5 h with anti-FLAG M2 affinity gel (Sigma Aldrich) pre-equilibrated in Lysis Buffer. After beads were washed twice with Wash Buffer (20 mM HEPES pH 7.5, 150 mM KOAc, 5 mM MgCl2, 1 mM DTT, 0.01% IGEPAL CA-630) and once with Elution Buffer (20 mM HEPES pH 7.5, 150 mM KOAc, 5 mM MgCl2, 1 mM DTT, 0.05% octaethylene glycol monododecyl ether) in batch, they were transferred to a 1 mL Mobicol (MoBiTec) spin column. Then, the beads were washed once more with Elution Buffer before elution for 1.5 h at 4 °C in Elution Buffer supplemented with 0.25 mg/mL HRV3C protease (homemade). The elution was collected by centrifugation at 450 x g for 1 min and either directly used for cryo-EM grid preparation or subjected to SDS-PAGE. Prominent protein bands were excised and subjected to mass spectrometry (MS) analysis by our in-house MS facility (Supplementary Data 1) to identify and annotate the respective proteins in Fig. 1a.

NAA40-NAC-METAP1-80S in vitro reconstitution
One hundred eighty nanomolar factor-free 80S were incubated with 1.08 µM NAC in Reconstitution Buffer (20 mM HEPES pH 7.5, 100 mM KOAc, 5 mM MgCl2, 1 mM DTT, 0.05% octaethylene glycol monododecyl ether) for 10 min, followed by the addition of 1.08 µM METAP1 and incubation for 10 min. Lastly, 1.08 µM NAA40-FLAG were added and incubated for another 30 min. Then, the reconstitution mixture was directly subjected to cryo-EM grid preparation.

Cryo-EM sample preparation
For all samples, 3.5 µL were applied to R3/3 copper grids with an additional 3 nm carbon layer (Quantifoil) using a Vitrobot (FEI) at 5 °C and 85–90% humidity. Grids were blotted for 3 s with 0 blot force and 45 s pre-blot time before plunge freezing in liquid ethane.

Cryo-EM data collection and processing
All cryo-EM datasets were collected on a Titan Krios (FEI) at 300 kV equipped with a Falcon 4i direct electron detector (Thermo Fisher) and a Selectris X energy filter (Thermo Fisher) at 5 eV slit width using EPU (v3.3.1) (Thermo Fisher). A nominal pixel size of 0.727 Å/pixel, a total dose of 40 e-/Å2 and a defocus range of 0.5–3.5 µm were used. Motion correction was performed using MotionCor2 40, and initial CTF parameters were determined using CTFFIND4 41. Micrographs with a resolution worse than 5 Å were removed.

For the native NAA40-NAC 80S sample, a total of 42,123 micrographs were used, and particles were picked using crYOLO 42 (v1.7.6) using the general model on lowpass filtered micrographs (Supplementary Fig. 1). Then, particles were extracted with 160 pixels box size, four times binned in Relion 43 (4.0.1) and imported in cryoSPARC 44 (v4.4.0) for 2D classification. Classes corresponding to 80S ribosomes (306,751 particles) were selected and refined using homogenous refinement with an ab initio generated volume as a reference. After 3D classification, all ribosome classes with distinct exit density were combined and refined together (239,101 particles). Using a small spherical mask, particles were classified for the presence of NAA40 and in a second classification step, sorted for the presence of stable NAA40-NAC complexes using a wider mask around the peptide exit in cryoSPARC. The resulting particles were re-imported into Relion and further classified with a mask around NAC. Particles with good density for NAC and NAA40 (47,197 particles) were re-extracted with an unbinned box size of 640 pixels. The un-binned particles resembling an inactive translation state were refined in cryoSPARC using homogenous refinement with global CTF and per particle defocus refinement to a resolution of 2.67 Å in cryoSPARC and subsequently local resolution filtered (Supplementary Fig. 2a-d upper panels, Supplementary Table 1). To isolate active ribosomes, a new round of unmasked 3D classification was performed in cryoSPARC, starting from the initial 3D refinement in cryoSPARC. A class with both tRNAs in the intersubunit space and density for NAC-NAA40 at the exit was selected (66,717 particles). Particles were then sorted for the presence of NAC-NAA40 with a wide mask around the peptide exit site. Particles containing stable NAC-NAA40 density (28,948 particles) were further sorted for their translational state with a mask around the intersubunit space, resulting in a defined P/PE/E POST-state (19,178 particles). The final active 80S-NAA40-NAC particles were re-extract un-binned with a box size of 640 pixels and refined using homogenous refinement with global CTF and per particle defocus refinement in cryoSPARC to a resolution of 2.91 Å (Supplementary Fig. 2a–d, lower panels and Supplementary Table 1), followed by local resolution filtering. To obtain nucleosome particles, classes excluded after the initial 2D classification were subjected to another round of reference-free 2D classification, which resulted in a few 2D classes representative for nucleosome side views (Supplementary Fig. 1; 11,799 particles). However, further processing or particle enrichment efforts were unsuccessful.

For the in vitro reconstituted METAP1-NAA40-NAC-80S sample, a total of 63,237 micrographs were collected. Initial particles were picked using crYOLO 42 (v1.7.6) with the general model on low-pass filtered micrographs (Supplementary Fig. 7). Particles were extracted four times, binned with a box size of 160 pixels in Relion 43 (v4.0.1) and imported into cryoSPARC 44 (v4.4.0) for 2D classification. Good 80S classes were selected (805,997 particles) and 3D refined using an ab initio reference. After 3D classification, particles corresponding to 80S with additional density at the peptide exit were selected (466,068 particles) and further sorted for exit site density with a wide mask. The resulting particles (167,604 particles) were re-imported to Relion and sorted for the presence of METAP1 with a spherical mask around NAA40 and the METAP1 binding site. Particles lacking METAP1 (72,590) were further classified with a wide spherical mask around the peptide exit site to obtain good density for NAA40-NAC (26,095 particles). The NAA40-NAC-80S particles were re-extract in Relion with an unbinned box size of 640 pixels. They were re-imported into cryoSPARC and refined using homogenous refinement with global CTF and per particle defocus refinement to a resolution of 2.72 Å (Supplementary Fig. 8a–d lower panels and Supplementary Table 1), followed by local resolution filtering. A class containing density for METAP1 from the second focused 3D classification (Supplementary Fig. 7) was further classified in Relion with a spherical mask around METAP1, resulting in two different conformations of METAP1. Each class was then separately classified again with a wide spherical mask around the exit for stable density, resulting in 11,809 particles for METAP1-State 1 (S1) and 15,772 particles for METAP1-State 2. For both states, particles were re-extracted with an unbinned box size of 640 pixels and refined in cryoSPARC using homogeneous refinement with global CTF and per particle defocus refinement to resolutions of 3.54 Å for METAP1-State 1 (Supplementary Fig. 8a–d upper panels and Supplementary Table 1) and 3.44 Å for METAP1-State 2 (Supplementary Fig. 8a–d middle panels and Supplementary Table 1). Finally, volumes were local resolution filtered.

