From the journal RSC Chemical Biology Peer review history

16-Dec-2021

Dear Prof Suga:

Manuscript ID: CB-ART-11-2021-000225
TITLE: Inner residues of macrothiolactone in autoinducer peptides-I/IV circumvents <i>S</i>-to-<i>O</i> acyl transfer to the upstream serine residue

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Prof Gonçalo Bernardes

Associate Editor, RSC Chemical Biology

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Reviewer 1

In this manuscript, the structures of the Staphylococcus aureus macrocyclic thiodepsipeptides (CTPs) AIP-I and AIP-IV are explored to elucidate why an S-to-O acyl transfer to the more chemically stable cyclized depsipeptide (CDP) does not occur. The authors first used their previously published method of one-pot ribosomal synthesis of CDPs via a CTP intermediate to confirm the S-to-O acyl transfer suppression in compounds AIP-I and AIP-IV. Following this, a mutational analysis of key positions in the AIP macrocycle revealed that glycine substitution of the Asp/Try residue in the STC(D/Y) motif and the C-terminal Met residue resulted in the transfer to the CDP form. Furthermore, expanding the thiolactone ring to six or seven residues maintained the CTP form, but extending it to eight residues resulted in the CDP form. The authors hypothesize that the alpha-L-chirality of the critical macrocyclic residues and ring constraints prevent the S-to-O acyl transfer from occurring.

This work confirms the well-established structures of S. aureus AIP-I and AIP-IV, and thus the structure validation is not entirely novel. However, the investigation into the nature of S-to-O acyl transfer suppression is of interest, and the experimental methodology is sound, although certain experimental choices could be elaborated on and details of quantitation methods are needed. I recommend that this manuscript be published in RSC Chemical Biology with minor revisions, as outlined below:

• The authors should elaborate on how they determined the thiolactone replacements in the compound 9-12GS. The D9G and M12G mutations are well-explained, but the choice of I11S was not clear, and F10G had previously been shown to not have an effect on CTP-CDP rearrangement. Furthermore, if the 10th and 11th positions are not important for this rearrangement, why did 9-12GS give more complete conversion to CDP than the D9G,M12G mutant? This was not clearly conveyed.

• An explanation for why a mutated thiolactone ring was used for the ring expansions, other than the addition of residues necessary to expand the ring. If this experiment is investigating the effect of ring size and not individual residue mutations, why were the original ring residues substituted for Gly and Ser residues? This was not explained and is confusing. If it was a matter of solubility or ring rigidity, that should be stated.

• The authors fail to describe how they quantitate CDP (and CTP) formation. Are the CDP products being formed, but only in a low percentage? In what ratios with CTP? Of if they form, is it always quantitative? They only show MS identification. The authors indicate 60 min for the shift in Figure 3, does this give complete conversion? Do certain mutations slow the transfer reaction rate? I would imagine so. General comments on cyclization/reaction rates and percent conversion, with details of the methods, are needed.

• The authors discuss their conclusion that because only Gly mutations to the Asp/Tyr residues resulted in the CDP form, the alpha-L-chirality of these residues plays a critical role in controlling the reactivity of the thioester bond. To further support this hypothesis, and to further strengthen this paper, analogs with the corresponding D-amino acid residues in the Asp/Tyr and Met positions could be synthesized (via standard SPPS if easier than in vitro) and their effect on transfer from CTP to CDP form investigated. This would elucidate the isolated effect of chirality without the additional macrocycle flexibility given by a glycine substitution, and provide a strong result to bring this study together.

• This paper would be strengthened by a longer discussion of the impact of the nature of the S-to-O acyl transfer suppression in the conclusion. This could cover potential applications of this discovery, or how this could shape structure-activity relationship studies of AIPs or other similar compounds. A sequence alignment of AgrD sequences, for example, to explore the feasibility of this shift in a broader range of species could be informative.

• In Table S1, there is no data in the column “MS signal of pre after 30 min translation” and it simply should be removed from the table for clarity. A sentence could be added in the table caption stating “no pre was detected after 30 min translation for any of the peptide products” if the authors feel that this information is important to convey.

