A protein adaptor mediating Ap4A-dependent control of protein acetylation

Domain: chemistry
Published: 2026-02-23
Editorial outcome: minor_revision

Abstract
Reversible lysine acetylation is a highly conserved post-translational modification across all domains of life controlling diverse cellular processes such as metabolism and gene expression. However, the regulation of protein acetylation remains poorly understood. Here, we report a regulatory system in Bacillus subtilis that controls the activity of the histone deacetylase (HDAC)-like protein AcuC, which has multiple substrates including acetyl-CoA synthetase and translation elongation factor. We show that AcuC is inhibited via formation of a stable complex with the hitherto uncharacterized protein AcuB. We furthermore demonstrate that the alarmone diadenosine tetraphosphate (Ap4A) binds to the cystathionine beta-synthase (CBS) domain of AcuB, thereby stabilizing AcuB and further enhancing the inhibition of AcuC. In summary, this study identifies AcuB as an Ap4A regulated deacetylation inhibitor, revealing an uncharacterized molecular mechanism to control HDAC-like proteins. Thus, the alarmone Ap4A modulates protein (de)acetylation, pointing towards a regulatory network that connects stress response, protein acetylation, and acetyl-CoA biosynthesis.

Introduction
Reversible lysine acetylation is an evolutionarily conserved post-translational modification, which plays a crucial role in a variety of cellular processes, including the regulation of metabolism and gene expression1–4. Lysine acetylation is catalyzed by different acetyltransferases, e.g., Gcn5-related N-acetyltransferases (GNATs), the p300/CBP family, and the MYST family, using acetyl-CoA (Ac-CoA) as acetyl donor. Lysine acetylation can also occur in the absence of a dedicated enzyme via acetyl-phosphate (AcP) or Ac-CoA3–5. Deacetylation is mediated by either of two classes of lysine deacetylases – (i) NAD-dependent sirtuins and (ii) Zn2+-dependent histone deacetylases (HDACs) and HDAC-like proteins6–9.

Advances in proteomics and mass spectrometry have uncovered pervasive lysine acetylation in diverse bacterial proteins2,10. However, how protein acetylation is regulated is poorly understood. One example is the reversible acetylation of the catalytic lysine in acetyl-CoA synthetases (Acs), which inactivates the enzyme11–13. In Bacillus subtilis, Acs (AcsA) acetylation is catalyzed by the GNAT AcuA, while the deacetylation (and thus reactivation) is mediated by the Zn2+-dependent HDAC-like protein AcuC13 (Fig. 1a). AcuA and AcuC are encoded in an operon that also contains acuB, a gene of unknown function13–16.

Nucleotide second messengers such as cyclic AMP (cAMP), guanosine-(penta)tetraphosphate ((p)ppGpp), and dinucleoside polyphosphates are crucial for stress signaling across bacteria and archaea17–21. Dinucleoside polyphosphates occur in all domains of life (e.g., as a side product of aminoacyl-tRNA synthetases) and have been proposed as evolutionary conserved alarmones22,23. In particular, diadenosine tetraphosphate (Ap4A) accumulates under various stress conditions in diverse bacteria such as Salmonella typhimurium, Escherichia coli, and Bacillus subtilis24–27. Previously, we demonstrated that Ap4A regulates GTP biosynthesis by binding to the CBS (Cystathionine-Beta-Synthase) domain of inosine-5′-monophosphate dehydrogenase (IMPDH) in B. subtilis27- a mechanism also found in the fungi28. Recent proteomic studies in human embryonic kidney cell lysates revealed nearly 100 potential Ap4A-binding proteins29, suggesting its role as an important signaling molecule also in the eukaryotes. However, only little mechanistic knowledge about Ap4A signaling exits.

In this work, we present that similar to IMPDH the B. subtilis protein of unknown function AcuB contains two CBS domains, which led us to hypothesize that Ap4A might interact directly with AcuB. Thus, we set out to explore the molecular function of AcuB. We demonstrate that it directly interacts with the HDAC-like deacetylase AcuC, thereby inhibiting deacetylation of various proteins, including AcsA, the elongation factor EF-Tu, the cell wall biosynthesis enzyme MurC, and the DNA replication regulator YabA. DRaCALA (direct radial capillary action of ligand assays) screening revealed that AcuB indeed binds Ap4A suggesting a potential connection between Ap4A and the bacterial acetylation–deacetylation machinery. The crystal structure of AcuB bound to Ap4A reveals that AcuB comprises three domains: two CBS domains and one ACT (aspartate kinase-chorismate mutase-tyrA) domain. The ACT domain mediates interaction with AcuC and blocks its active site. The CBS domains bind Ap4A with high affinity, increasing its thermal stability and binding to AcuC thereby accelerating the inhibitory effect of AcuB on AcsA dependent Ac-CoA production. Together, our findings introduce a regulatory mechanism that connects alarmone signaling to protein acetylation and biosynthesis of Ac-CoA.

Results
AcuB interacts with AcuC
The enzyme activity of B. subtilis AcsA is tightly regulated through reversible acetylation of its catalytic lysine residue (K549), mediated by the opposing actions of the acetyltransferase AcuA and the deacetylase AcuC (Fig. 1a). AcuA and AcuC reside within an operon together with AcuB, a protein of unknown function (Fig. 1b). We and others have shown that AcsA can form a stable complex with AcuA but not with AcuB16,30.

To identify interaction partners of AcuB, we performed in vivo pull-down assays in B. subtilis using Streptavidin II-tagged AcuB expressed from the replicative plasmid pLike_Rep35. The empty plasmid pLike_Rep expressing only the tag served as a negative control. Comparative mass spectrometry analysis of eluates identified AcuC as one of the most abundant proteins specifically enriched in the AcuB samples (Supplementary Fig. 1, Supplementary datasets 1 and 2) suggesting a physical interaction between AcuB and AcuC in B. subtilis.

To confirm the interaction of AcuB and AcuC, we recombinantly produced both B. subtilis proteins in Escherichia coli BL21(DE3) and purified both proteins via a two-step protocol consisting of a nickel-affinity chromatography followed by a size-exclusion chromatography (SEC). During purification, B. subtilis AcuB aggregated severely. As proteins from thermophilic organisms often display improved biochemical properties and stability31, we instead used the AcuB homolog from Geobacillus thermodenitrificans NG80-2 (Gt) (Supplementary Fig. 2). Indeed, (Gt)AcuB did not aggregate allowing the purification of soluble (Gt)AcuB with a high yield. For consistency reasons, we purified the (Gt)AcuC homolog as well (Supplementary Fig. 3). To validate complex formation, we performed an analytical SEC experiment, which demonstrated the formation of a stable heteromeric (Gt)AcuB/C complex (Fig. 1c, Supplementary Fig. 4).

(Gt)AcuB inhibits the deacetylase activity of (Gt)AcuC towards multiple targets including AcsA
Next, we assessed the influence of (Gt)AcuB binding on (Gt)AcuC deacetylase activity. It was known that (Gt)AcuC removes the acetyl group from the catalytic lysine 549 of AcsA, thereby reactivating AcsA to synthesize Ac-CoA13. To examine the effect of (Gt)AcuB on the activity of (Gt)AcuC, we prepared acetylated AcsA (AcsA_K549Ac) and confirmed that it lost its ability to synthesize Ac-CoA as previously described30 (Fig. 1d). As expected, incubation of (Gt)AcuC with AcsA_K549Ac in the presence of Zn2+ restored the production of Ac-CoA, confirming that AcuC deacetylates AcsA_K549Ac to reactivate the enzyme. However, when (Gt)AcuB was premixed with (Gt)AcuC and then incubated with AcsA_K549Ac, Ac-CoA production decreased by approximately 75% demonstrating that (Gt)AcuB inhibits the activity of (Gt)AcuC (Fig. 1d).

A previous study showed that in B. subtilis, the translation elongation factor TufA (EF-Tu) is acetylated by a yet unknown acetyltransferase or non enzymatical manner and subsequently deacetylated by (Bs)AcuC32. To investigate the impact of (Gt)AcuB on the deacetylation activity of (Gt)AcuC towards TufA, we overexpressed (Bs)TufA in E. coli and purified the His-tagged fusion protein via nickel affinity chromatography and gel filtration. Immunoblot analysis revealed that TufA was not acetylated under these conditions (Fig. 1e). Since high Ac-CoA concentrations are sufficient to acetylate proteins at least in vitro32, we incubated (Bs)TufA with 1 mM Ac-CoA overnight, yielding acetylated elongation factor (Bs)TufAAc. The acetylation was confirmed by a strong anti-acetyl-lysine antibody signal (Fig. 1e). Incubation with (Gt)AcuC strongly reduced this signal, indicating (Gt)AcuC was able to remove the acetylation from (Bs)TufA. Importantly, pre-incubation of (Gt)AcuC with (Gt)AcuB prior to adding (Bs)TufAAc resulted in a much stronger signal, showing that AcuB also inhibits deacetylation activity of (Gt)AcuC on the elongation factor TufA (Fig. 1e).

Another putative target of AcuC is MurC, a protein involved in cell wall biosynthesis33. Previous work showed that depletion of the acetyltransferase AcuC reduced MurC acetylation. We successfully generated acetylated MurC (MurCAc) and confirmed that it is a substrate of AcuC (Fig. 1f). Again, addition of AcuB inhibited the deacetylation of MurCAc (Fig. 1f). A Yeast two-hybrid assay performed previously suggested an interaction of AcuB and the DNA replication regulator YabA34. Although we could not establish an AcuB/YabA interaction with the purified proteins (Supplementary Fig. 5), we observed that acetylated (Bs)YabA (YabAAc) is deacetylated by (Gt)AcuC (Fig. 1g), and again (Gt)AcuB inhibited the AcuC-dependent deacetylation of (Bs)YabA (Fig. 1g). In summary, our experiments demonstrate that AcuC can efficiently remove acetyl groups from various proteins and this activity is regulated by AcuB.

The ACT-domain of AcuB interacts with AcuC
Our attempts to obtain a high-resolution structure of the AcuB-AcuC complex using X-ray crystallography or cryo-electron microscopy were unsuccessful. AlphaFold35 analysis revealed that AcuB consists of two CBS domains at the N-terminus and one ACT domain at the C-terminus (Fig. 2a). AcuC essentially consists of one domain resembling the fold of a typical histone deacetylase (HDAC) class I domain (HDAC_AcuC_like domain)36 (Fig. 2a). To identify the interaction interface between the two proteins, we applied hydrogen-deuterium exchange mass spectrometry (HDX-MS). The HDX-MS results uncovered the interaction interface between AcuB and AcuC. In (Gt)AcuB, HDX protection was observed in the ACT domain (residues R184–Y202) and the linker region between the ACT and CBS domains (residues Q129–I140), indicating that they are part of the complex interface (Supplementary Fig. 6). For (Gt)AcuC, significant HDX protection was detected in multiple regions, including residues A84–H100, S119–A152, V167–F181, S191–L233, A260–Y276, A297–W306, and A368–Y379. These regions correspond to areas surrounding the active site cavity (Fig. 2b, Supplementary Fig. 7), as seen in the structure PDB ID: 1ZZ137 of the homolog from Alcaligenaceaebacterium, in which the inhibitor suberoylanilide hydroxamic acid (SAHA) was bound in the active center. Our HDX-MS data are corroborated by the AlphaFold model, which also predicts that the ACT domain of AcuB interacts with the regions surrounding the active site of AcuC (Fig. 2b, Supplementary Fig. 8).