All cryo-EM densities were visualized using ChimeraX 45 (v1.9).

Model building
For the molecular model of the ex vivo combined inactive 80S-NAA40-NAC reconstruction, PDB: 6Z6M 20 was used as the base model for the ribosome, SERBP1 and eEF2. The ribosome-bound NAC globular domain and NACB ribosome anchor were taken from PDB: 7QWR 27 and rigidly docked into the densities. For NAA40 with the NACA-UBA domain, our AF2 multimer model was used and a rigid body fit.

For the model of the ex vivo active 80S-NAA40-NAC reconstruction, the ribosomal model from PDB: 6Y2L 46 was used. Models for ribosomal proteins eS12 and eS31 were taken from PDB: 8GLP 47. The P- and E-site tRNAs from PDB: 6GZ5 19 were used as placeholders for our tRNAs. Placeholders for mRNA and nascent chain were built de novo.

For the NACB ribosome anchor and the NAC globular domain PDB: 7QWR 27 was used again, and both NAC parts were independently fit initially. NAA40 with the NACA-UBA domain from our AF2 multimer model were rigidly docked.

For all in vitro reconstituted 80S complexes, the initial factor-free 80S model from PDB: 8YOO 38, the AF2 multimer prediction for NAA40 with the NACA-UBA domain and the AF2 multimer prediction for the NAC globular domain and the NACB ribosome anchor were used. All ribosome-bound factors were rigidly docked. For 80S-NAA40-METAP1-NAC State 1 and State 2, the AF2 database 23 model for METAP1 was rigid-body fitted, and the zinc finger domain was removed manually.

All models were real-space refined in Phenix 48 (v1.19.2-4158 and v1.21.2-5419) and manually adjusted in Coot 49 (v0.9.8). Molecular models were visualized with ChimeraX 45 (v1.9).

RNA extraction and quantitative Real Time PCR (qRT-PCR)
Total RNA was extracted using the RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. An amount of 0.5 μg total RNA was then reverse transcribed to complementary DNA using the PrimeScript RT reagent kit (Takara) with random primers. qRT-PCR was carried out using KAPA SYBR Green (SYBR Green Fast qPCR Master Mix) and the Biorad CFX96 Real-Time System. Expression data were normalized to the mRNA levels of the β-actin housekeeping gene and calculated using the 2−ΔΔCt method. Primer sequences were obtained from IDT (Supplementary Table 2).

Protein extraction
For whole-cell protein extraction, cell pellets were resuspended in 1x PBS at a ratio of two volumes of 1x PBS to one volume of cell pellet. Protein extracts were obtained by adding by adding an equal volume of 2x lysis buffer to the resuspended cells (0.1 M Tris-HCl, pH 7, 4% SDS, and 12% β-mercaptoethanol), resulting in a final 1x concentration. The samples were then boiled at 95 °C for 10 min, followed by centrifugation at 12,000 x g for 10 min at 4 °C to isolate proteins. Total protein concentration was quantified using a Nanodrop ND-1000. For histone acid extraction, cells were lysed in hypotonic lysis buffer (10 mM Tris-HCL, pH 8, 1 mM KCL, 1.5 mM MgCl2, 0.1% Triton X-100 and 1x protease inhibitor cocktail) and incubated for 30 min with constant agitation at 4 °C. Isolated nuclei were then washed once in hypotonic lysis buffer and, after centrifugation at 6500 x g for 10 min, were resuspended in 0.2 M HCL (4 × 107 nuclei per mL) and incubated overnight with constant rotation at 4 °C. Histones were isolated by centrifugation at 6500 x g for 10 min, and the pH was neutralized with 2 M NaOH at 1/10 of the volume of the supernatant.

Biochemical fractionation
Ten million cells were harvested in 1x PBS and lysed in Buffer S (10 mM HEPES, 10 mM KCL, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol) plus 0.1% Triton X-100 and 1x protease inhibitor cocktail) on ice for 10 min. Following centrifugation at 1300 x g for 5 min at 4 °C, the supernatant S1 was centrifuged at maximum speed for 10 min, and the supernatant S2 was taken as the cytoplasmic fraction. Pellet was washed with 5× pellet volume nuclear extraction Buffer A (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl) plus 100 mM NaCl and 1 mM DTT, followed by centrifuge at 3000 x g for 5 min at 4 °C.

In order to purify the nuclear fractions, pellets were then resuspended in a low-salt lysis buffer (20 mM HEPES, 1.5 mM MgCl2, 150 mM NaCl, 0.2 mM EDTA, 25% glycerol) plus 0.1% v/v NP-40, 0.2 mM DTT and protease inhibitors (0.5 mM PMSF and 1x protease inhibitor cocktail) and treated with 200 units/mL of benzonase (Sigma E1014) for 15 min at 37 °C. Following sonication for 2 × 5 s pulses with a Bioruptor (Diagenode), samples were centrifuged at 16,000 x g for 10 min at 4 °C to obtain the supernatant that represents the low-salt nuclear fraction.