Reviewer 2

Prof. Suga and co-workers use the genetic code reprogramming technology to synthesize autoinducing peptides featuring the cyclic thiodepsipeptide (CTP) skeleton, which contain the SX1CX2 motif known to undertake S-to-O acyl transfer to cyclic depsipeptides. However, as a series of S. aureus autoinducing peptides contanning such a motif do not undertake the S-to-O acyl transfer, the author carried out a study to determine which internal residues make possible the conversion into cyclic depsipeptides (CDP).
The article is interesting in the sense that provide new insights into the structural factor determining the stability of the thiolactone moiety. Specifically, it was found that mutations at the neighboring positions of the thiolactone moiety by Gly allow the S-to-O acyl transfer to take place.
The article is very well conducted and flows well taking the reader to those important aspects of the research. The methodology is not new, because this laboratory is an actual pioneer in the one-pot ribosomal synthesis approach, but the results are indeed of interest for the chemical biology audience.
My only concern is the following:
Throughout the manuscript, authors claim that the alleviation of the conformational rigidity of the thiolactone macrocycle ring after Gly mutation at the 9th and/or 12th position is the reason for the CDP formation. I am not entirely opposed to this, but other structural elements should be mentioned as well.
My point is that the S-to-O acyl transfer mediated by the nucleophilic attack of the hydroxyl group of S6 to the thioester is not a transannular process where conformational rigidity is crucial, this is an attack of an exocyclic group. Author noticed that neither F10G nor I11G mutation induced the conversion of CTP to CDP, but changing those restudies by Gly also leads to a significantly reduced rigidity of the macrocyclic ring, so it is not the conformational rigidity. The actual reason is the reduction of steric hindrance around the thiolactone moiety due to the change of L-chiral amino acids to the non-substituted Gly. The attack of Ser side chain is an acyl nucleophilic substitution, which is known to be very sensitive to steric hindrance, so it is actually the alleviation of the steric hindrance around the thiolactone which favors that attack, and not the alleviation of the conformational rigidity, because inserting Gly at positions 10th and 11th also makes the macrocycle much more flexible, but do not favor the S-to-O acyl transfer.
Authors should consider focusing the explanation in the discussion section on the diminished steric hindracen when placing Gly at the neighboroing positions of the thiolactone, and not the conformational ridigity of the macrocycle. Please notice that, if, the Ser would be endocyclic, then the situation would be different and the macrocycle's rigidity/flexibility would matter much more, but, in my opinion, not in this case.
Only in the Conclusions, the authors refer to this point when they state: ‘Since mutations from the Asp/Tyr residue to arbitrary amino acids other than Gly do not alter the resistance, their alfa-L-chiral center plays a critical role in controlling the reactivity of the thioester bond to the hydroxyl group of the upstream Ser’.
But the same line of explantion is not followed in the discussion section.
With this minor revision, I consider the paper is suitable for publication in this journal.

[R1-1 to R-6] : Revierer-1’s comments
[R1-1 to R-2] : Revierer-2’s comments

Modified sentences in the manuscript are highlighted in yellow.

Reviewer-1
In this manuscript, the structures of the Staphylococcus aureus macrocyclic thiodepsipeptides (CTPs) AIP-I and AIP-IV are explored to elucidate why an S-to-O acyl transfer to the more chemically stable cyclized depsipeptide (CDP) does not occur. The authors first used their previously published method of one-pot ribosomal synthesis of CDPs via a CTP intermediate to confirm the S-to-O acyl transfer suppression in compounds AIP-I and AIP-IV. Following this, a mutational analysis of key positions in the AIP macrocycle revealed that glycine substitution of the Asp/Try residue in the STC(D/Y) motif and the C-terminal Met residue resulted in the transfer to the CDP form. Furthermore, expanding the thiolactone ring to six or seven residues maintained the CTP form, but extending it to eight residues resulted in the CDP form. The authors hypothesize that the alpha-L-chirality of the critical macrocyclic residues and ring constraints prevent the S-to-O acyl transfer from occurring.