More detailed analysis revealed that the interface between (Gt)AcuB and (Gt)AcuC is stabilized by multiple interactions, including salt bridges between (Gt)AcuB_E4–(Gt)AcuC_R362, (Gt)AcuB_D89–(Gt)AcuC_K378, and (Gt)AcuB_E92–(Gt)AcuC_R197, as well as π–π and hydrophobic interactions between (Gt)AcuB_W206–(Gt)AcuC_Y197 and (Gt)AcuB_M188–(Gt)AcuC_F200/F143 (Supplementary Fig. 9A). To validate these predicted interfacial contacts, we constructed single-point mutants (Gt)AcuB_D89K, E92R, M188A and (Gt)AcuC_R197E, Y198A, R362E, and performed pulldown assays (Supplementary Fig. 9B). The mutations D89K, E92R, and M188A in (Gt)AcuB reduced complex formation, while R197E and Y198A of (Gt)AcuC moderately weakened the interaction. In contrast, (Gt)AcuC_R362E showed minimal effect. These results confirm the key interfacial residues mediating the (Gt)AcuB–AcuC interaction.

In addition, we conducted an in vitro pull-down assay. We fused the (Gt)AcuB_ACT domain to a GST-tag, overexpressed this fusion protein in E. coli, and used (Gt)AcuC as prey. A band corresponding to (Gt)AcuC (approximately 45 kDa) was observed when incubated with (Gt)AcuB_ACT, confirming a direct interaction (Fig. 2c). Hence, the ACT domain of AcuB contains the binding site for AcuC.

AcuB binds to the alarmone Ap4A
Our finding that the ACT domain of AcuB is the primary binding site of AcuC suggests that the N-terminal CBS domains of AcuB fulfill another function. CBS domains are evolutionarily conserved and occur in a wide range of species from bacteria to humans. Typically they come in pairs and bind adenosine-containing molecules such as AMP, ATP38 or Ap4A (Fig. 2d)27. Using tagged ORF libraries comprising >5000 B. anthracis genes overexpressed in E. coli39, we performed a DRaCALA39 with in vitro synthesized 32P-Ap4A to identify potential binding targets. Notably, AcuB appeared as a binder of Ap4A in Bacillus anthracis just as IMPDH, which we have confirmed previously27 (Fig. 2e). To test this screening result more directly, we performed isothermal calorimetry (ITC) experiments with different adenosine-containing nucleotides, such as AMP, ADP, ATP, Ap3A and Ap4A. Purified (Bs)AcuB protein showed moderate binding affinity for Ap4A, with a dissociation constant (KD) value of 0.39 ± 0.07 µM (Fig. 2f). (Gt)AcuB exhibits a slightly weaker binding affinity for Ap4A, with a KD value of 2.81 ± 0.49 µM. The other nucleotides that were tested in ITC, showed no binding or only weak affinity to (Gt)AcuB (Fig. 2f, Supplementary Fig. 10). Thus, AcuB binds to Ap4A probably via its CBS domain-containing N-terminus.

During the purification of (Bs)AcuB, the protein showed slower precipitation if Ap4A was added compared to our original attempts (see above). This suggests that Ap4A stabilizes the protein and prevents its denaturation. To quantify this effect, we assessed the thermal stability of (Bs)AcuB using Nano Differential Scanning Fluorimetry (nanoDSF). Remarkably, the presence of Ap4A raised the melting point of (Bs)AcuB by approximately 30 °C, from 38.0 °C to 68.0 °C (Fig. 2g), compared to (Bs)AcuB alone. Next, we investigated the stability of the variant (Bs)AcuB_R33E (which lost the binding affinity to Ap4A, see below). This variant was slightly more thermostable than the wild-type protein. Furthermore, the addition of Ap4A did not result in a similar increase in thermostability (38.3 °C – 43.8 °C; Supplementary Fig. 11A). These results suggest that Ap4A binding substantially stabilizes AcuB under conditions where Ap4A levels are elevated, such as during heat stress. Supporting this notion, we observed that (Gt)AcuB, derived from the moderately thermophilic bacterium Geobacillus thermodenitrificans, showed only a modest increase in thermostability upon Ap4A addition—approximately 5 °C (from 63.1 °C – 68.4 °C). (Supplementary Fig. 11B). It is worth mentioning that ADP also shows a µM binding affinity to (Bs)AcuB. To further investigate the binding specificity of (Bs)AcuB to Ap4A, we performed competition experiments using a thermoshift assay. In these experiments, AcuB was incubated with both ADP and Ap4A, with ADP concentrations set 5 times higher than Ap4A. Despite the excess ADP, the thermal shift profile was nearly identical to that observed when Ap4A was present alone, confirming that Ap4A preferentially binds to (Bs)AcuB also in competitive binding conditions. As ADP binds to (Bs)AcuB and changes its thermal stability – albeit to a lesser extent – it is possible that variation of ADP levels regulate AcuB stability in non-stressed cells, a phenomenon which merits future investigation. Together our data show that AcuB is an Ap4A stabilized AcuC inhibitor.

Structural basis of Ap4A-binding to AcuB
To elucidate the binding mode of Ap4A to AcuB, we attempted to crystallize both (Gt)AcuB and (Bs)AcuB in the presence of Ap4A. Crystals of (Bs)AcuB were obtained in the presence of Ap4A. The crystal structure of (Bs)AcuB in complex with Ap4A was determined at a resolution of 2.4 Å (PDB ID: 9QS7, Supplementary Table 1). The overall structure of (Bs)AcuB consists of two CBS domains in the N-terminus and one ACT domain in the C-terminus (Fig. 3a). (Bs)AcuB forms a homodimer, with the two chains cooperating in a configuration similar to that of its homolog TTHA0829 from Thermus thermophilus (PDB ID: 5AWE)40 (Fig. 3b, Supplementary Fig. 12). This suggests that TTHA0829 also represents an AcuB protein. During data processing of the electron density map for AcuB, two additional electron densities were detected between its four CBS domains, which could be identified as Ap4A molecules (Supplementary Fig. 13). They bind into extended surface grooves present on the opposite sides of the CBS-domain part of the AcuB homodimer (Fig. 3b). Given the high structural alignment between (Bs)AcuB and the (Gt)AcuB AlphaFold model (Supplementary Fig. 14), we infer that two Ap4A molecules also bind within the CBS domains of (Gt)AcuB. The results from HDX-MS experiments align well with the X-ray structure, confirming that Ap4A binds to and stabilizes the CBS domain (see below, Supplementary Fig. 6).

Further inspection using LigPlot41,42 reveals the highly symmetrical coordination of the two Ap4A molecules and provides a molecular model explaining how they might stabilize the AcuB homodimer (Fig. 3b, c). Each Ap4A molecule binds to both units of the homodimer in a crisscross fashion. One adenosine moiety forms hydrogen bonds with the backbone carbonyl groups of His34 and Ile12 from one monomer, while the other adenosine moiety binds to the backbone carbonyls of Cys105 and Val183 from the other monomer. These interactions are reciprocal in the symmetric dimer, with the second Ap4A molecule binding in the opposite orientation to the pair of monomers. In addition, the two ribose moieties of each Ap4A molecule interact with Asp122 through van der Waals forces (Fig. 3c). Moreover, the positively charged side chains of Arg33, Arg51, Lys120, His34, along with the backbone of Gly104 from both monomers form hydrogen bonds with the phosphate moieties of the two Ap4A molecules (Fig. 3b, c). Notably, Arg33 and His34 from both monomers bridge the two Ap4A molecules via hydrogen bonds (Fig. 3d). These interactions indicate a tight binding mode where Ap4A stabilizes and induces significant conformational changes in the (Bs)AcuB structure.

Next, variations of Arg33, Arg51 into glutamate, and Asp122 into alanine were introduced in (Bs)AcuB to assess their impact on Ap4A binding via ITC. The R33E variant abolished binding, highlighting its critical role (Fig. 3f, Supplementary Fig. 10). The R51E and D122A variants showed reduced binding affinities with KDs of 28.6 ± 15.0 µM and 0.72 ± 0.08 µM, respectively, compared to the wild type protein (Supplementary Figs. 10 and 15). Homology analysis of 200 AcuB sequences revealed that R33 and His34 are highly conserved residues, implying that AcuB orthologs are conserved Ap4A binders (Fig. 3e)43. Together, our data show that the CBS domains of the AcuB homodimer coordinate two Ap4A molecules in asymmetric fashion.

Ap4A binding induces conformational changes in AcuB and enhances its inhibitory function
To examine how Ap4A binding affects AcuB structure and function, we analyzed the effects of Ap4A on (Gt)AcuB using HDX-MS (Supplementary Table 2 and Supplementary Dataset 3). This method revealed significant Ap4A-induced changes in the CBS domain of AcuB. Residues V3–L14, V48–F61, D81–F90, and G116–T121 exhibited reduced deuterium uptake, indicating stabilization of these regions upon Ap4A binding. In contrast, residues D122–L127, encompassing a helix and the loop connecting the CBS and ACT domains, showed increased deuterium uptake, suggesting enhanced flexibility in this area (Fig. 4a, Supplementary Fig. 6). Thus, Ap4A binding induces significant conformational changes in the CBS domain of AcuB and increases the flexibility of the linker. The dynamic structural rearrangements of AcuB upon Ap4A binding suggest that Ap4A may impact the inhibitory action of AcuB on the AcuC deacetylase. However, we did not observe any HDX changes in the (Gt)AcuB-AcuC complex with or without Ap4A, suggesting that Ap4A does not directly induce large conformational changes (Supplementary Figs. 6 and 7).

To assess the precise role of Ap4A binding, we investigated the effect of Ap4A on both the AcuB/AcuC interaction and the inhibitory role of AcuB on AcuC. To validate the interaction between AcuB and AcuC, we performed pull-down assays using strep-tagged (Gt)AcuB as bait and (Gt)AcuC as prey. A band corresponding to (Gt)AcuC was only observed in the presence of (Gt)AcuB, confirming the formation of a (Gt)AcuB-AcuC complex (Fig. 4b). Notably, the presence of Ap4A increased the intensity of the (Gt)AcuC band compared to (Gt)AcuC alone, whereas other nucleotides, such as ATP, ADP, and AMP did not show this increase (Fig. 4b). Interestingly, when Ap4A was added to the (Gt)AcuB_ACT/(Gt)AcuC mixture, no increase in (Gt)AcuC band intensity was observed (Fig. 2c). This finding indicates that while the (Gt)AcuB_ACT domain can interact with (Gt)AcuC (see also above), Ap4A does not affect this interaction, likely due to its specific binding to the CBS domain of AcuB (Figs. 3b, 4a). In addition, we employed mass photometry (MP) to better assess the effect of Ap4A on the stability of the (Gt)AcuB-AcuC complex. When 500 nM (Gt)AcuB, 500 nM (Gt)AcuC, and 1 mM Ap4A were premixed, resulting in final protein concentrations of 25 nM and a final Ap4A concentration of 50 µM for MP analysis, the intensity of the complex (100 kDa peak) increased approximately 2.5-fold (Fig. 4c). No increase was observed upon the addition of AMP, ADP, or ATP (Fig. 4c, Supplementary Fig. 16), which shows a similar stability to the (Gt)AcuB–AcuC complex in the absence of any nucleotide (approximately 10% of the total protein forms a complex in this assay). Such enhancement of complex formation was not observed in the (Gt)AcuB_R33E mutant upon Ap4A addition (Fig. 4c). These results confirm that (Gt)AcuB forms a stable complex with (Gt)AcuC, and that this interaction is enhanced by Ap4A.