Immunoblotting
Twenty micrograms of protein extract or three micrograms of histone extract were separated on SDS-PAGE and then transferred to a nitrocellulose membrane (GE Healthcare). After blocking with 3% TBS-T/BSA for 1 h at RT, the membranes were incubated with the primary antibodies overnight at 4 °C. The primary antibodies used were as follows: H2A/H4S1Ph (1:1,000, Abcam, ab177309, LOT: GR214941-1, Clone EPR18184), H4 (1:1,000, Millipore, 05-858, LOT: 3836545, Clone 62-141-13), H2A (1:1,000, Millipore, 07-146, LOT: 1970740, Polyclonal), ACTIN (1:1,000, Abcam, ab8227, LOT: 1065644-1, Polyclonal), V5 (1:1,000, Abcam, ab27671, LOT: GR322548-8, Clone SV5-Pk1), GAPDH (1:2,000, Abcam, ab9485, LOT: GR3318490-1, Polyclonal), NAA40 (1:1,000, Abcam, ab106408, LOT: GR3409009-8, Polyclonal), FLAG (1:1,000, Sigma-Aldrich, F1804, Clone: M2). For secondary antibody, a horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:30,000, Thermo Scientific, 31462, LOT: 095113I, Polyclonal), or an HRP-conjugated goat anti-mouse IgG (1:20,000, Thermo Scientific, A16078, LOT: 86-132-110221, Polyclonal) was used.

Transient RNA interference
HCT116 cell lines were seeded in antibiotic-free medium and grown to 60% confluence at the time of transfection. Subsequently, the cells were transiently transfected with 50 nM of siNACA (L-027161-00-0005, Horizon discovery) or the negative control (D-001810-10-05, Horizon discovery) for 72 h using DharmaFECT 1 Transfection Reagent (Horizon discovery) according to the manufacturer’s instructions.

Transient exogenous expression for NACA complementation
Human siRNA-resistant pcDNA5/FRT/TO-FLAG-NACA and pcDNA5/FRT/TO-FLAG-NACA-ΔUBA plasmids were transfected into HCT116 cells. In brief, cells were seeded in antibiotic-free medium and grown to 60% confluence at the time of the transfection. Cells were transfected with 2 μg plasmid DNA carrying human FLAG-NACA or FLAG-NACA ΔUBA using FuGENE HD (Promega Corporation) according to the manufacturer’s instructions. Cells were harvested 48 h post-transfection.

Generation of Nt-Ac antibody
Rabbit polyclonal antibodies were raised in collaboration with LifeTein LLC (USA). To generate the Nt-Ac antibody, the animal was immunized with a Keyhole Limpet Hemocyanin-conjugated synthetic tetravalent Multiple Antigenic Peptide (MAP4-C) containing the acetylated sequence Ac-SGRGK on a lysine-based core. The peptides used were not biotinylated. Serum was affinity-purified by the manufacturer against peptides with and without N-terminal acetylation to enrich for recognizing N-terminal acetylation. After the purification, antibody specificity was determined by enzyme-linked immunosorbent assay using the specific and non-specific peptides as antigens.

Dot blot analysis
Synthesized biotinylated peptides with at least 95% purity (BIO-SYNTAN GmbH) were dissolved in water, and drops containing 25, 20, or 15 μΜ were deposited on a PVDF membrane, and allowed to air-dry for 1 h. The membrane was then submerged in 100% methanol for 1 min and 1x PBS for another minute. After blocking with 3% TBS-T/BSA for 1 h at RT, the membranes were incubated with the primary antibodies overnight at 4 °C. The primary antibodies used were as follows: Biotin (1:1,000, Santa Cruz, sc-101339, LOT: J1019, Clone 33) and the Nt-Ac antibody (1:1,000) generated in collaboration with LifeTain LLC (USA). For secondary antibody, an HRP-conjugated goat anti-rabbit IgG (1:30,000, Thermo Scientific, 31462, LOT: 095113I, Polyclonal) was used.

Molecular cloning
For overexpression of NAA40-3C cleavage site-3×FLAG in human cells, the coding sequence was amplified from cDNA and cloned into a pcDNA5/FRT/TO-3C-3×FLAG plasmid using BamHI and XhoI restriction sites. For expression of FLAG-NACA and -NACA-ΔUBA expression in human cells, the NACA coding sequence or the ΔUBA fragment was amplified from cDNA and cloned into a pcDNA5/FRT/TO-FLAG plasmid using BamHI and XhoI restriction sites. For siRNA-resistant NACA and NACA-ΔUBA expression constructs, fragments from the respective pcDNA5 plasmids were amplified with mutations and complementary overhangs by PCR and assembled using FastCloning 50. For recombinant purification of NAA40- and NAA40S-FLAG, the NAA40 coding sequence and a C-terminal FLAG-tag were simultaneously inserted into a pET-28a plasmid (Novagen) with an N-terminal His6-SUMO tag using FastCloning 50. NAA40S was inserted the same way into the expression plasmid. For recombinant purification of METAP1, the coding sequence was inserted into a pET-28a plasmid (Novagen) with an N-terminal His6-SUMO tag using FastCloning 50. For recombinant purification of NAC or NAC-ΔUBA complexes, the sequences for NACA or NACA-ΔUBA (1–173 aa) were amplified and cloned into a pET-28a plasmid (Novagen) with an N-terminal His6-thrombin cleavage site-tag using NdeI and BamHI restriction sites. In the resulting plasmids, the coding sequence for NACB isoform 2 with a preceding ribosome binding site was cloned into the same vector using EcoRI and NotI restriction sites. Primer sequences are listed in Supplementary Table 2.