This work confirms the well-established structures of S. aureus AIP-I and AIP-IV, and thus the structure validation is not entirely novel. However, the investigation into the nature of S-to-O acyl transfer suppression is of interest, and the experimental methodology is sound, although certain experimental choices could be elaborated on and details of quantitation methods are needed. I recommend that this manuscript be published in RSC Chemical Biology with minor revisions, as outlined below:

--> We thank the reviewer for the comments and constructive critiques below.

[R1-1]
The authors should elaborate on how they determined the thiolactone replacements in the compound 9-12GS. The D9G and M12G mutations are well-explained, but the choice of I11S was not clear, and F10G had previously been shown to not have an effect on CTP-CDP rearrangement. Furthermore, if the 10th and 11th positions are not important for this rearrangement, why did 9-12GS give more complete conversion to CDP than the D9G, M12G mutant? This was not clearly conveyed.

--> Thank you for the comment. The sequence SCGGSGCO- in macrothiolactone of 9-12GS is derived from a previous report, where underwent migration to a corresponding 7-ring CDP in a near-quantitative manner (ref-11). To see whether a similar near-quantitative migration occurs, we grafted the same thiolactone sequence into a 5-residual ring of AIP-I mutant (the compound 9-12GS).
As the reviewer pointed out, single glycine mutations at F10 and I11 positions were not influential. Since D9 and M12 are located adjacent to the thioester forming Cys8, thus sidechains of these two residues may directly influence to induce steric hindrance avoiding nucleophilic attack by Ser6.

We added the following sentence highlighted in yellow in the section of Gly scanning:
“More drastic replacement of the DFIM thiolactone sequence with GGSG (9–12GS) gave a nearly complete conversion to the CDP form (Fig. 4a and b, Fig. S3e, ESI), which was consistent with our previous report11.”

“Likely, G mutation at the 9th or 12th position in the AIP-Ia alleviates the conformational rigidity of the thiolactone ring and reduces the steric hindrances around the carbonyl group of the thioester, allowing for the nucleophilic attack of the hydroxyl group of S6 to the thioester bond and the formation of CDP via the S-to-O acyl transfer.”

[R1-2]
An explanation for why a mutated thiolactone ring was used for the ring expansions, other than the addition of residues necessary to expand the ring. If this experiment is investigating the effect of ring size and not individual residue mutations, why were the original ring residues substituted for Gly and Ser residues? This was not explained and is confusing. If it was a matter of solubility or ring rigidity, that should be stated.

--> Comparing to the single Gly substitution of Asp9 and Met12, the AIP mutant, 9–12GS, showed near-quantitative conversion to a corresponding CDP (Figure 4). This suggests that a conformationally flexible macrolactone substituted by less bulky residues could likely be suited to macrolactonization via S-to-O acyl transfer. Based on this observation, we designed the macrothiolactone sequences to include relatively flexible Gly-Ser repeat other than Asp9 and Met12, aiming to expedite an assessment of ring-expansion against the suppression of the S-to-O acyl transfer.

We added the following sentence highlighted in yellow in the section of ring-expansion:
“We thus prepared 6, 7, and 8-residual ring CTPs, where more structurally relaxed peptide sequences consisting of Gly and Ser residues were introduced to between Asp9 and Met12 in the thiolactone ring (Fig. 5a and b, Fig. S5, Table S1c, ESI).”


[R1-3]
The authors fail to describe how they quantitate CDP (and CTP) formation. Are the CDP products being formed, but only in a low percentage? In what ratios with CTP? Of if they form, is it always quantitative? They only show MS identification. The authors indicate 60 min for the shift in Figure 3, does this give complete conversion? Do certain mutations slow the transfer reaction rate? I would imagine so. General comments on cyclization/reaction rates and percent conversion, with details of the methods, are needed.