Next, we examined if Ap4A binding has an impact on the function of AcuB as AcuC inhibitor. (Gt)AcuC efficiently deacetylates AcsA_K549Ac, resulting in an increase in Ac-CoA production over time (Figs. 4d, 1d). However, when (Gt)AcuB and (Gt)AcuC were premixed, Ac-CoA production was notably reduced compared to (Gt)AcuC alone. Adding Ap4A to the (Gt)AcuB and (Gt)AcuC mixture further suppressed Ac-CoA production (Fig. 4d). In contrast, (Gt)AcuB_R33E, which cannot bind Ap4A anymore (Fig. 3f), did not inhibit Ac-CoA production beyond the effect of (Gt)AcuB_WT, even in the presence of Ap4A (Fig. 4d) demonstrating that Ap4A inhibits deacetylation activity of (Gt)AcuC on AcsA_K549Ac through its interaction with (Gt)AcuB.

Mechanistic insight into mediated AcuB inhibition and the effect of Ap4A
Bioinformatic analysis revealed that AcuC is a class I HDAC homolog – it shares conserved catalytic residues and catalyzes deacetylation in a very similar way (Supplementary Figs. 17 and 18)2,44. The predicted AlphaFold model of (Gt)AcuC resembles the structures of Homo sapiens HDAC (Hs)HDACs 1–3 and 845–48, respectively. Key residues such as D259, D170, and H172 coordinate the Zn2+ ion crucial for binding to the acetyl moiety of acetylated lysine. Additionally, the proton donor/acceptor residues Y303, H133, and H134 are conserved in both AcuC and the (Hs)HDACs, suggesting a similar deacetylation mechanism (Fig. 5a, Supplementary Fig. 17). Notably, the active center residues exhibited protection in the HDX experiment investigating the (Gt)AcuB-(Gt)AcuC complex indicating that (Gt)AcuB affects key residues in the active center of (Gt)AcuC (Supplementary Figs. 6 and 7).

Interestingly, the HDAC inhibitor SAHA was previously co-crystallized with the (Hs)HDAC848 (Fig. 5b). In the superimposed structures of the (Gt)AcuC–AcuB complex and the (Hs)HDAC8–SAHA complex, AcuB_M188 aligns closely with the SAHA binding site, suggesting that AcuB may occlude the substrate entry/exit tunnel of AcuC (Fig. 5c). This interaction was supported by the HDX experiment, which revealed protection in the loop containing AcuB_M188 and the active sites of AcuC in the HDX analysis (Fig. 5f).

To investigate how Ap4A enhances the inhibitory activity of AcuB on AcuC, we conducted molecular dynamics (MD) simulations. We used the AlphaFold model as the starting structure, as it showed high confidence in the predicted complex with high Per-residue predicted Local Distance Difference for all chains (pLDDT > 85, Supplementary Fig. 19). Initially, a 200 nanoseconds (ns) accelerated MD simulation was performed at 300 K on the (Gt)AcuB dimer. The results indicate that when two Ap4A molecules bind to the active site, the RMSF (Root Mean Square Fluctuation) values are significantly lower compared to the apo state (Fig. 5d), suggesting that ligand binding reduces conformational flexibility and stabilizes the protein. This finding aligns well with our data obtained in the thermostability assay (Fig. 2g). The increase in stability may facilitate the binding of the (Gt)AcuB dimer to (Gt)AcuC. To further explore this effect, we increased the simulation temperature to 450 K and conducted MD simulations on the (Gt)AcuB-AcuC complex to compare the two states. It should be noted that the simulations at 450 K were not intended to mimic experimental conditions but rather to accelerate conformational sampling and enable comparison of the relative stability of the apo and Ap4A-bound complexes. The results reveal that in the presence of Ap4A, the distance between AcuB_M188 and the Zn²⁺ ion in the active site of AcuC is shorter than in the apo state (Fig. 5e, g, h), which correlates with the observed enhancement of Ap4A-mediated inhibition of AcuC (Fig. 4e). Likewise, variation of AcuB M188 to alanine diminished complex formation, supporting the important role of this residue for the AcuB-AcuC interaction (Supplementary Fig. 9).

To further characterize the conformational space explored during the simulations, we performed principal component analysis (PCA) on the concatenated trajectories after removing overall translational and rotational motions. A mass-weighted covariance matrix was constructed and diagonalized, and the first two principal components (PC1 and PC2), accounting for approximately 50% of the total variance, were used as reaction coordinates. Free energy landscapes were then derived through Boltzmann inversion of kernel density–estimated conformational probabilities (Supplementary Fig. 20). Both apo and Ap4A-bound systems exhibited two main basins, indicating the presence of two dominant conformational states. The relatively flat energy profiles suggest low energy barriers between these states, while subtle shifts in local minima imply that Ap4A binding may allosterically stabilize specific local conformations of AcuB that enhance its inhibitory interaction with AcuC. Together, these results suggest that Ap4A binding stabilizes the AcuB dimer and strengthens its association with AcuC through conformational restriction and local allosteric effects.

Methods
Protein Production and Purification
The genes encoding B. subtilis proteins (Bs)AcuB, (Bs)AcuC, and (Bs)AcsA were amplified from genomic DNA of strain NCIB3610 (DK1042), while (Gt)acuB and (Gt)acuC genes were amplified from genomic DNA of G. thermodenitrificans NG80-2. Open reading frames were cloned into a pET24d-based plasmid using N- or C-terminal hexa-histidine tags via a Golden Gate cloning approach, utilizing BsaI restriction sites. For GST-tagged proteins, a similar cloning method was employed. The point mutants were generated by fusion PCR. Two overlapping PCR fragments containing the desired mutation were amplified using (Bs or Gt)acuB as the template and mutation-specific primers (Supplementary Tables 3 and 4). The fragments were subsequently fused in a second round of PCR to yield the full-length mutated gene. The resulting PCR product was cloned into the corresponding expression vector using a Golden Gate assembly approach as described above.

All proteins were overexpressed in E. coli BL21(DE3) (Novagen) in LB (Luria-Bertani) medium (tryptone: 10 g/L, yeast extract: 5 g/L, NaCl: 10 g/L), supplemented with 10 g/L D(+)-lactose monohydrate, 50 μg/mL kanamycin, or 100 μg/mL ampicillin (depending on the vector’s resistance) for 16–20 h at 30 °C with vigorous shaking. After cultivation, cells were harvested by centrifugation at 4500 × g for 20 min at 4 °C. The cell pellet was then resuspended in buffer A (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 20 mM MgCl2, and 40 mM imidazole) and lysed using an LM10 microfluidizer (Microfluidics) at 18,000 psi. The lysate was subsequently centrifuged at 47,850 × g for 20 min at 4 °C.

The supernatant was applied to a HisTrap HP 1 mL column (Cytiva) equilibrated with buffer A. After washing with 10 mL of buffer A, proteins were eluted with 10 mL of buffer B (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 20 mM MgCl2, and 250 mM imidazole). The eluted proteins were concentrated to 2 mL using Amicon Ultracel-30K (Millipore) and subjected to size-exclusion chromatography (SEC) on a HiLoad 26/600 Superdex 200pg column (Cytiva) equilibrated with 50 mM HCl pH 7.5, 150 mM NaCl, and 20 mM MgCl2. Fractions containing the target proteins were further concentrated using Amicon Ultracel-30K (Millipore), snap-frozen in liquid nitrogen, and stored at −80 °C for subsequent analysis. Protein concentration was measured photometrically using a NanoDrop Lite (Thermo Fisher).

Thermostability assays
NanoDSF was used to measure the thermal stability of proteins using a Prometheus NT.48 instrument (NanoTemper Technologies, Germany). Protein samples were prepared at 1 mg/mL in SEC and with/without 500 μM Ap4A. Approximately 2 μL of each sample was loaded into UV-transparent capillaries (NanoTemper Technologies). The temperature was increased from 20 °C to 95 °C with a velocity of 1 °C per minute, while the intrinsic fluorescence at 330 nm and 350 nm was monitored. The unfolding transition temperature (Tm) was determined from the change in the fluorescence ratio (350/330 nm) using the ThermControl software (NanoTemper).

In vitro pull down assays
Strep-Tag pull down assays were carried out using spin columns and filters from MobiTec. A 20 μL suspension of Strep-Tactin® coated magnetic beads (IBA) was placed in an eppi tube and resuspended in 450 μL of SEC buffer. After centrifuging at 4500 rpm for 1 minute, 200 μg of Strep-tagged protein was added to the beads and incubated on a rotating mixer for 15 min. The mixture was then centrifuged under the same conditions. The immobilized protein was washed with 500 μL of SEC buffer, followed by another centrifugation. Subsequently, 200 μg of the putative interaction partners were added in 400 μL of SEC buffer and incubated with the Strep-tagged protein on the rotating mixer for 30 minutes. After three washes with 500 μL of SEC buffer, the bound proteins were eluted using 50 μL of SEC buffer containing 50 mM D-Biotin (pH 7.5, Carbosynth). The samples were analyzed by SDS-PAGE stained with Coomassie blue. GST pull down assays were performed similarly using MobiTec spin columns and filters. A 20 μL suspension of GST-Sepharose beads (GE Healthcare) was added to a spin column and resuspended in 450 μL of SEC buffer. After 1 minute of centrifugation at 4500 rpm, 200 μg of GST-tagged protein was added and incubated on a rotating mixer for 15 min. The mixture was centrifuged again, and the immobilized protein was washed with 500 μL of SEC buffer. Next, 200 μg of the interaction partner was added in 400 μL of SEC buffer and incubated with the GST-tagged protein on the rotating mixer for 30 min. Following three washes with 500 μL of SEC buffer, the bound proteins were eluted with 50 μL of SEC buffer containing 20 mM GSH (pH 8.0). The samples were separated by SDS-PAGE and stained with Coomassie blue for analysis.

Isothermal titration calorimetry (ITC)
ITC experiments were conducted at a temperature of 25 °C using a MicroCal PEAQ-ITC instrument (Malvern Panalytical). Ligands and proteins were diluted in a buffer containing 20 mM HEPES-HCl, 20 mM MgCl2, 20 mM KCl, 200 mM NaCl, pH 7.5. To measure the interaction between (Bs)AcuB or (Gt)AcuB and the different nucleotides, the purified protein was titrated into the sample cell up to final concentrations between 40 and 120 μM. The protein concentrations were pre-determined by measuring absorbance at 280 nm. The ligand was placed in the titration syringe at a nominal concentration of 0.4−1 mM to saturate the protein sample during the titrations. Nucleotides were obtained from Jena Bioscience with a purity of greater than 95%. The assay consisted of 13 injections (first 0.4 μl, and then 3 μl per injection) with 150 seconds of spacing between each injection. The raw data were processed with the MicroCal PEAQ-ITC Analysis Software using the “one binding site” model and plotted using GraphPad Prism.

Protein crystallization, X-ray diffraction, and structure determination
For Ap4A-bound (Bs)AcuB, a mixture of 300 μM (Bs)AcuB and 1 mM Ap4A was incubated for 10 min in ice. Initial crystallization trials were performed in SWISS CI MRC 2-well plates (Jena Bioscience) with a 30 μL reservoir, where 0.25 μL of protein was mixed with an equal volume of precipitant solution. Crystals appeared between two and seven days. To optimize crystallization conditions, hanging drop vapor diffusion was employed with a 1 mL reservoir. Drops were setup by mixing 1 μL of precipitant solution with varying volumes of protein solution (0.5 μL, 1 μL, and 2 μL). Ap4A-bound (Bs)AcuB crystals were successfully grown in a solution containing 0.2 M sodium chloride, 0.1 M imidazole (pH 8.0), and 1.0 M potassium/sodium tartrate.