Statistics and reproducibility
Representative biochemical experiments were repeated at least once with highly similar results. Cryo-EM data for ex vivo and in vitro samples were collected once. For quantification of Western Blots and knockdown validation by qPCR, statistical tests and number of biological replicates are indicated. Mean values and individual data points are displayed. Error bars representing the standard error of the mean (SEM) are shown.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Guan et al. combine immunopurification, cryo-electron microscopy, and biochemical assays to reveal how NAA40 engages the ribosome during translation. They show that NAA40 associates with ribosomes in vivo and that this interaction depends on the nascent polypeptide–associated complex (NAC). Structural analyses place NAA40 and NAC adjacent to the peptide exit tunnel, where the positively charged α0 helix of NAA40 functions as a ribosome anchor in a manner reminiscent of NatA/E. Functional experiments demonstrate that the UBA domain of NACA is essential for stable NAA40 recruitment and efficient histone acetylation. Moreover, reconstitution assays reveal that NAA40 can form a multienzyme assembly with methionine aminopeptidase 1 (MetAP1) and NAC, suggesting coordinated removal of the initiator methionine and subsequent acetylation. Together, the authors delineate the molecular architecture and cofactors underlying co-translational histone N-terminal acetylation by NAA40 and highlight the dual potential of NAA40 for both co- and post-translational activity.\nThis study provides new mechanistic insights into NatD function and establishes NAC as a general recruiter of co-translational modifying enzymes. The work will be of interest to structural biologists, by extending our knowledge of ribosome-bound modification factors; to epigeneticists, by clarifying the timing and regulation of histone acetylation; and to the broader field of protein biogenesis and translational control, by illustrating how multiple enzymatic activities are coordinated at the ribosomal exit site. The manuscript is clearly written and the figures well presented. We recommend publication after the following points are addressed.\nGeneral points:\nWhile the structural and biochemical data convincingly reveal how NAA40 can be recruited to the ribosome and coordinated with NAC and METAP1, much of the evidence relies on in vitro reconstitution. The authors do point this out in the text (e.g. lines 254–257, 280–283), but it would be helpful if they more clearly emphasized the current lack of full in vivo confirmation and discussed how future work might address this.\nMinor points\n1. Please check whether referencing in the abstract complies with Nat. Comms. guidelines.\n2. Line 49: write out the abbreviations “Ph” and “me” in full (or in parentheses) at first mention.\n3. Fig. 1a: Clarify how histones, XRCC5/6, and PRKDC were identified (e.g. MS, immunoblotting) and provide supporting data. In addition, there is no negative control IP (e.g. FLAG-empty, untagged, or unrelated FLAG-protein) to assess specificity.\n4. Line 88-90 and line 92 is referenced to Supplementary Fig. 1a and b, but should be 2a and b. In the text is written AF2, in the figure legend AF3.\n5. Fig. 3a: there is a quite striking difference between lane 7 and 9. From the presented data, it seems likely, NACΔUBA occludes NAA40’s weak direct binding to the ribosome. This could be tested by adding increasing amounts of NAC-deltaUBA to see if it displaces NAA40 from the ribosomes. Alternatively, titrating NAC vs. NAC-deltaUBA would strengthen the case that UBA-dependent binding is specific, not an artefact. Rephrase then the statement that NAC-ΔUBA “abolishes” ribosome binding accordingly. The assay is qualitative; additional controls would strengthen the data (e.g. NatA) or quantification or maybe mutants in the N-terminus of NAA40. Shift the legend in 3a slightly upward for clarity.\n6. Antibody validation (Fig. 5e): Were the peptides biotinylated? It is nowhere stated in the methods and not clear. Was the antibody further purified to ensure Ac-binding , e.g. against free-Nt peptides binding and the FT against Ac-Nt binding?\n7. Line 186: Residual Nt-acetylation in NACA-KD cells may also result from incomplete knockdown.\n8. There is missing one H.S. in Supplementary Fig. 5h", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "This study provides compelling structural evidence for NAC-mediated recruitment of NAA40 to ribosomes. Combined with biochemical data demonstrating that NACA knockdown reduces H4/H2A acetylation, these findings significantly advance our understanding of co-translational histone modification. Overall, this is an excellent study with high quality in experimental data and data presentation. However, the following major and minor issues require clarification in order to strengthen the manuscript.\nMajor Issues:\n1. NAA40 binds both hibernating and active ribosomes, yet the cryo-EM structures show no engagement of its catalytic domain with the cognate substrate motif (Ser-Gly-Arg-Gly). This may arise from non-physiological NAA40 overexpression, permitting indiscriminate ribosome association irrespective of nascent chain identity. To ensure substrate binding ability of NAA40., I recommend the authors to perform AlphaFold-Multimer predictions of NAA40 bound to H2A/H4 N-termini and check whether this binding mode conflicts with resolved NAA40-ribosome interactions.\n2. The NAA40 α0 helix mediates ribosome anchoring via rRNA (H19/H24/H46)—an interaction that is nascent chain-independent and topologically similar to NatA/E complexes. Given that NATs compete for overlapping ribosomal sites, I recommend the authors to discuss how NAC orchestrates histone-specific modification despite non-specific ribosome anchoring of NAA40.\nMinor Issues:\n1. Include pLDDT coloring and PAE metrics for all AlphaFold-predicted interactions (e.g., NAA40-NAC interfaces) to validate model confidence.\n2. \"Horseradish peroxide\" should be corrected to horseradish peroxidase (HRP) in Methods.", "reviewer_4": "The paper provides novel structural insights into the interaction of the shortest Nat protein NatD to the ribosome. In contrast to other Nats this protein consists of a single subunit, NAA40, which specifically acetylates Histones H2A and H4 after their processing by MetAP1. NAA40 thereby is recruited close to the ribosome exit tunnel by interaction with NAC in a similar manner as shown previously for NatA and NatE. A critical role in recruitment seems to play the highly charged N-domain of NAA40 and the NACA Uba domain. Based on their structural data, the authors suggest that MetAP1 and NAA40 might be recruited concomitantly to the exit tunnel, possibly indicating a coordinated cleavage and acetylation reaction. The conclusions drawn from the structural data are supported by a variety of different biochemical experiments. Both, the structural data and the biochemical data, are of very high quality and seem to be consistent with the proposed mechanisms. The manuscript is very well written and most of the Figures are very well prepared. Together with the importance of the NAA40 for its possible function in various tumors and our still limited understanding of the N-terminal modification reactions I strongly recommend this manuscript for publication in Nature Communications.