--> To address the reviewer’s comment, we carried out a semi-quantitative analysis on D9G mutant of AIP-1a by LC-MS, and the results are now added in Figure S3. Since our in vitro-translation usually takes around 20 min to get a steady-state, translated products at 30 min and 60 min incubation were analyzed by LC-MS. Translated product was quenched by MeOH precipitation, and the supernatant was subjected to LC-MS. In the case of the product at 30 min incubation, extracted ion chromatogram (EIC) of D9G-CTP ([M+H]: m/z = 1332.5541) appeared two separated peaks, which were corresponding to CDP and CTP, in 22 % and 78 % conversion yield, respectively. Extending translation time to 60 min increased a peak area corresponding to D9G-CDP to 45 %, suggesting the S-to-O acyl transfer in D9G-CTP slowly occurred. Furthermore, post-translational alkylation by iodoacetamide (IAA) of the translated product diminished the peak derived from D9G-CDP. It was found as a new peak in the EIC chromatogram of D9G-CDP-AA ([M+H]: m/z = 1389.5755) in 54 % conversion yield. These data clearly demonstrate the ratio in CDP/CTP form with or without IAA alkylation and further support results gained via MALDI-TOF MS analysis. A detail of the experimental method is added in the section of Experimental.

We added the following paragraph in the main text:
“To determine the ratio of CDP/CTP form in D9G mutant, the translated product was semi-quantitatively analyzed by LC-MS. A positive control peptide, AIP-1a, was subjected to LC-MS, and then extracted ion chromatogram (EIC) was evaluated. We observed no separated peak and no alkylated product by IAA (Fig. S3a, ESI). On the other hand, when the same experiment was performed on D9G-CTP, EIC chromatogram gave two peaks in 19 % (11.0 min) and 81 % (11.6 min), which were assigned to D9G-CDP and D9G-CTP, respectively (Fig. S3b, ESI). Extended translation time from 30 min to 60 min increased the CDP product from 19 % to 45 %, suggesting that the S-to-O acyl transfer in D9G-CTP slowly proceeded. The IAA alkylation of D9G-CDP (a peak observed at 11.0 min) afforded a fraction of D9G-CDP-AA (the peak observed at 10.5 min) in an approximately 54 % yield. These results confirmed the occurrence of the S-to-O acyl transfer of D9G mutant.”

[R1-4]
The authors discuss their conclusion that because only Gly mutations to the Asp/Tyr residues resulted in the CDP form, the alpha-L-chirality of these residues plays a critical role in controlling the reactivity of the thioester bond. To further support this hypothesis, and to further strengthen this paper, analogs with the corresponding D-amino acid residues in the Asp/Tyr and Met positions could be synthesized (via standard SPPS if easier than in vitro) and their effect on transfer from CTP to CDP form investigated. This would elucidate the isolated effect of chirality without the additional macrocycle flexibility given by a glycine substitution, and provide a strong result to bring this study together.

--> Thank you for the comments. We apologize that our conclusion, in which “the importance of alpha-L-chirality” of Asp/Tyr residues for tolerance of S-to-O acyl transfer, was overstated since we do not have clear experimental evidence to state this. Based on AIP’s biosynthesis, which lacks an epimerase domain in their genome, the mutation experiment using D-amino acid may not be much meaningful. Thus, we removed the words “alpha-L-chirality of Asp/Tyr residues” from the main text and abstract. Instead, we modified it to “the importance of steric hindrance induced by the sidechain of the amino acids”.

We added the following sentence highlighted in yellow in the section of abstract and conclusion, respectively:
“This suggests that the steric hindrances originating from the alpha-substituted sidechain in these two amino acids in the AIP-I/IV thiolactone likely play a critical role in controlling the resistance against the macrolactone rearrangement to the upstream Ser residue.”

“Since mutations from the Asp/Tyr residue to arbitrary amino acids other than Gly do not alter the resistance, the steric hindrances originating from the alpha-substituted sidechain likely play a critical role in controlling the reactivity of the thioester bond to the hydroxyl group of the upstream Ser.”