X-ray data were collected under cryogenic conditions at the ID23–1 beamline of the European Synchrotron Radiation Facility (ESRF) and at the P13 beamline of DESY. Data processing, integration, and scaling were performed with XDS and XSCALE60. The structures of Ap4A-bound (Bs)AcuB were solved by molecular replacement with PHASER61, employing the crystal structure of a hypothetical protein, TTHA0829 from Thermus thermophilus HB8 (PDB-ID: 5AWE40). This hypothetical protein shared approximately 30% sequence identity (Supplementary Fig. 12). The structure was refined to RWork/RFree values of 0.2384 and 0.2832, respectively (Supplementary Table 1). Model building was conducted in Coot62, and refinement was performed with PHENIX63. Figures were generated with PyMOL64.

In vivo pull down assay and identification of interaction partners
Overnight LB cultures were used to inoculate 20 mL of MC medium to an OD600 of 0.08, which was then grown to an OD600 of 1.2–1.4 at 37 °C. MC medium was prepared by combining 2 mL of MC-Buffer 10×, 0.67 mL of 1 M MgSO4, and sterile double-distilled water up to 20 mL. The MC-Buffer 10× was made by dissolving K2HPO4·3H2O (35.099 g), KH2PO4 (13.0975 g), glucose (50 g), 25 mL of 300 mM Na3-citrate, 2.5 mL of 22 mg/mL ammonium ferric citrate, casein (2.5 g), and potassium glutamate (5 g) in 250 mL, followed by sterile filtration and storage in frozen aliquots. The cells were mixed with 0.5–1.5 μg of DNA and incubated for 2 h. After centrifugation at 5000 × g for 3 min, the pellet was plated on LB agar with antibiotics and incubated overnight at 37 °C. MC medium was prepared by combining MC-buffer with 1 M MgSO4 and sterile water, while MC-buffer 10x was made by dissolving various components and sterilizing the solution. For expression testing with pLIKErep, overnight LB+MLS cultures with 1 μg/mL erythromycin were used to inoculate 10 mL LB+MLS, grown to OD600 0.4–0.5, and induced with 27 μg/mL bacitracin. The cultures were harvested, lysed in buffer, and analyzed by SDS-PAGE. Strep-Tactin® coated magnetic beads (IBA) were used to capture Strep-tagged AcuB, which was then eluted with 50 mM D-biotin. The eluate was analyzed by SDS-PAGE and mass spectrometry.

Trypsin (0.1 μg solubilized in 50 mM NH4HCO3) was added to each sample and samples were incubated at 37 °C over-night. Peptides were desalted and concentrated using Chromabond C18WP spin columns (Macherey-Nagel, Part No. 730522). Finally, peptides were dissolved in 25 μL of water with 5% acetonitrile and 0.1% formic acid. Peptide concentration was measured using a Nanodrop (Thermo Scientific) and samples were diluted accordingly to 100 ng of peptides per μL. The mass spectrometric analysis of the samples was performed using a timsTOF Pro mass spectrometer (Bruker Daltonic). A nanoElute HPLC system (Bruker Daltonics), equipped with an Aurora C18 RP column (25 cm × 75 μm ID) filled with 1.7 μm beads (Ion Opticks, Australia) was connected online to the mass spectrometer. A portion of 2 μL of the peptide solution was injected directly on the separation column. Sample Loading was performed at a constant pressure of 800 bar. Separation of the tryptic peptides was achieved at 60 °C column temperature with the following gradient of water/0.1% formic acid (solvent A) and acetonitrile/0.1% formic acid (solvent B) at a flow rate of 400 nL/min: Linear increase from 2% B to 17% B within 18 min, followed by a linear gradient to 25% B within 9 min and linear increase to 37% solvent B in additional 3 min. Finally, B was increased to 95% within 10 min and hold at 95% for additional 10 minutes. The built-in “DDA PASEF-standard_1.1sec_cycle time” method developed by Bruker Daltonics was used for mass spectrometric measurement. Protein identification was performed using Proteome Discoverer 2.4 (Thermo Fisher Scientific, Germany).

DRaCALA screen
Two open reading frame proteome overexpression libraries, each consisting of 5139 ORFs from B. anthracis str. Ames, were used for the DRaCALA screen. The distinction between the two libraries was the protein expression vector and N-terminal protein tag (pVL791-AmpR with 10x His tag; pVL847-GentR with 10x His-MBP tag). The libraries had previously been constructed and overexpressed in Escherichia coli BL21lacIq, then lysed and prepared as described in ref. 39. Screening for binding targets of Ap4A was conducted by differential radial capillary action of ligand assay (DRaCALA). 32P-labelled Ap4A was diluted to 15 μM total concentration in binding buffer (10 mM Tris-Cl pH 7.5, 100 mM NaCl, 5 mM MgCl2), added to lysates at 1:1 volume, incubated for 10 min with shaking, and then spotted onto nitrocellulose paper. The spotted DRaCALA reactions were dried, then exposed to a phospho screen and imaged with Typhoon FLA 9000 scanner.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS)
Samples of AcuB, AcuC and AcuB/AcuC complexes were purified by size-exclusion chromatography prior HDX-MS and used at 50 μM stock concentration. Where indicated, Ap4A was employed at 1 mM stock concentration.

Preparation of samples for HDX-MS was supported by a two-arm robotic autosampler (LEAP Technologies) essentially as previously described in ref. 65. In brief, HDX reactions were initiated by 10-fold dilution of the proteins (50 μM) with buffer (20 mM Tris pH 7.4, 150 mM NaCl, 20 mM MgSO4) prepared in D2O and incubated for 10, 30, 100, 1000 or 10,000 s at 25 °C. The exchange was stopped by mixing with an equal volume of pre-dispensed quench buffer (400 mM KH2PO4/H3PO4, 2 M guanidine-HCl; pH 2.2) kept at 1 °C, and 100 μl of the resulting mixture injected into an ACQUITY UPLC M-Class System with HDX Technology66. Non-deuterated samples were generated by a similar procedure through 10-fold dilution with buffer prepared with H2O. The injected HDX samples were washed out of the injection loop (50 μL) with water + 0.1% (v/v) formic acid at a flow rate of 100 μL/min and guided to a column containing immobilized porcine pepsin kept at 12 °C. The resulting peptic peptides were collected on a trap column (2 mm × 2 cm), that was filled with POROS 20 R2 material (Thermo Scientific) and kept at 0.5 °C. After three minutes, the trap column was placed in line with an ACQUITY UPLC BEH C18 1.7 μm 1.0 × 100 mm column (Waters) and the peptides eluted with a gradient of water + 0.1% (v/v) formic acid (eluent A) and acetonitrile + 0.1% (v/v) formic acid (eluent B) at 30 μL/min flow rate as follows: 0–7 min/95–65% A, 7–8 min/65–15% A, 8–10 min/15% A. Eluting peptides were guided to a Synapt G2-Si mass spectrometer (Waters) and ionized with by electrospray ionization (capillary temperature and spray voltage of 250 °C and 3.0 kV, respectively). Mass spectra were acquired with the software MassLynX MS version 4.1 (Waters) over a range of 50–2000 m/z in enhanced high-definition MS (HDMSE)67,68 or high-definition MS (HDMS) mode for non-deuterated and deuterated samples, respectively. Lock mass correction was conducted with [Glu1]-Fibrinopeptide B standard (Waters). During separation of the peptides on the ACQUITY UPLC BEH C18 column, the pepsin column was washed three times by injecting 80 μL of 0.5 M guanidine hydrochloride in 4% (v/v) acetonitrile. Blank runs (injection of double-distilled water instead of the sample) were performed between each sample. All measurements were carried out in triplicates. Peptides were identified and evaluated for their deuterium incorporation with the softwares ProteinLynx Global SERVER 3.0.1 (PLGS) and DynamX 3.0 (both Waters). Peptides were identified with PLGS from the non-deuterated samples acquired with HDMSE employing low energy, elevated energy and intensity thresholds of 300, 100 and 1000 counts, respectively and matched using a database containing the amino acid sequences of AcuB, AcuC, porcine pepsin and their reversed sequences with search parameters as follows: Peptide tolerance = automatic; fragment tolerance = automatic; min fragment ion matches per peptide = 1; min fragment ion matches per protein = 7; min peptide matches per protein = 3; maximum hits to return = 20; maximum protein mass = 250,000; primary digest reagent = non-specific; missed cleavages = 0; false discovery rate = 100. For quantification of deuterium incorporation with DynamX, peptides had to fulfil the following criteria: Identification in at least 6 of the 12 non-deuterated samples; the minimum intensity of 10,000 counts; maximum length of 40 amino acids; minimum number of products of two and 0.1 products per residue; maximum mass error of 25 ppm; retention time tolerance of 0.5 min.

All spectra were manually inspected and omitted, if necessary, e.g., in case of low signal-to-noise ratio or the presence of overlapping peptides disallowing the correct assignment of the isotopic clusters. Residue-specific deuterium uptake from peptides identified in the HDX-MS experiments was calculated with the software DynamX 3.0 (Waters). In the case that any residue is covered by a single peptide, the residue-specific deuterium uptake is equal to that of the whole peptide. In the case of overlapping peptides for any given residue, the residue-specific deuterium uptake is determined by the shortest peptide covering that residue. Where multiple peptides are of the shortest length, the peptide with the residue closest to the peptide C-terminus is utilized. Raw data of deuterium uptake by the identified peptides and residue-specific HDX are provided in Supplementary Table 2 and Supplementary Dataset 3.

Activity assay for Ac-CoA recovery with AcsAAc
The acetylated AcsA (AcsA_K549Ac) was prepared as described in ref. 30. The AcsA activity assay was performed by preincubating 12 μg of (Gt)AcuC with 4 μg of (Gt)AcuB at room temperature for 10 minutes. To assess the effects of Ap4A, 400 μM of Ap4A was added to the (Gt)AcuC-(Gt)AcuB mixture. The reaction was initiated by adding AcsA_K549Ac to a 200 μL reaction mixture containing 0.5 mM CoA, 2 mM ATP, 0.1 mM DTT, and 5 mM acetate. The reaction proceeded at room temperature, and 20 μL samples were taken at intervals of 1, 3, 6, 10, 30, and 60 minutes. The reaction was quenched by adding 40 μL of acetonitrile, followed by centrifugation at 16,200 x g to remove any precipitates. The resulting supernatant was analyzed using High-Performance Liquid Chromatography (HPLC) on an Agilent 1200 series system equipped with a Metrosep A Supp 5–150/4.0 column (Metrohm). Isocratic elution was performed with 90 mM (NH4)2CO3 (pH 9.25) at a flow rate of 0.6 mL/min for 20 minutes, with detection carried out at 260 nm.

Mass photometry
Mass photometry (MP) measurements were performed using a TwoMP mass photometer (Refeyn Ltd., UK) and controlled with the AcquireMP v2023 R1.1 software (Refeyn Ltd., UK). Microscope coverslips (1.5 H, 24×60 mm, Carl Roth) and CultureWell Reusable Gaskets (CW-50R-1.0, 3×1 mm; Grace Bio-labs, USA) were cleaned by alternating rinses with isopropanol and ultrapure water (Milli-Q, Merck, Germany) three times, followed by air-drying under a compressed air stream. The cleaned coverslips and gaskets were assembled and mounted on the mass photometer using immersion oil (ImmersolTM 519F; Carl Zeiss, Germany). For each MP measurement, a drop of 19 μL of SEC buffer was applied to one well of the gasket and focused using the “Droplet dilution” option. This was followed by adding 1 μL of pre-diluted protein or protein mixture (details below) to reach a final concentration of 25 nM in the drop. The mass photometer was calibrated using a custom molecular weight standard containing proteins with known sizes ranging from 86 kDa to 344 kDa. MP measurements were recorded for 60 seconds at a rate of 100 frames per second. Data analysis was conducted using DiscoverMP v2023 R.1.2 software (Refeyn Ltd., UK). Peaks in the data were manually selected and fitted with a Gaussian model. The total number of counts predicted by the Gaussian fit, along with percentage values relative to the total number of binding events, were extracted to quantify and compare the oligomeric states of the proteins under study.