\nThere are only some minor queries that should be addressed by the authors.\nI would appreciate, if the authors could discuss the unique substrate specificity of NAA40 for H2A and H4 in some more detail.\nGiven the copurification of XRCC5, XRCC6 and PRKDC a more global analysis of the extract shown in Fig. 1a by qMS might provide deeper and novel insights into the substrate specificity of NAA40. It would also be interesting if gammaH2A is present in the sample.\nline 289 and 290: If the N-terminal helix of NAA40 is a major determinant for ribosome binding and also harbors the nuclear localization signal, it is unlikely that the N-terminally truncated version serves in “potential nuclear post translational activity”. The authors should better explain what they mean here.\nMinor:\n1: some of the Figures are too small to really see details and should be enlarged by the authors. This includes Figure 2a,3g, 4c, Extended Data Fig 1e, Extended Data Fig 2e, Extended Data Figure 3g and 3h and the Extended Data Figure 6b to 6e.\n2: On top of Figure 3a the legends table needs to be adjusted.\n3: Not sure what the authors mean with the question mark in Figure 3g. This should be removed or described in the legend.\n4: Figure 4a and b. I suppose the shown maps are filtered, which should be indicated in the text of the legend.\n5: Figure 5: the colors of NACB and A are too pale and should be bit darkened to provide better contrast to the gray large subunit.\n6: Legend to Extended Data Figure 3: (line 797) f should be substituted by h\n7: there happened some misassignment of Figures in the text: e.g. line 89/90 Extended Data Figure 1a actually refers to 2a: line 95 Extended Data Fig. 1a…;\n8: I suppose the sentence in lines 214/215 refers to the experimental data in Fig. 3 and not to the structural data as indicated.\n9: Line 140: references missing for NatA/E"}, "author_response": "Point-by-point response\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nRemarks to the authors:\nGuan et al. combine immunopurification, cryo-electron microscopy, and biochemical assays to\nreveal how NAA40 engages the ribosome during translation. They show that NAA40 associates\nwith ribosomes in vivo and that this interaction depends on the nascent polypeptide–associated\ncomplex (NAC). Structural analyses place NAA40 and NAC adjacent to the peptide exit tunnel,\nwhere the positively charged α0 helix of NAA40 functions as a ribosome anchor in a manner\nreminiscent of NatA/E. Functional experiments demonstrate that the UBA domain of NACA is\nessential for stable NAA40 recruitment and efficient histone acetylation. Moreover, reconstitution\nassays reveal that NAA40 can form a multienzyme assembly with methionine aminopeptidase 1\n(MetAP1) and NAC, suggesting coordinated removal of the initiator methionine and subsequent\nacetylation. Together, the authors delineate the molecular architecture and cofactors underlying\nco-translational histone N-terminal acetylation by NAA40 and highlight the dual potential of\nNAA40 for both co- and post-translational activity.\nThis study provides new mechanistic insights into NatD function and establishes NAC as a general\nrecruiter of co-translational modifying enzymes. The work will be of interest to structural\nbiologists, by extending our knowledge of ribosome-bound modification factors; to\nepigeneticists, by clarifying the timing and regulation of histone acetylation; and to the broader\nfield of protein biogenesis and translational control, by illustrating how multiple enzymatic\nactivities are coordinated at the ribosomal exit site. The manuscript is clearly written and the\nfigures well presented. We recommend publication after the following points are addressed.\nWe are happy about this reviewer’s overall very positive evaluation.\nGeneral points:\nWhile the structural and biochemical data convincingly reveal how NAA40 can be recruited to the\nribosome and coordinated with NAC and METAP1, much of the evidence relies on in vitro\nreconstitution. The authors do point this out in the text (e.g. lines 254–257, 280–283), but it\nwould be helpful if they more clearly emphasized the current lack of full in vivo confirmation and\ndiscussed how future work might address this.\nWe agree with the reviewer that the lack of in vivo confirmation should be clearly stressed,\nespecially since all current data on multi-enzyme/-factor assembly like METAP1-NAA40, METAP1-\nNatA or METAP2-NatA heavily rely on in vitro data. Therefore, we expanded the second chapter\nof the discussion where we already touch on the subject further (lines 305-309) highlighting\nfuture advances in in situ cryo-EM and in cell (sm)FRET.\nMinor points\n1. Please check whether referencing in the abstract complies with Nat. Comms. guidelines.\nWe thank the reviewer for the advice. To comply with Nat. Comms. formatting guidelines we\nremoved all references from the abstract.\n2. Line 49: write out the abbreviations “Ph” and “me” in full (or in parentheses) at first mention.\nWe wrote out the two abbreviations at first mention in the introduction in parentheses.\n3. Fig. 1a: Clarify how histones, XRCC5/6, and PRKDC were identified (e.g. MS, immunoblotting)\nand provide supporting data. In addition, there is no negative control IP (e.g. FLAG-empty,\nuntagged, or unrelated FLAG-protein) to assess specificity.\nHistones, XRCC5/6 and PRKDC were identified by MS of SDS-PAGE gel cutouts of prominent bands\nobserved after Coomassie staining. MS was performed and results analyzed by our in-house MS\nfacility. This is now more clearly stated in the Methods section (lines 439-441) and indicated in\nthe Figure legend. Additionally, a supplementary table with the MS results is attached as\nSupplementary Data Table 1. In the case presented in Fig. 1a, no negative control is necessary\nsince no quantitative assessment has been made, simply an inventory of co-enriched proteins.\nFurthermore, (over-) expression of FLAG-tagged proteins followed by native co-IP has been\nperformed multiple times from human cells with different bait proteins before throughout\nliterature (e.g. Thoms M. et al. Science 2020; ; Huso V. L. et al. Nature 2025; Fiorentino F. et al.