[R1-5]
This paper would be strengthened by a longer discussion of the impact of the nature of the S-to-O acyl transfer suppression in the conclusion. This could cover potential applications of this discovery, or how this could shape structure-activity relationship studies of AIPs or other similar compounds. A sequence alignment of AgrD sequences, for example, to explore the feasibility of this shift in a broader range of species could be informative.

--> Thank you for the comments. We explored known AIPs bearing a thiolactone and added a list of alignments (Table S2). So far, at least 41 AIPs-thiolactones from 30 different species have been known. Including S. aureus AIP-I and IV in this study, 10 out of 41 AIPs possess a SX1CX2 motif. Still, no Gly residue(s), where is found as critical position avoiding S-to-O acyl transfer, is observed in their thiolactone sequence. Moreover, all the 10 AIP thiolactones are commonly composed of constrained 5-residual ring, which is also thought to be relevant to suppression of the acyl transfer. Thus, based on our findings, such other AIPs with SX1CX2 motif maintain a CTP form. This will be useful information to identify unexplored AIP molecules.

We added the following sentence highlighted in yellow in the conclusion:
“So far, more than 40 AIPs with a thiolactone have been reported (Table S2, ESI)9,22, and among them 8 AIPs have the SX1CX2 motif in addition to the S. aureus AIP-I/IV described in this study. However, none of them have a Gly residue at the critical positions near the thioester bond, implying that these naturally occurring AIPs are also resistant to the S-to-O acyl transfer, i.e., maintain the CTP form. This information is useful to predict the naturally occurring thiolactone structure of AIP(ref 23–25), giving a guide for the study on the structure-activity relationship in quorum sensing modulators.(ref 13,26)”

Reference
9 M. Thoendel, J. S. Kavanaugh, C. E. Flack and A. R. Horswill, Chem. Rev., 2011, 111, 117–151.
22 D. N. Mcbrayer, D. Cameron, Y. Tal-gan and C. Cameron, Org. Biomol. Chem., 2020, 18, 7273–7290.
23 B. H. Gless, M. S. Bojer, P. Peng, M. Baldry, H. Ingmer and C. A. Olsen, Nat. Chem., 2019, 11, 463–469.
24 B. H. Gless, B. S. Bejder, F. Monda, M. S. Bojer, H. Ingmer and C. A. Olsen, J. Am. Chem. Soc., 2021, 143, 10514–10518.
25 E. M. Molloy, M. Dell, V. G. Hänsch, K. L. Dunbar, R. Feldmann, A. Oberheide, L. Seyfarth, J. Kumpfmüller, T. Horch, H. D. Arndt and C. Hertweck, Angew. Chemie - Int. Ed., 2021, 60, 10670–10679.
26 T. Yang, Y. Tal-gan, A. E. Paharik, A. R. Horswill and H. E. Blackwell, ACS Chem Biol, 2016, 11, 1982–1991.

[R1-6]
In Table S1, there is no data in the column “MS signal of pre after 30 min translation” and it simply should be removed from the table for clarity. A sentence could be added in the table caption stating “no pre was detected after 30 min translation for any of the peptide products” if the authors feel that this information is important to convey.

--> Thank you for the comments. We have corrected Table S1 accordingly.


Reviewer-2
Prof. Suga and co-workers use the genetic code reprogramming technology to synthesize autoinducing peptides featuring the cyclic thiodepsipeptide (CTP) skeleton, which contain the SX1CX2 motif known to undertake S-to-O acyl transfer to cyclic depsipeptides. However, as a series of S. aureus autoinducing peptides contanning such a motif do not undertake the S-to-O acyl transfer, the author carried out a study to determine which internal residues make possible the conversion into cyclic depsipeptides (CDP).
The article is interesting in the sense that provide new insights into the structural factor determining the stability of the thiolactone moiety. Specifically, it was found that mutations at the neighboring positions of the thiolactone moiety by Gly allow the S-to-O acyl transfer to take place.
The article is very well conducted and flows well taking the reader to those important aspects of the research. The methodology is not new, because this laboratory is an actual pioneer in the one-pot ribosomal synthesis approach, but the results are indeed of interest for the chemical biology audience.