Western blot
The YabAAc, TufAAc, MurCAc were acetylated by incubating the proteins with 1−5 mM Ac-CoA overnight. The reaction mixture was then diluted and concentrated using Amicon® Ultra Centrifugal Filter concentrators with a 10 kDa or 30 kDa molecular weight cut-off until the Ac-CoA concentration was reduced to below 100 nM. For activity assays, 20 μg of (Gt)AcuC was pre-incubated with 5 μg of (Gt)AcuB for 10 minutes. To evaluate the effects of Ap4A, 400 μM of Ap4A was added to the (Gt)AcuC-(Gt)AcuB mixture. The acetylated proteins (10 μg) were then added to the enzyme mixture, and the reaction was carried out at 37 °C. Samples were collected after 1 and 2 hours. The samples were subjected to SDS-PAGE and transferred to a PVDF membrane using the BIO-RAD Trans-Blot Turbo system (7 minutes, 1.3 A, 25 V). The membrane was blocked for 1 hour at room temperature with 10% non-fat dry milk (NFDM) in 1x TBST (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% (v/v) Tween-20). Anti-acetyl-lysine antibodies (1:1500) were incubated overnight at a temperature of 4 °C, followed by three washes with 1x TBST. The membrane was then incubated with anti-rabbit IgG-alkaline phosphatase antibodies (1:1500) for 1 hour at 4 °C. Signals were detected using the ECL Prime system and visualized with a Fusion-SL chemiluminescent imager (Peqlab). Full scan blots are provided in the the Source Data file.

Analytical size-exclusion chromatography (SEC)
Purified N-Strep-(Gt)AcuB-C-His and N-His-(Gt)AcuC were prepared at 100 μM in a buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 20 mM MgCl2, and incubated for 10 minutes at room temperature. Subsequently, 100 μL of the mixture were injected at 10 °C onto a pre-equilibrated S75 300/10 GL analytical size-exclusion column (GE Healthcare, Munich, Germany) using an Äkta system (UNICORN 7.6; Cytiva). The data were plotted using GraphPad Prism (GraphPad Prism Corp., San Diego). Single proteins were also injected and processed using the same procedure.

Accelerated MD Simulations of the AcuB Dimer at 300 K. The three-dimensional structures of the AcuB dimer in both the apo and Ap4A-bound states were generated using SWISS-MODEL69, with the crystal structure of the (Bs)AcuB–Ap4A complex resolved in this study serving as a template. The Ap4A ligand was docked into the active site of AcuB using LeDock70 software. For molecular dynamics (MD) simulations, the AMBERff14SB71 force field was applied to the protein, while the General Amber Force Field (GAFF)72 was used for the Ap4A ligand. The systems were solvated in an explicit TIP3P73 water box with a minimum buffer distance of 16 Å, and sodium ions were added to neutralize the system. Energy minimization was performed in two stages: initial relaxation of solvent and ions, followed by minimization of the entire system. The system was gradually heated to 300 K over 50 picoseconds (ps) using the NVT ensemble, followed by 500 ps of equilibration under NPT conditions. Production simulations were conducted using AMBER22 for 200 ns under accelerated MD (aMD)74 conditions to enhance sampling efficiency. Long-range electrostatics were treated using the Particle Mesh Ewald (PME) method75, with a cutoff of 12 Å for nonbonded interactions. Covalent bonds involving hydrogen atoms were constrained using the SHAKE algorithm76, and the timestep was set to 2 femtoseconds (fs).