\nNucleic Acids Research 2025; Ameismeier M. et al. Nature 2020; etc.) showing no unspecific co-\npurification of histones, XRCC5/6 or PRKDC, especially not at a Coomassie stainable level. Most\nimportantly, this study is not focussing on expanding or completing the set of interactors for\nNAA40 but specifically on characterizing the co-translational activity of NAA40. Regardless,\nbacked by MS and a recent publication that finds involvement of the yeast NAA40 homolog in\nDNA damage signaling (Klavaris A. et al. Epigenetics Chromatin 2025), we feel that co-enrichment\nof XRCC5/6 and PRKDC is noteworthy and might even provide the community with starting points\nfor further investigations for the role of NAA40 in e.g. DNA damage or its nuclear role besides co-\ntranslational modification in the cytoplasm.\n4. Line 88-90 and line 92 is referenced to Supplementary Fig. 1a and b, but should be 2a and b. In\nthe text is written AF2, in the figure legend AF3.\nWe apologize for the mistake. Now, Supplementary Figures 3a-c and 3d are correctly referenced\nin lines 92-93 and line 95. We also corrected the Supplementary Figure legend to AF2 and 3 for\neither NAC-NAA40 or NAC.\n5. Fig. 3a: there is a quite striking difference between lane 7 and 9. From the presented data, it\nseems likely, NACΔUBA occludes NAA40’s weak direct binding to the ribosome. This could be\ntested by adding increasing amounts of NAC-deltaUBA to see if it displaces NAA40 from the\nribosomes. Alternatively, titrating NAC vs. NAC-deltaUBA would strengthen the case that UBA-\ndependent binding is specific, not an artefact. Rephrase then the statement that NAC-ΔUBA\n“abolishes” ribosome binding accordingly. The assay is qualitative; additional controls would\nstrengthen the data (e.g. NatA) or quantification or maybe mutants in the N-terminus of NAA40.\nShift the legend in 3a slightly upward for clarity.\nAs suggested by the reviewer we performed an additional binding assay following our original\nexperimental setup but in which we titrate NAC-wt vs NAC-deltaUBA. As expected, with\nincreasing amounts of NAC-deltaUBA the co-precipitation/binding of ribosomes decreased until\nagain no ribosomes were bound with NAC-deltaUBA alone (see new Supplementary Fig. 6e).\nInversely, the amount of non-bound ribosomes in the flow-through fraction increases with the\nfraction of NAC-deltaUBA added (see new Supplementary Fig. 6f). We added a description to the\nResults section as requested (lines 163-170). While we cannot provide a definitive answer to this\nobservation of apparent competitive binding, we speculate that the loss of ribosome binding of\nNAA40 with the NAC-deltUBA might be caused by the flexible positioning of the NAC globular\ndomain around the exit tunnel site leading to potential steric overlaps with NAA40 (more\nspecifically its GNAT domain), and might therefore prohibit the interaction of (on its own weakly\nassociated) NAA40 with the ribosome. This way, the UBA domain might provide an “allosteric\neffect” that mitigates the proper positioning of NAC on one hand and also enhances the binding\nof NAA40 on the other.\n6. Antibody validation (Fig. 5e): Were the peptides biotinylated? It is nowhere stated in the\nmethods and not clear. Was the antibody further purified to ensure Ac-binding , e.g. against free-\nNt peptides binding and the FT against Ac-Nt binding?\nWe thank the reviewer for the comment. The peptides used for antibody generation and\nspecificity testing were not biotinylated. The antibody was affinity-purified against peptides with\nand without N-terminal acetylation to ensure strict specificity for the N-terminally acetylated\nepitope. We also clarified that the peptides used for the Dot Blot Analysis were biotinylated\n(Supplementary Fig. 6g). This information has now been added in the revised Methods section\nand Figure legend.\n7. Line 186: Residual Nt-acetylation in NACA-KD cells may also result from incomplete knockdown.\nWe thank the reviewer for this comment. qPCR analysis demonstrates that NACA transcript levels\nare reduced almost entirely in the knock-down cells (Supplementary Fig. 6i). While these data\nindicate that the knock-down is very effective, we cannot exclude the possibility that residual\nNACA protein or remaining NAC function contributes to the observed residual Nt-acetylation. We\nhave clarified this point in the revised manuscript.\n8. There is missing one H.S. in Supplementary Fig. 5h\nWe apologize for the oversight. H.S. was added to the Western blot annotation in Supplementary\nFig. 6j.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nSee replies to other reviewers. Thank you for your efforts in reviewing this manuscript.\nReviewer #3 (Remarks to the Author):\nThis study provides compelling structural evidence for NAC-mediated recruitment of NAA40 to\nribosomes. Combined with biochemical data demonstrating that NACA knockdown reduces\nH4/H2A acetylation, these findings significantly advance our understanding of co-translational\nhistone modification. Overall, this is an excellent study with high quality in experimental data and\ndata presentation. However, the following major and minor issues require clarification in order to\nstrengthen the manuscript.\nWe are happy about this reviewer’s overall very positive evaluation.\nMajor Issues:\n1. NAA40 binds both hibernating and active ribosomes, yet the cryo-EM structures show no\nengagement of its catalytic domain with the cognate substrate motif (Ser-Gly-Arg-Gly). This may\narise from non-physiological NAA40 overexpression, permitting indiscriminate ribosome\nassociation irrespective of nascent chain identity. To ensure substrate binding ability of NAA40., I\nrecommend the authors to perform AlphaFold-Multimer predictions of NAA40 bound to H2A/H4\nN-termini and check whether this binding mode conflicts with resolved NAA40-ribosome\ninteractions.\nWe agree with the reviewer that overexpression can generally lead to increased indiscriminate\nribosome association as becomes obvious with the large portion of NAA40-NAC bound\nhibernating ribosomes in our ex vivo cryo-EM data. However, in the manuscript we use the\nsmaller subset of actively translating NAA40-NAC ribosomes to estimated the nascent chain\nlength requirements for NAA40 mediated nascent histone acetylation (Fig. 1d). Since we cannot\nconfidently assign density of the nascent chain within the ribosome or NAA40 to either of the (at\nleast) two histone substrates we use a substrate analog bound crystal structure (Supplementary\nFig. 3g) overlaid with our structure as reference. This crystal structure is essentially identical with\nanother published crystal structure of N-terminal histone H2A/H4 peptide bound NAA40\n(Magin R. S. et al, Structure 2015; PDB: 49UW). Since both substrate and substrate analog bound\nNAA40 structures were already determined experimentally at high resolution (which is in\nagreement with our structures) we don’t think a computationally calculated structure model\nwould add more insights. As illustrated by the dashed line in Fig. 1d connecting the nascent\npeptide density from the ribosome peptide tunnel exit to the NAA40 substrate cavity/catalytic\nsite we do not observe any conflicts regarding substrate cavity accessibility when fitted to our\ncryo-EM data. To the contrary, as stated in the manuscript the cavity is facing towards the peptide\nexit site potentially allowing NAA40 to readily engage emerging substrates.