--> We thank the reviewer for positive comments.

[R2-1]
My only concern is the following:
Throughout the manuscript, authors claim that the alleviation of the conformational rigidity of the thiolactone macrocycle ring after Gly mutation at the 9th and/or 12th position is the reason for the CDP formation. I am not entirely opposed to this, but other structural elements should be mentioned as well.
My point is that the S-to-O acyl transfer mediated by the nucleophilic attack of the hydroxyl group of S6 to the thioester is not a transannular process where conformational rigidity is crucial, this is an attack of an exocyclic group. Author noticed that neither F10G nor I11G mutation induced the conversion of CTP to CDP, but changing those restudies by Gly also leads to a significantly reduced rigidity of the macrocyclic ring, so it is not the conformational rigidity. The actual reason is the reduction of steric hindrance around the thiolactone moiety due to the change of L-chiral amino acids to the non-substituted Gly. The attack of Ser side chain is an acyl nucleophilic substitution, which is known to be very sensitive to steric hindrance, so it is actually the alleviation of the steric hindrance around the thiolactone which favors that attack, and not the alleviation of the conformational rigidity, because inserting Gly at positions 10th and 11th also makes the macrocycle much more flexible, but do not favor the S-to-O acyl transfer.
Authors should consider focusing the explanation in the discussion section on the diminished steric hindracen when placing Gly at the neighboroing positions of the thiolactone, and not the conformational ridigity of the macrocycle. Please notice that, if, the Ser would be endocyclic, then the situation would be different and the macrocycle's rigidity/flexibility would matter much more, but, in my opinion, not in this case.

--> Thank you for your attentive reading and the suggestion. We agree that steric hindrance around the sidechain thioester of Cys8 generated by the sidechain of D9/Y9 and M12 in the thiolactone would be important to suppress the S-to-O acyl transfer. However, conformational rigidity of the macrothiolactone also would be relevant since a ring-expansion study of the thiolactone, which works reduction of the ring-constrain, allowed S-to-O acyl transfer to take place. The macrothiolactone ring constraints possibly forced the thioester to a wrong orientation against the acyl transfer of Ser6, which requires a 12-membered ring transition state to take place.
We added the sentence below to follow up the explanation of the role of D9/Y9 and M12 residues in the discussion section and the conclusion.
“Likely, G mutation at the 9th or 12th position in the AIP-Ia alleviates the conformational rigidity of the thiolactone ring and reduces the steric hindrances around the carbonyl group of the thioester, allowing for the nucleophilic attack of the hydroxyl group of S6 to the thioester bond and the formation of CDP via the S-to-O acyl transfer.”

“We propose that not only the steric hindrances by the inner residues adjacent to the thioester bond in the thiolactone but also the ring constrains in the AIP-I/IV effectively prevent the undesired ring-expansion21 to CDP.”

[R2-2]
Only in the Conclusions, the authors refer to this point when they state: ‘Since mutations from the Asp/Tyr residue to arbitrary amino acids other than Gly do not alter the resistance, their alfa-L-chiral center plays a critical role in controlling the reactivity of the thioester bond to the hydroxyl group of the upstream Ser’.
But the same line of explantion is not followed in the discussion section.
With this minor revision, I consider the paper is suitable for publication in this journal.

--> Thank you for the comment. We modified the sentence in the conclusion to accord with the contents of the discussion as below:
“Since mutations from the Asp/Tyr residue to arbitrary amino acids other than Gly do not alter the resistance, the steric hindrances originating from the alpha-substituted sidechain likely play a critical role in controlling the reactivity of the thioester bond to the hydroxyl group of the upstream Ser.”

23-Jan-2022

Dear Prof Suga:

Manuscript ID: CB-ART-11-2021-000225.R1
TITLE: Inner residues of macrothiolactone in autoinducer peptides-I/IV circumvents <i>S</i>-to-<i>O</i> acyl transfer to the upstream serine residue

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