MD Simulations of the AcuB2-AcuC1 Trimer at 450 K
To further investigate the interaction between AcuB and AcuC, a 2AcuB_1AcuC trimer model was generated using AlphaFold335. The coordination environment of the Zn²⁺ ion in the active site of AcuC was parameterized using the “MCPB.py” modeling tool of AmberTools2277, ensuring proper metal-ligand interactions. The protonation states of all titratable residues were determined based on their pKa values, which were calculated using the PROPKA software78. The system was prepared using the AMBERff14SB force field for proteins and GAFF for Ap4A, while partial atomic charges were obtained from the RESP79 method using the B3LYP/6-31G* level of theory. The system was solvated in a TIP3P water box with a 16 Å buffer, and neutralizing ions were added. Following the same two-stage minimization protocol, the system was gradually heated to 450 K under NVT conditions, followed by 500 ps of NPT equilibration. A 200 ns production run was performed using AMBER22 with periodic boundary conditions. Electrostatic interactions were handled using the PME method, and a 12 Å cutoff was applied for van der Waals interactions. The SHAKE algorithm was used to constrain bonds involving hydrogen atoms, allowing for a 2 fs timestep. Trajectory analysis was performed using Visual Molecular Dynamics (VMD)80 software to evaluate the structural dynamics of the simulated systems. Root Mean Square Fluctuation (RMSF) values of Cα atoms were computed to assess conformational flexibility across the simulation. For molecular dynamics trajectories see Supplementary Dataset 4. Summary and checklist tables of MD simulations are shown as Supplementary tables 5 and 6, respectively. Evidence for system equilibration is shown in Supplementary Fig. 23.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Reversible lysine acetylation is a highly conserved post-translational modification that regulates a wide range of cellular processes in organisms. In bacteria, acetylation level precisely controls critical processes such as metabolic adaptation and virulence, which holds significant implications for understanding the physiological functions of protein acetylation and designing antibacterial targets. This study primarily reveals the previously uncharacterized protein AcuB, which is stabilized by the alarmone diadenosine tetraphosphate (Ap4A), leading to the functional inhibition of the deacetylase AcuC. The physiological significance of AcuB is emphasized.\nThrough a series of experiments, including pulldown assays, protein purification, HDX-MS, isothermal titration calorimetry and so on, the interaction domains of AcuC-AcuB and Ap4A-AcuB complex were determined. Specifically, during stress conditions, two Ap4A molecules bind symmetrically to the cystathionine beta-synthase domain of AcuB, thereby enhancing the stability of AcuB and subsequently inhibiting the deacetylase activity of AcuC. This inhibition affects the deacetylation of multiple substrates, including acetyl-CoA synthetase and translation elongation factors.\nOverall, this study is highly intriguing and innovative, with rigorous experimental design and reliable results. It unveils a regulatory network connecting stress response, protein acetylation, and acetyl-CoA biosynthesis, and innovatively identifies the physiological role of the previously uncharacterized protein AcuB. However, the study has the following main limitations:\nMajor comments:\n1. It lacks a global acetylome analysis to validate the hypothesis that protein acetylation increases under stress conditions.\n2. Proteomics techniques should be employed to systematically screen and identify potential substrate proteins of AcuC, further elucidating its function and related regulatory networks.\n3. The study primarily focuses on Bacillus species; the universality of this mechanism in other organisms should be thoroughly explored. In Geobacillus thermodenitrificans, the addition of Ap4A results in a relatively modest increase in the thermal stability of AcuB. These results raise the possibility that this process is controlled by species-specific regulatory mechanisms.\n4. The authors demonstrate that under stress conditions, Ap4A stabilizes AcuB and inhibits AcuC's deacetylase activity. Whether this mechanism can be applied to antibacterial drug design or used to optimize microbial metabolic efficiency for industrial production through engineered acetylation regulatory circuits remain to be explored.\n5. The term \"(Bs)AcuB_R33E\" is mentioned for the first time in Supplementary Figure 8a, but its origin (e.g., mutation rationale, experimental design) has not been explained.\nMinor comments:\n1. The horizontal axis label is missing in Figure 3e.\n2. The phrase \"in the (Gt)AcuC_R33E mutant\" should be corrected to \"in the (Gt)AcuB_R33E mutant\" on page 6, line 40.", "reviewer_2": "The manuscript \"A protein adaptor mediating Ap4A-dependent control of protein acetylation\" by Bange, Wang and collaborators, demonstrates the interaction both mesophilic and thermophilic homologs between AcuB and AcuC, and suggests the relevance of Ap4A as an alarmone able to modulate such binding and, as a consequence, downregulating the deacetylase activity of AcuC.\nThe article is well written and organized, providing a number of convicing experimental data that confirms the proposed model of interaction of Ap4A with CBS domains in AcuB, as well as the distinctive stability of the latter and its interaction with AcuC.\nThe main concern of this referee is in the use of too simplistic (despite fully standard) and importantly limited molecular dynamics simulations to explain the molecular mechanism of AcuB/AcuC binding, as well as the effect of the presence of Ap4A. Despite the simulation apparently confirming the experimental results, only partial information is provided with respect to the differential stability of the apo and Ap4A bound states, and they are mostly based in the distance between the sulfur atom in AcuB_M188 and the catalytic zinc ion in AcuC. It is not clear than a simgle 200ns trajectory can be determinant for the conclusions claimed in Fig 5e, 5g and 5h and further analysis, at least, of the simulation are needed. For example, how good are the docking calculations? what other conformations (if any) are explored during the MD run for each system? Giving the fact that two apparently distant conformations are obtained in the simulation, a proper analysis of the free energy lansdcape using tolls like Markov models or alike would enhance the interest of the simulations in this article. It is obvious that such analysis would require larger sampling either by extending the simulation or by adding additional replicas. Otherwise, this section of the article does not seems to be justifiable.\nMinor issues:\n-in page 3, lines 10-19, it appears confusing information in the labelling of AcuC and AcuB, with (Gt) prefixes in some sentences and not present in others. It is not clear if this difference is intentional, reflecting generic vs specific concepts, or just a typo.\n-page 3. line 20-22. The sentence seems to lack correct grammar.\n-page 4. lines 26-40. A value of Kd = 0.39 micromolar is said to be a \"strong binding affinity\", when a most realistic description would be moderate binding affinity.\n-page 5. line 2. The variant (Bs)AcuB_R33E is first mentioned and it was not mentioned \"above\".\n-page 5. lines 9-10. I suggest using the sentence \"Thus, AcuB is an Ap4A stabilized AcuC inhibitor\"", "reviewer_3": "The paper by Zheng et al., addresses the mechanism of action of AcuB whose functions have remained unknown for decades. AcuB is a regulator of AcuC which deacetylates a broad spectrum of substrates. Based on this broad specificity, the authors proposed that AcuC has a global role in controlling the acetylome. The interaction of AcuB with AcuC is mediated by the second messenger alarmone A4pA. Binding of the alarmone stabilises AcuB and favours the interaction with AcuC. The authors proposed based on HDX-MS measurements and pulldown assays that while the CBS domain of AcuB is the one that interacts with A4pA, it is the ACT domain of AcuB that engages AcuC and blocks the active site of the enzyme.\nThis is a solid and well written manuscript that deconstructs different aspects of the inhibition of AcuC by AcuB and the regulation of AcuB by the A4pA alarmone.\nGeneral comments:\n- While the interaction between Ap4A and AcuB is well supported by biochemical, biophysic and structural data the interaction between AcuB and AcuC relies only on HDX-MS data, therefore the predicted binding interface should be further supported by point mutations.\n- It is undisputed that the protein binds preferentially Ap4A, however the affinity of AcuB for ADP is not negligible. In this context, the Kd differential between these molecules is less relevant, how do the authors rationalise this? Can the authors provide evidence from competition experiments?\n- Is there a technical reason why ITC could not be used here? The reported methods are both qualitative and don’t much mechanistic information that could explain the role of Ap4A on the stabilization of the AcuB-AcuC complex\n- From the calorimetry experiments shown in Fig 3 : Can the authors clarify the molar ratio from the ITC measurement? From the figure it looks like a molar ratio of 0.5 Ap4A : 1 AcuB, suggesting that only one molecule of Ap4A is bound to the dimer. This seems to differ with the crystal structure which contains 2 Ap4A…\n- In the Discussion section, perhaps should tone down the first section, the importance of the discovery is very clear, so there is no need to repeat this in three different ways. In addition, in my opinion the separation of the Discussion into small sections reduces somewhat the impact of the finding since all the results are only presented in a smaller context. I believes a more overarching text that combines all sections may address this issue.\nMinor comments:\n- Page 5, line 18: Perhaps the shape of the protein should be renamed for non Start Trek connoisseurs …\nPage 8 line 22: Clostridium perfringens should be in Italic.\n- The figures with the HDX differences plotted on the structures are not very clear, could the authors plot the HDX in surface representation as well? With the ribbons and the shadows is a rather difficult to analyse the figures.", "reviewer_4": "In this manuscript Zheng et al describes a novel regulatory pathway linking protein (de)acetylation and stress response via the alarmone Ap4A. Authors report interaction of previously uncharacterized protein AcuB with the AcuC deacetylase, which results in the inhibition of its activity. They demonstrate that otherwise unstable AcuB is stabilized by binding of alarmone Ap4A. They report the structure of AcuB bound to Ap4A, showing extensive interaction with CBS domain which results in stabilization of the homodimer and further impacts the stability of AcuB/AcuC complex. Moreover, this represents yet another unique mode of Ap4A binding, different from other (few) examples. Overall, this study is well designed and quite complete, it characterizes unprecedented function of AcuB and uncovers the pathway in which it is involved. It is novel and well written and will certainly be of great interest across the fields of post-translational modifications, stress response and metabolism.\nThe only weakness of this study is that different parts are performed using Acu proteins from two different bacteria. For example, the affinity of Ap4A for Bacillus AcuB is much bigger than that of Geobacillus, and the stabilizing effect is most likely much stronger. Hence, while Ap4A effect is much stronger in the Bacillus model, the studies on conformational changes and inhibitory function were performed in Geobacillus model. Maybe this is the reason, why the impact on interaction seen by pulldown (figure 4c, lane 3) is not very big as compared to condition without Ap4A. In the printed version of draft, it is barely visible. Nevertheless, this limitation is due to a technical constraint, since authors cannot work with AcuB from Bacillus which aggregates during purification. However, since the Geobacillus AcuB is already relatively stable, I am wondering if the interpretation of the Ap4A effect on its interaction with AcuC is comparable to the case of Bacillus. This issue could be better communicated in the text. Otherwise, I have only minor suggestions:\nPage 2, Lines 16-19 – I missed the transition between these two paragraphs. Authors statement is very broad, i.e. that they set out to fill the gap of knowledge of Ap4A mechanisms across domains of life, but they actually detail one particular case. Although the argument is valid, it is also detached from what comes immediately after, since authors first talk about interactions between Acu proteins, and only later present role of Ap4A. I would consider smoother transition, maybe including explanation why authors selected Acu pathway for investigation. I suppose the inspiration came from DRaCALA experiments and presence of ACT domain in AcuB.\nPage 2, Line 21 – abbreviation or description for ACT domain should be provided at first mention.\nFigure 1 d - Authors should be more precise in the legend of what is represented in this panel. Typically the minus sign would mean that there is no enzyme, but here it means inactivated (acetylated AcsA_K549ac), which is also present in all the conditions, but further supplemented with Zn2+ and AcuC and AcuC/AcuB (also with Zn2+?). If authors indicated directly on the figure that AcsA_K549 was present in all conditions, the figure would be immediately easier to interpret.\nAuthors should provide SEC calibration curve (Mw standards) to appreciate the sizes/size differences of the peaks in Figure 1 c.\nSupplementary figures 5 and 6 – the first conditions addressed in the text are the ones with AcuB/AcuC, and only later those with Ap4A. The HDX figures could follow (top to bottom) this outline as well.\nPage 4, Line 15 – the comparison with the structure of homolog from Alcaligenacea bacterium is very brief and its relevance is not very clear. Was it solved with ligand? If this comparison is useful to define the active site of (Gt)AcuC, authors should be clearer in the text and could also provide supplementary figure with structure alignment indicating the predicted active site, maybe also in Figure 2b. Currently, the active site is only discussed much later in the last part of results.\nPage 5, line 2, the description of R33E mutant is not provided above, it is given below at the line 38.\nPage 5, line 18 – not being a fan of Star Trek, I personally had to google the term to understand what is “starship enterprise”. As for TTHA0829 protein, it is worth mentioning that it is a protein of an unknown function whose structure was previously reported.\nFigure 3 d – for clarity, it would be better that carbons of both of the Ap4A molecules would differ from carbons of AcuB. Now one of right Ap4A molecule has the same color as one of the AcuB’s.\nPage 6, line 4 – for consistency please use the same type of abbreviations for amino acids, either one letter code or three letter code.\nFigure 4c, it would be easier to interpret the figure if y axis would be called more specifically, i.e. molar ratio of which proteins?\nFigure 4d, same comment as for figure 1d – it would be easier if the presence of AcsA_K549ac would appear in the legend or axis name, for example “AcCoA (peak area) in presence of AcsA_K549ac”.\nPage 7, line 27 – what is the reason to perform MD simulations at 300K and then have a drastic shift to 450K? Wouldn’t proteins denature at this temperature? As for non-expert in MD simulations, it sounds odd to me.\nFigure 5 d – for easier interpretation legend to what is red, or blue could be provided directly in the figure panel.\nFigure 6 a seems to be slightly alien to the study, maybe figure 6 b is sufficient to make to point.\nPage 8, line 29 – In this study, the kD of Ap4A interaction with AcuB was measured to be about 0.4 uM. Authors could put this measurement in the biological context here, i.e. is it known what concentration of Ap4A in the cell under steady state conditions or upon stress?\nFigure 6 c – I am not sure what is the reason to represent the AcuC in ribbon, and AcuB in volume? Esthetically it would be better to use one type of representation. Ap4A molecule could also be shown. The scheme could present the stabilizing effect of Ap4A on AcuB more clearly."