\n2. The NAA40 α0 helix mediates ribosome anchoring via rRNA (H19/H24/H46)—an interaction\nthat is nascent chain-independent and topologically similar to NatA/E complexes. Given that NATs\ncompete for overlapping ribosomal sites, I recommend the authors to discuss how NAC\norchestrates histone-specific modification despite non-specific ribosome anchoring of NAA40.\nThe issue brought up by the reviewer is highly interesting but also highly complex and so far\npoorly understood. Indeed, some of the known nascent chain interacting/modifying factors and\ncomplexes share similar and/or overlapping binding sites on the ribosome, e. g. SRP and NAA40\nor NatA/E, METAP2 and NAC, NMT1 and METAP1/2. Additionally, as ribosome binding is possible\nfor any of these factors regardless of the presence of a nascent polypeptide, at least in vitro or\nunder overexpression conditions in cells. As inferred by the reviewer, one would expect a degree\nof coordination for the different factors to be confidently directed to their target substrates within\nthe competitive environment of the ribosome peptide exit site. Furthermore, not only is there an\noverlap of ribosome binding sites between NAA40 and NatA/E or SRP, but all of them benefit from\nthe same NACA UBA-domain interaction which adds an extra level of complexity. To entangle the\ncoordination and interplay between these multiple factors and other not fully characterized ones\nlike human NatB and NatC (especially in cells) will undoubtedly be one of the upcoming challenges\nfor the field. To that end we can only speculate whether there is a constant exchange of competing\nfactors on the ribosome until the correct factor and nascent chain match or whether there is\nindeed a more defined mechanism that relies on some sort of substrate pre-recognition by NAC\nthat is then relayed by recruitment of the correct modifying enzyme. We prefer a hypothesis\nwhich would predict constant sampling of ribosomes of these factors and a change of off-rates\ndependent on substrate recognition. In any case, since we briefly pick up this topic in the first\nchapter of the discussion we now elaborate more on these possibilities and challenges there\n(lines 282-288).\nMinor Issues:\n1. Include pLDDT coloring and PAE metrics for all AlphaFold-predicted interactions (e.g., NAA40-\nNAC interfaces) to validate model confidence.\nAs requested we included pLDDT coloring and PAE matrices for all AlphaFold-predicted\ninteractions.\n2. \"Horseradish peroxide\" should be corrected to horseradish peroxidase (HRP) in Methods.\nWe corrected that mistake. The text now states: “horseradish peroxidase (HRP)”.\nReviewer #4 (Remarks to the Author):\nThe paper provides novel structural insights into the interaction of the shortest Nat protein NatD\nto the ribosome. In contrast to other Nats this protein consists of a single subunit, NAA40, which\nspecifically acetylates Histones H2A and H4 after their processing by MetAP1. NAA40 thereby is\nrecruited close to the ribosome exit tunnel by interaction with NAC in a similar manner as shown\npreviously for NatA and NatE. A critical role in recruitment seems to play the highly charged N-\ndomain of NAA40 and the NACA Uba domain. Based on their structural data, the authors suggest\nthat MetAP1 and NAA40 might be recruited concomitantly to the exit tunnel, possibly indicating\na coordinated cleavage and acetylation reaction. The conclusions drawn from the structural data\nare supported by a variety of different biochemical experiments. Both, the structural data and the\nbiochemical data, are of very high quality and seem to be consistent with the proposed\nmechanisms. The manuscript is very well written and most of the Figures are very well prepared.\nTogether with the importance of the NAA40 for its possible function in various tumors and our\nstill limited understanding of the N-terminal modification reactions I strongly recommend this\nmanuscript for publication in Nature Communications.\nWe are happy about this reviewer’s overall very positive evaluation.\nThere are only some minor queries that should be addressed by the authors.\nI would appreciate, if the authors could discuss the unique substrate specificity of NAA40 for H2A\nand H4 in some more detail.\nThis issue has been analyzed and explained in great detail in R.S. Magin, et al. Structure 2015,\nwhere the details of NAA40 substrate binding were investigated by X-ray crystallography and\ncombined with mutational analysis. We added a short explanation to the introduction highlighting\nthe difference in substrate recognition between Naa40 and other NATs with broader substrate\nrange such as NatA and NatE (lines 45-47).\nGiven the copurification of XRCC5, XRCC6 and PRKDC a more global analysis of the extract shown\nin Fig. 1a by qMS might provide deeper and novel insights into the substrate specificity of NAA40.\nIt would also be interesting if gammaH2A is present in the sample.\nWe agree with the reviewer that the co-purification of DNA damage response factors is intriguing\nas well as the question whether the substrate range of NAA40 extends from canonical histones\nH4/H2A. In the meantime, we have published a study demonstrating that H2A.X is a bona fide\nsubstrate of NAA40 (Klavaris A. et al. Epigenetics Chromatin 2025), further supporting a potential\nlink between NAA40 and DNA damage related pathways (We have included this information in\nthe Introduction (line 44) and updated paragraph 3 of the discussion (lines 321-323)). However,\nwhile we feel that the co-immunoprecipitation of XRCC5, XRCC6 and PRKDC is worthwhile\nmentioning in the manuscript to potentially provide initial evidence to further investigate the role\nof NAA40 in the nucleus and/or in DNA damage signaling (see Reviewer #1 Minor Point 3), it is\nnot the focus of this study. Therefore, we believe that performing qMS to get deeper insights into\nNAA40 substrate specificity or interactors aside from NAC and the ribosome is beyond the scope\nof this study.\nline 289 and 290: If the N-terminal helix of NAA40 is a major determinant for ribosome binding\nand also harbors the nuclear localization signal, it is unlikely that the N-terminally truncated\nversion serves in “potential nuclear post translational activity”. The authors should better explain\nwhat they mean here.\nIn the discussion portion mentioned by the reviewer only the canonical NAA40 is discussed but\nnot the short form. However, as mentioned in the discussion, the cellular distribution of canonical\nNAA40 leaves certain questions for future research. For example, the full-length NAA40 is found\nmainly in the nucleus compared to the short version that is mainly located in the cytoplasm\n(PMID: 33916271 and compare Supplementary Fig. 6j: main and lower band for NAA40). This is\ncounterintuitive since the α0 extension is functioning as main ribosome anchor as well as\npotential NLS, but the short version that lacks the N-terminal anchor and cannot associate with\nthe ribosome is predominantly located in the cytoplasm. This distribution and its regulation,\nespecially of full-length NAA40, might have a profound impact on its activity and would warrant\nfurther investigation in future work. Since the synthesis of histone proteins is for the most part\ntied to the cell cycle, one could for example imagine that the localization of NAA40 might also be\nlinked to cell cycle progression.\nSo, when we mention in the results that NAA40S could take part in post-translational acetylation\nwe still talk about activity within the cytoplasm (lines 183-185). To clarify this, we added “in the\ncytosol” to the sentence in question (now line 183-185).\nMinor:\n1: some of the Figures are too small to really see details and should be enlarged by the authors.\nThis includes Figure 2a,3g, 4c, Extended Data Fig 1e, Extended Data Fig 2e, Extended Data Figure\n3g and 3h and the Extended Data Figure 6b to 6e.\nAs requested, we enlarged the figure panels in question and adjusted the figures to accommodate\nthe larger panels. For Supplementary Fig. 1e and 6b-e, we split the figures into one containing the\ncryo-EM sorting scheme and the other containing the reconstruction validation panels, each.\nThese are now represented by Supplementary Fig. 1 and 2 or 7 and 8, respectively. Please note\nthat splitting Supplementary Fig. 1 results in an incremental shift of +1 in numbering for all other\nSupplementary Figures thereafter that has been adopted within the text. We have enlarged all\nother panels in question to improve readability. We also want to note that lower resolution\nversions of the figures were provided to keep file sizes reasonable. The final figures will be\nprovided as higher resolution versions which should also increase the readability.\n2: On top of Figure 3a the legends table needs to be adjusted.\nThe table legend for Figure 3a has been adjusted and should now be better understandable.\n3: Not sure what the authors mean with the question mark in Figure 3g. This should be removed\nor described in the legend.\nFigure 3g shows an overlay of our NAA40-NAC assembly on the peptide tunnel exit site at the 60S\nribosome overlaid with a surface representation of METAP1 taken from a previously reported\nstructure. The idea behind the figure is to give the reader a picture of our hypothesis why\nconcomitant binding of the three factors at the ribosome might be possible from a structural\npoint of view instead of plainly describing the possibility in the text. We think this greatly helps\nreaders - especially those who are not as familiar with structural data or previous publications on\nthe topic - to visualize und understand a key point of why the following in vitro reconstitution\nexperiments and cryo-EM analysis were conducted. We added the question mark for two reasons:\n1) To indicate that we were questioning or wondering about the possibility of NAA40-NAC and\nMETAP1 assembling together at the peptide exit. 2) While the panel serves as a visualization aid\nfor our rational we do not want readers to accidentally mistake the overlay shown for actual data.\nTherefore, we think it is justified to keep the question mark in the panel but we added an overall\nmore detailed explanation to the figure legend as requested.\n4: Figure 4a and b. I suppose the shown maps are filtered, which should be indicated in the text\nof the legend.\nThe figure panels show local resolution filtered densities. As requested, we added the description\nto the figure legend.\n5: Figure 5: the colors of NACB and A are too pale and should be bit darkened to provide better\ncontrast to the gray large subunit.\nWe adjusted the colors of all factors represented in the cartoon in Fig. 5 to give better contrast to\nthe large ribosomal subunit.\n6: Legend to Extended Data Figure 3: (line 797) f should be substituted by h\nWe corrected this mistake.\n7: there happened some misassignment of Figures in the text: e.g. line 89/90 Extended Data\nFigure 1a actually refers to 2a: line 95 Extended Data Fig. 1a…;\nWe apologize and corrected the wrong assignment in formely lines 89/90, Supplementary Fig. 3a-\nc is now correctly referenced. Formerly line 95 now correctly references Supplementary Fig. 1 and\n3e,f. 1 being the cryo-EM sorting scheme showing how the subclass of actively translating 80S\nwas derived and 3e,f showing overviews of the local resolution filtered map and the molecular\nmodel as well as the density for the cut through views shown in main Fig. 1c where density has\nbeen replaced with surface representations of the molecular model for a clearer representation\nfor the reader.\n8: I suppose the sentence in lines 214/215 refers to the experimental data in Fig. 3 and not to the\nstructural data as indicated.\nWe corrected that mistake.\n9: Line 140: references missing for NatA/E\nWe added relevant references to the text passage in question.\nPoint-by-point response\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have sadisfactorily addressed our concerns, and the mansucript is suitable for\npublication in Nature Communications.\nWe thank the reviewer for their positive assessment of our revised manuscript.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nWe thank the reviewer for their efforts in co-reviewing this manuscript.\nReviewer #3 (Remarks to the Author):\nThe authors have addressed my concerns. Overall, this is a very important study and warrants the\nrapid publication.\nWe are glad to hear that we could address the reviewer’s concerns in the revised manuscript.\nReviewer #4 (Remarks to the Author):\nThe manuscript significantly improved. I do not have further queries.\nWe are pleased to hear that we could improve the manuscript and address the reviewer’s remarks."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have sadisfactorily addressed our concerns, and the mansucript is suitable for publication in Nature Communications.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The authors have addressed my concerns. Overall, this is a very important study and warrants the rapid publication.", "reviewer_4": "The manuscript significantly improved. I do not have further queries."}, "author_response": ""}]

Decision letter