}, "author_response": "We appreciate this insightful suggestion. We have attempted to perform a global acetylome analysis under stress conditions. Unfortunately, due to the limited quality of the obtained data, we were not able to reach a conclusive result as also stated in the discussion section of the initial submission: “This is likely to lead to a global increase in protein acetylation. Testing this hypothesis would require comprehensive acetylome profiling, although technical challenges have hindered such analyses so far”. We have not included our preliminary findings in the current version of the manuscript.\n2. Proteomics techniques should be employed to systematically screen and identify potential substrate proteins of AcuC, further elucidating its function and related regulatory networks.\nWe thank the reviewer for this valuable suggestion. We agree that proteomics approaches are a possible venue for a more systematic identification of AcuC substrates and for elucidation of the broader regulatory network underlying these pathways. However, in the current study, we focus primarily on the regulatory mechanism of AcuB toward AcuC. Our results provide an initial indication that AcuC can act on multiple targets, laying the groundwork for future comprehensive proteomic investigations, which we feel are outside the scope of the current more mechanistic manuscript. Please note that we have previously shown, that AcsA – a well-established substrate of AcuC is encoded in close proximity in the genome – however it does not pull-down AcuC1. Thus, a tight physiological interaction may not be mandatory for the deacetylation to occur. We will probably need a workaround e.g. a catalytic inactive AcuC variant in future attempts to find more AcuC substrates in vivo.\n3. The study primarily focuses on Bacillus species; the universality of this mechanism in other organisms should be thoroughly explored. In Geobacillus thermodenitrificans, the addition of Ap4A results in a relatively modest increase in the thermal stability of AcuB. These results raise the possibility that this process is controlled by species-specific regulatory mechanisms.\nOur bioinformatics analysis shows that the acuA, acuB, acuC operon mostly occurs in Bacillota phylum. The key binding residues (R33 and H34) for Ap4A are conserved across species (more than 200 species have been investigated, Fig. 3e). In the two tested bacteria the function of AcuB and also its preferential binding to Ap4A are conserved features, although both Bacillus species are adapted to a very different life-styles. This makes us believe that the Ap4A dependent regulation mechanism is conserved across species in principle. Please note that we detect Ap4A dependent stabilization of the AcuA-AcuB complex for G. thermodenitrificans proteins, something which we cannot measure for the B. subtilis proteins due to the instability of (Bs)AcuB.\n4. The authors demonstrate that under stress conditions, Ap4A stabilizes AcuB and inhibits AcuC's deacetylase activity. Whether this mechanism can be applied to antibacterial drug design or used to optimize microbial metabolic efficiency for industrial production through engineered acetylation regulatory circuits remain to be explored.\nWe thank the reviewer for this valuable comment. We agree with the reviewer at this point, but think that addressing this point is probably outside the scope of our study.\n5. The term \"(Bs)AcuB_R33E\" is mentioned for the first time in Supplementary Figure 8a, but its origin (e.g., mutation rationale, experimental design) has not been explained.\nWe have added this part in the method section\nMinor comments:\n1. The horizontal axis label is missing in Figure 3e.\nWe have added axis label as reviewer suggested.\n2. The phrase \"in the (Gt)AcuC_R33E mutant\" should be corrected to \"in the (Gt)AcuB_R33E mutant\" on page 6, line 40.\nWe add the axis label in Figure 3e and change (Gt)AcuC_R33E to (Gt)AcuB_R33E as well\nReviewer #2 (Remarks to the Author):\nThe manuscript \"A protein adaptor mediating Ap4A-dependent control of protein acetylation\" by Bange, Wang and collaborators, demonstrates the interaction both mesophilic and thermophilic homologs between AcuB and AcuC, and suggests the relevance of Ap4A as an alarmone able to modulate such binding and, as a consequence, downregulating the deacetylase activity of AcuC.\nThe article is well written and organized, providing a number of convicing experimental data that confirms the proposed model of interaction of Ap4A with CBS domains in AcuB, as well as the distinctive stability of the latter and its interaction with AcuC.\nThe main concern of this referee is in the use of too simplistic (despite fully standard) and importantly limited molecular dynamics simulations to explain the molecular mechanism of AcuB/AcuC binding, as well as the effect of the presence of Ap4A. Despite the simulation apparently confirming the experimental results, only partial information is provided with respect to the differential stability of the apo and Ap4A bound states, and they are mostly based in the distance between the sulfur atom in AcuB_M188 and the catalytic zinc ion in AcuC. It is not clear than a simgle 200ns trajectory can be determinant for the conclusions claimed in Fig 5e, 5g and 5h and further analysis, at least, of the simulation are needed. For example, how good are the docking calculations? what other conformations (if any) are explored during the MD run for each system? Giving the fact that two apparently distant conformations are obtained in the simulation, a proper analysis of the free energy lansdcape using tolls like Markov models or alike would enhance the interest of the simulations in this article. It is obvious that such analysis would require larger sampling either by extending the simulation or by adding additional replicas. Otherwise, this section of the article does not seems to be justifiable.\nWe sincerely thank the reviewer for this insightful comment. To further validate the robustness of our MD-based conclusions, we performed three independent MD simulations for both the apo and Ap4A-bound 2AcuB_1AcuC complexes. The results consistently revealed that in the presence of Ap4A, the distance between the sulfur atom of AcuB_M188 and the catalytic Zn²⁺ ion in AcuC was markedly shorter than in the apo state (average distances of 7.13 ± 0.87 Å and 11.5 ± 2.56 Å, respectively). More importantly, mutation of AcuB_M188A significantly reduced the AcuB-AcuC complex formation. These consistent findings strongly support the robustness of the conclusions drawn in the original manuscript (Figs. 5e, 5g, and 5h).\nFor the conformational analysis, each MD trajectory was preprocessed by removing overall translational and rotational motions. A mass-weighted covariance matrix was then constructed and diagonalized to obtain the principal components (PCs). The first two PCs, accounting for 50% of the total variance, were used as reaction coordinates. Using kernel density estimation, conformational probability distributions were computed, which were then converted to free energy landscapes via Boltzmann inversion. The resulting landscapes (Supplementary Fig. 19) provide a clear visualization of the conformational energy distributions for both apo and Ap4A-bound systems.\nThe free energy surfaces of both systems exhibit two shallow basins, indicating that the overall motion patterns are similar. However, subtle differences in local conformations suggest that the presence of Ap4A may allosterically stabilize regions critical for AcuC inhibition, consistent with our biochemical data.\nTogether, these results demonstrate that Ap4A binding stabilizes the AcuB dimer and strengthens its association with AcuC through conformational restriction and local allosteric effects. Although the current simulations were limited to hundreds of nanoseconds, the convergence of replicated trajectories and consistent distance and PCA analyses support the robustness of these conclusions. In future studies, extended simulations or enhanced sampling methods, such as Markov state modeling, could be employed to further resolve the detailed kinetic pathways underlying Ap4A-mediated regulation of AcuC activity.\nTherefore, we added some more details to the revised manuscript:\n“To further characterize the conformational space explored during the simulations, we performed principal component analysis (PCA) on the concatenated trajectories after removing overall translational and rotational motions. A mass-weighted covariance matrix was constructed and diagonalized, and the first two principal components (PC1 and PC2), accounting for approximately 50% of the total variance, were used as reaction coordinates. Free energy landscapes were then derived through Boltzmann inversion of kernel density–estimated conformational probabilities (Supplementary Fig. 19). Both apo and Ap4A-bound systems exhibited two main basins, indicating the presence of two dominant conformational states. The relatively flat energy profiles suggest low energy barriers between these states, while subtle shifts in local minima imply that Ap4A binding may allosterically stabilize specific local conformations of AcuB that enhance its inhibitory interaction with AcuC…….”\nMinor issues：\n-in page 3, lines 10-19, it appears confusing information in the labelling of AcuC and AcuB, with (Gt) prefixes in some sentences and not present in others. It is not clear if this difference is intentional, reflecting generic vs specific concepts, or just a typo.\nWe have added the relevant species\n-page 3. line 20-22. The sentence seems to lack correct grammar.\nWe have made the change\n-page 4. lines 26-40. A value of Kd = 0.39 micromolar is said to be a \"strong binding affinity\", when a most realistic description would be moderate binding affinity.\nWe have made the change accordingly\n-page 5. line 2. The variant (Bs)AcuB_R33E is first mentioned and it was not mentioned \"above\".\nWe have made the change\n-page 5. lines 9-10. I suggest using the sentence \"Thus, AcuB is an Ap4A stabilized AcuC inhibitor\"\nWe have made the change\nReviewer #3 (Remarks to the Author):\nThe paper by Zheng et al., addresses the mechanism of action of AcuB whose functions have remained unknown for decades. AcuB is a regulator of AcuC which deacetylates a broad spectrum of substrates. Based on this broad specificity, the authors proposed that AcuC has a global role in controlling the acetylome. The interaction of AcuB with AcuC is mediated by the second messenger alarmone A4pA. Binding of the alarmone stabilises AcuB and favours the interaction with AcuC. The authors proposed based on HDX-MS measurements and pulldown assays that while the CBS domain of AcuB is the one that interacts with A4pA, it is the ACT domain of AcuB that engages AcuC and blocks the active site of the enzyme.\nThis is a solid and well written manuscript that deconstructs different aspects of the inhibition of AcuC by AcuB and the regulation of AcuB by the A4pA alarmone.\nGeneral comments:\n- While the interaction between Ap4A and AcuB is well supported by biochemical, biophysic and structural data the interaction between AcuB and AcuC relies only on HDX-MS data, therefore the predicted binding interface should be further supported by point mutations.\nWe thank the reviewer for raising this important point. We analyzed the interface of the (Gt)AcuB–AcuC complex supported by HDX-MS data, which revealed several interfacial interactions, including salt bridges between (Gt)AcuB_E4 and (Gt)AcuC_R362, (Gt)AcuB_D89 and (Gt)AcuC_K378, and (Gt)AcuB_E92 and (Gt)AcuC_R197, as well as π–π and hydrophobic interactions between (Gt)AcuB_W206 and (Gt)AcuC_Y197, and between (Gt)AcuB_M188 and (Gt)AcuC_F200/F143 (Supplementary Figure 7). Based on these analyses, we constructed several point mutants, including (Gt)AcuB_D89K, E92R, and M188A, as well as (Gt)AcuC_R197E, Y198A, and R362E. Subsequently, pulldown assays were performed to validate the predicted interactions. Among the AcuB mutants, D89K, E92R, and M188A showed markedly reduced complex formation; R197E and Y198A caused a moderate decrease; while R362E had little to no effect. These results are consistent with the predicted (Gt)AcuB–AcuC interface (Supplementary Figure 7).\nThe corresponding data have been added as Supplementary Figure 7, and the relevant description has been incorporated into the main text as follows: “More detailed analysis revealed that the interface between (Gt)AcuB and (Gt)AcuC is stabilized by multiple interactions, including salt bridges between (Gt)AcuB_E4–(Gt)AcuC_R362, (Gt)AcuB_D89–(Gt)AcuC_K378, and (Gt)AcuB_E92–(Gt)AcuC_R197, as well as π–π and hydrophobic interactions between (Gt)AcuB_W206–(Gt)AcuC_Y197 and (Gt)AcuB_M188–(Gt)AcuC_F200/F143 (Supplementary Fig. 9A). To validate these predicted interfacial contacts, we constructed single-point mutants (Gt)AcuB_D89K, E92R, M188A and (Gt)AcuC_R197E, Y198A, R362E, and performed pulldown assays (Supplementary Fig. 9B). The mutations D89K, E92R, and M188A in AcuB significantly reduced complex formation, while R197E and Y198A moderately weakened the interaction. In contrast, R362E showed minimal effect. These results confirm the key interfacial residues mediating the (Gt)AcuB–AcuC interaction.\n- It is undisputed that the protein binds preferentially Ap4A, however the affinity of AcuB for ADP is not negligible. In this context, the Kd differential between these molecules is less relevant, how do the authors rationalise this? Can the authors provide evidence from competition experiments?\nWe have conducted competition assays using a ThermoShift assay to address the concern regarding the relative affinity of AcuB for ADP and Ap4A. The data clearly demonstrate that even when ADP is present at concentrations 5 times higher than Ap4A, the thermal shift induced by the binding of (Bs)AcuB remains comparable to that observed with Ap4A alone. This suggests that Ap4A preferentially binds to (Bs)AcuB, even in the presence of excess ADP. The fact that the thermal shift response is similar indicates that Ap4A is the dominant ligand, reinforcing its higher affinity for (Bs)AcuB. These results support the notion that, despite the presence of ADP, the binding of Ap4A is more energetically favorable and thus drives the interaction. Nevertheless, our data does not exclude a function for ADP binding in the absence of stress/Ap4A.\nWe have added following description in the main text: “It is worth mention that ADP also shows a µM binding affinity to (Bs)AcuB. To further investigate the binding specificity of (Bs)AcuB to Ap4A, we performed competition experiments using a thermoshift assay. In these experiments, AcuB was incubated with both ADP and Ap4A, with ADP concentrations set 5 times higher than Ap4A. Despite the excess ADP, the thermal shift profile was nearly identical to that observed when Ap4A was present alone, confirming that Ap4A preferentially binds to (Bs)AcuB, even in competitive binding conditions. As ADP binds to (Bs)AcuB and changes its thermal stability – albeit to a lesser extent – it is possible that variations of ADP levels regulate AcuB activity in non-stressed cells, a phenomenon which merits future investigation ”\n- Is there a technical reason why ITC could not be used here? The reported methods are both qualitative and don’t much mechanistic information that could explain the role of Ap4A on the stabilization of the AcuB-AcuC complex\nWe also attempted to use ITC to investigate the role of Ap4A in stabilizing the AcuB–AcuC complex. However, under the conditions tested, we were unable to obtain a reliable Kd for the AcuB–AcuC interaction (Fig. R1). Unfortunately, ITC was not suitable to characterize this interaction, which is why we relied on complementary more qualitative methods such as pulldown and MP to evaluate the effect of Ap4A on complex stabilization.\nFigure R1. ITC analysis of (Gt)AcuB–AcuC interaction measured at different protein concentrations.\n- From the calorimetry experiments shown in Fig 3 : Can the authors clarify the molar ratio from the ITC measurement? From the figure it looks like a molar ratio of 0.5 Ap4A : 1 AcuB, suggesting that only one molecule of Ap4A is bound to the dimer. This seems to differ with the crystal structure which contains 2 Ap4A.\nWe appreciate this point. We noted that both (Gt)AcuB and (Bs)AcuB are not completely stable in solution and tend to precipitate at room temperature – even within 20 minutes, which might have affected the observed molar ratio in the ITC experiments, resulting in an apparent ratio of ~0.5 Ap4A per AcuB dimer. Given our structural analysis we think this is the most likely explanation\n- In the Discussion section, perhaps should tone down the first section, the importance of the discovery is very clear, so there is no need to repeat this in three different ways. In addition, in my opinion the separation of the Discussion into small sections reduces somewhat the impact of the finding since all the results are only presented in a smaller context. I believes a more overarching text that combines all sections may address this issue.\nWe thank the reviewer for this suggestion and revised the first paragraph. However, we kept the different paragraphs as we believe it is helpful to discuss the different aspects of our finding.\nMinor comments:\n- Page 5, line 18: Perhaps the shape of the protein should be renamed for non Start Trek connoisseurs\nWe change the description, as following:\n“(Bs)AcuB forms a homodimer, with the two chains cooperating in a configuration similar to that of its homolog TTHA0829 from Thermus thermophilus (PDB ID: 5AWE) (Fig. 3b, Supplementary Fig. 9).”\nPage 8 line 22: Clostridium perfringens should be in Italic.\nWe have made the change\n- The figures with the HDX differences plotted on the structures are not very clear, could the authors plot the HDX in surface representation as well? With the ribbons and the shadows is a rather difficult to analyse the figures.\nWe changed the representation of HDX projected onto structures for enhanced clarity. The shadows are omitted as suggested and alpha helices are now shown as cylinders. We decided against a surface representation as this obstructed the view onto critical parts, i.e., the Ap4A binding cleft of AcuB and the active site of AcuC.\nReviewer #4 (Remarks to the Author):\nIn this manuscript Zheng et al describes a novel regulatory pathway linking protein (de)acetylation and stress response via the alarmone Ap4A. Authors report interaction of previously uncharacterized protein AcuB with the AcuC deacetylase, which results in the inhibition of its activity. They demonstrate that otherwise unstable AcuB is stabilized by binding of alarmone Ap4A. They report the structure of AcuB bound to Ap4A, showing extensive interaction with CBS domain which results in stabilization of the homodimer and further impacts the stability of AcuB/AcuC complex. Moreover, this represents yet another unique mode of Ap4A binding, different from other (few) examples. Overall, this study is well designed and quite complete, it characterizes unprecedented function of AcuB and uncovers the pathway in which it is involved. It is novel and well written and will certainly be of great interest across the fields of post-translational modifications, stress response and metabolism.\nThe only weakness of this study is that different parts are performed using Acu proteins from two different bacteria. For example, the affinity of Ap4A for Bacillus AcuB is much bigger than that of Geobacillus, and the stabilizing effect is most likely much stronger. Hence, while Ap4A effect is much stronger in the Bacillus model, the studies on conformational changes and inhibitory function were performed in Geobacillus model. Maybe this is the reason, why the impact on interaction seen by pulldown (figure 4c, lane 3) is not very big as compared to condition without Ap4A. In the printed version of draft, it is barely visible. Nevertheless, this limitation is due to a technical constraint, since authors cannot work with AcuB from Bacillus which aggregates during purification. However, since the Geobacillus AcuB is already relatively stable, I am wondering if the interpretation of the Ap4A effect on its interaction with AcuC is comparable to the case of Bacillus. This issue could be better communicated in the text. Otherwise, I have only minor suggestions:\nWe thank the reviewer for the overall positive comments. We have discussed potential adaptations in both organisms in the revised paper. However, we are confident that key features are conserved. Both AcuB variants bind Ap4A and AcuC. While we indeed see stronger stabilization of (Bs)AcuB through Ap4A, we still observe increased interaction for (Gt)AcuB with AcuC upon Ap4A treatment. Both stabilization of AcuB and interaction AcuC might foster inhibition of AcuC in vivo, thus it appears to be a conserved regulatory scheme at least in principle.\nWe added the following paragraph to the discussion section:\nSpecies specific adaptation of this process appears likely given that the effects of Ap4A on thermal stability in investigated AcuB orthologs is variable. Although we only observe a modest increase in stability for (Gt)AcuB in the presence of Ap4A, we detect a significantly increased interaction of AcuB with AcuC specifically in response to Ap4A treatment (Fig. 4c).\nPage 2, Lines 16-19 – I missed the transition between these two paragraphs. Authors statement is very broad, i.e. that they set out to fill the gap of knowledge of Ap4A mechanisms across domains of life, but they actually detail one particular case. Although the argument is valid, it is also detached from what comes immediately after, since authors first talk about interactions between Acu proteins, and only later present role of Ap4A. I would consider smoother transition, maybe including explanation why authors selected Acu pathway for investigation. I suppose the inspiration came from DRaCALA experiments and presence of ACT domain in AcuB.\nWe thank the reviewer for pointing this out. We agree that the transition between the two paragraphs was abrupt and have now improved the flow of the introduction. Specifically, we have added a clearer rationale for selecting the acu operon for investigation which resulted from the CBS domain in AcuB, which we suspected to be an Ap4A binder also confirmed by our DRaCALA sreening. The revised text now provides a smoother transition.\nPage 2, Line 21 – abbreviation or description for ACT domain should be provided at first mention.\nWe have made the change\nFigure 1 d - Authors should be more precise in the legend of what is represented in this panel. Typically the minus sign would mean that there is no enzyme, but here it means inactivated (acetylated AcsA_K549ac), which is also present in all the conditions, but further supplemented with Zn2+ and AcuC and AcuC/AcuB (also with Zn2+?). If authors indicated directly on the figure that AcsA_K549 was present in all conditions, the figure would be immediately easier to interpret.\nWe thank the reviewer for this valuable suggestion. We agree that indicating the presence of AcsA_K549Ac in all conditions will improve clarity. We have now revised the figure to explicitly include AcsA_K549Ac in each condition. In addition, we confirm that Zn²⁺ was supplemented in the AcuC reaction, and have updated the figure and legend accordingly.\nAuthors should provide SEC calibration curve (Mw standards) to appreciate the sizes/size differences of the peaks in Figure 1 c.\nWe have now included the molecular weight standards in Figure 1c to better illustrate the peak size differences and have added the corresponding details to the figure legend. To simplify the Figure, we provide the standard curve as Supplementary Fig 4.\nSupplementary figures 5 and 6 – the first conditions addressed in the text are the ones with AcuB/AcuC, and only later those with Ap4A. The HDX figures could follow (top to bottom) this outline as well.\nWe amended the order in Supplementary Figs 5 and 6 as suggested.\nPage 4, Line 15 – the comparison with the structure of homolog from Alcaligenacea bacterium is very brief and its relevance is not very clear. Was it solved with ligand? If this comparison is useful to define the active site of (Gt)AcuC, authors should be clearer in the text and could also provide supplementary figure with structure alignment indicating the predicted active site, maybe also in Figure 2b. Currently, the active site is only discussed much later in the last part of results.\nWe thank the reviewer for this insightful comment. We have expanded the description of the structural comparison with the homolog from Alcaligenaceae bacterium (PDB ID: 1ZZ1) to better explain its relevance in defining the putative active site of (Gt)AcuC. In addition, we have added a structural alignment of (Gt)AcuC with 1ZZ1 as a new supplementary figure (Supplementary Fig. 8), and we now reference this comparison more clearly in the main text, including in the context of Figure 2b and the active-site discussion.\nPage 5, line 2, the description of R33E mutant is not provided above, it is given below at the line 38.\nWe have supplied the information in the method section.\nPage 5, line 18 – not being a fan of Star Trek, I personally had to google the term to understand what is “starship enterprise”. As for TTHA0829 protein, it is worth mentioning that it is a protein of an unknown function whose structure was previously reported.\nWe have changed the description as suggested.\nFigure 3 d – for clarity, it would be better that carbons of both of the Ap4A molecules would differ from carbons of AcuB. Now one of right Ap4A molecule has the same color as one of the AcuB’s.\nWe have changed the color as suggested.\nPage 6, line 4 – for consistency please use the same type of abbreviations for amino acids, either one letter code or three letter code.\nWe have changed all with one letter code\nFigure 4c, it would be easier to interpret the figure if y axis would be called more specifically, i.e. molar ratio of which proteins?\nWe have specified y axis in the Figure 4c legend, as following:\n“The y axis molar ratio represents the N counts (peak of (Gt)AcuB-AcuC complex)/N counts (total) in percent”\nFigure 4d, same comment as for figure 1d – it would be easier if the presence of AcsA_K549ac would appear in the legend or axis name, for example “AcCoA (peak area) in presence of AcsA_K549ac”.\nWe have added the details in legend as following: The peak area (Ac-CoA) is produced in the presence of AcsA_K549Ac\nPage 7, line 27 – what is the reason to perform MD simulations at 300K and then have a drastic shift to 450K? Wouldn’t proteins denature at this temperature? As for non-expert in MD simulations, it sounds odd to me.\nWe thank the reviewer for this valuable comment. Indeed, proteins would denature at 450 K under experimental conditions. However, in MD simulations, transient heating is often employed not to reproduce physiological conditions but to accelerate conformational sampling and help the system overcome local energy barriers within a limited simulation time.\nIn our study, simulations at 300 K were used to evaluate the conformational stability of the AcuB dimer under near-physiological conditions, whereas simulations at 450 K were designed to enhance sampling efficiency and probe the relative stability and interaction tendencies of the AcuB–AcuC complex. Similar high-temperature MD protocols have been widely applied in previous studies to investigate protein–protein interaction stability, ligand dissociation, and transition pathways2-4. Importantly, our conclusions are based on comparative trends between the apo and Ap4A-bound systems rather than on the absolute structural integrity of proteins at 450 K.\nWe have added following explanation in the main text: “It should be noted that the simulations at 450 K were not intended to mimic experimental conditions but rather to accelerate conformational sampling and enable comparison of the relative stability of the apo and Ap4A-bound complexes.”\nFigure 5 d – for easier interpretation legend to what is red, or blue could be provided directly in the figure panel.\nThe color have now been added directly to Figures 5d and 5e\nFigure 6 a seems to be slightly alien to the study, maybe figure 6 b is sufficient to make to point.\nWe have moved both Fig 6a and 6b to supplementary Figure as Supplementary Fig 19.\nPage 8, line 29 – In this study, the kD of Ap4A interaction with AcuB was measured to be about 0.4 uM. Authors could put this measurement in the biological context here, i.e. is it known what concentration of Ap4A in the cell under steady state conditions or upon stress?\nWe have now included additional context regarding cellular concentrations of Ap4A under both steady-state and stress conditions (around 60 µM). This information has been added to the text\nFigure 6 c – I am not sure what is the reason to represent the AcuC in ribbon, and AcuB in volume? Esthetically it would be better to use one type of representation. Ap4A molecule could also be shown. The scheme could present the stabilizing effect of Ap4A on AcuB more clearly.\nWe thank the reviewer for this helpful suggestion. We agree that the stabilizing effect of Ap4A on AcuB should be presented more clearly and have revised Figure 6c accordingly, now also including the Ap4A molecule in the visualization.\nRegarding the representation style, our intention in using a surface (volume) view for AcuB and a ribbon view for AcuC was to better illustrate the specific region of AcuB that inserts into the active site of AcuC, which we feel is more easily appreciated in this mixed representation. We hope the reviewer will agree that this choice improves the clarity of the interaction interface.\nReference"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The author's response is purely textual and does not include experimental data, such as a global acetylome analysis or proteomics screening, which are typically used to identify AcuC's substrate proteins. The omission of such data is likely due to technical challenges or other constraints.\nTherefore, the response does not fully resolve or rebut the core concerns raised in my original query.", "reviewer_2": "The response from the authors and their improvements to the manuscript are acceptable for this referee", "reviewer_3": "As mentioned in my previous report, this is a very interesting and well constructed work.\nThe authors have addressed all my concerns and comments in their revision and I am satisfied with the final manuscript.", "reviewer_4": "In the revised version of the manuscript authors have improved clarity of the manuscript and strengthened some parts of the study with additional results, such as mutations at the protein interaction surface and additional MD simulations. Authors have fully responded to my requests and suggestions and to majority of other reviewers concerns. I have no further comments, and I believe that this manuscript will be valuable contribution to several fields of studies as I have mentioned in my initial review."}, "author_response": "Point-by-point response le/er\nReviewer #1 (Remarks to the Author):\nThe author's response is purely textual and does not include experimental data, such as a global acetylome analysis or proteomics screening, which are typically used to iden9fy AcuC's substrate proteins. The omission of such data is likely due to technical challenges or other constraints. Therefore, the response does not fully resolve or rebut the core concerns raised in my original query.\nWe thank the reviewer for their valuable comments. We agree that global acetylome analysis or proteomics-based screening would be highly informa9ve and represents an important direc9on for future follow-up studies.\nReviewer #2 (Remarks to the Author):\nThe response from the authors and their improvements to the manuscript are acceptable for this referee\nWe thank the reviewer for great comments and are pleased that the reviewer agree to publish our paper\nReviewer #3 (Remarks to the Author):\nAs men9oned in my previous report, this is a very interes9ng and well constructed work.\nThe authors have addressed all my concerns and comments in their revision and I am sa9sfied with the final manuscript.\nWe thank the reviewer for excellent comments and are pleased that all concerns have been addressed.\nReviewer #4 (Remarks to the Author):\nIn the revised version of the manuscript authors have improved clarity of the manuscript and strengthened some parts of the study with addi9onal results, such as muta9ons at the protein interac9on surface and addi9onal MD simula9ons. Authors have fully responded to my requests and sugges9ons and to majority of other reviewers concerns. I have no further comments, and I believe that this manuscript will be valuable contribu9on to several fields of studies as I have men9oned in my ini9al review.\nWe thank the reviewer for their construc9ve feedback and posi9ve evalua9on of the revised manuscript."}]

Decision letter