PONE-D-24-30056Mutational analysis of Phanerochaete chrysporium´s purine transporterPLOS ONE

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5. Address the kinetical and structural concerns pointed, including  the generation of 3D models of WT and mutants of PhZ employing AlphaFold.

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Comments to the Author

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Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

Reviewer #3: No

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

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Reviewer #1: Comments to article PONE-D-24-30056

The article PONE-D-24-30056 evaluates, by mutational analysis, the amino acid residues important for purine transport in the PhZ protein of Phanerochaete chrysosporium. This work is based on the premise that the NAT transporter family has 2 different types of protein groups, the UapA/C-like ones that are specific for the transport of uric acid, xanthine, oxypurinol and allopurines and the AzgA-like ones that are transporters of hypoxanthine, guanine, 8-azaguanine, 6-mercaptopurine. Based on structural comparisons of AzgA-like proteins, conserved motifs in proteins of different taxonomic ranks are chosen to make changes in residues that could participate in purine transport, choosing the phZ gene to make the site-directed mutations that lead to the desired amino acid changes. Both the unmutated PhZ protein (WT) and mutant versions of the PhZ protein were heterologously expressed in an Aspergillus nidulans strain with mutations in purine transporters (ΔZAC). In this genetic background, the ability of these mutant proteins to transport purines was evaluated by assessing the growth of strains with purines as the sole nitrogen source, the uptake of radiolabeled hypoxanthine, the intracellular localization of the fusions of the WT PhZ protein and its variants, and western blot analysis. As a result of all these analyses, three-dimensional models of the PhZ protein with hypoxanthine and 8-azaguanine are established.

The approach of this work is very interesting; however, it is important to address certain inconsistencies in some points of the manuscript in order to transmit the information more efficiently.

General remarks

1. The aim of the study should be made clear, as well as the reason why PhZ was chosen for mutational analysis, as this is very ambiguous in the introduction and leads to many questions that could affect the interpretation of the overall work.

2. The description of the results should be carefully revised and rewritten. At several points in the manuscript, the description of the results seems more like a methodological description and in the figure captions the results are described.

3. In the results of the structural model, the description should focus more on the results obtained and not on comparisons with other models, as the notion of the real contribution of the work is lost. Comparisons with the results of other authors should be moved to the discussion.

4. Express decimal amounts with periods and not commas, for example, 0.8 instead of 0,8 for clarity.

Specific comments

Line 34. Replace "purines" with "purine".

Line 74. Has the structure of the AzgA protein of Aspergillus nidulans or any other microorganism been previously crystallized?

Lines 91-104. The objective of the study is not well defined, so the following questions are raised:

1. Why was the mutational analysis not performed directly on the AzgA protein of Aspergillus nidulans? Is its structure and the residues involved in purine transport already known? If so, this should be indicated in the introduction.

2. Why was PhZ chosen among the 11 AzgA-type proteins? What is the importance of analyzing this protein?

If the goal is to understand the structural differences between UapA/C and AzgA proteins that lead to substrate differences, as inferred in lines 82-84, why not perform a mutational analysis of UapA/C residues involved in purine transport, or of differential residues with AzgA, if the crystal structure of UapA/C is known

Lines 91-114. I recommend rewriting this paragraph to give fewer details of the results and to concisely describe the contribution of the work.

Lines 111-112. The hypothesis is very ambiguous

Table S1. Correct the description of the strain phZ_V58A*.

Line 126. Put the formula for ammonium tartrate in parentheses, not just ammonium.

Line 127. Indicate which purines and where they were acquired from.

Line 128. Indicate why these concentrations of 8-azaguanine and oxypurinol were used to assess toxicity. If a reference exists as background, include it. If not, provide adequate justification.

Line 166. Recommended by...?

Line 257. The representation of the conserved motifs in a table in Figure S2 is very confusing and difficult to understand. I suggest generating a figure with the alignment of the sequences.

Line 264. The figure caption in Figure 1 is too long. It should be more concise and describe the figure in the text in more detail instead of too much description in the figure caption. I suggest splitting Figure 1 into 2 parts so that Figure 1 B is Figure 2 so that it can be analyzed separately as it discusses the mutants generated.

Line 303-311. Part of the information in this section corresponds to the methodology.

Line 334. I consider that figure 2 should be better described in the text, not only in the figure caption, since the figure caption indicates why only the photos of the hx results are included and not those of adenine and guanine, but this is not mentioned in the text, or I suggest changing figure 2 for figure S3 but including the control in which AzgA is expressed.

Photographs of the growth of the WT strain of Aspergillus nidulans under the different conditions should also be included in this figure.

Line 372. Fig.3. Indicate what %V means.

Line 400. Indicate with which substrate differences were observed.

Reviewer #2: The authors present a complete characterization of several single mutants of purine transporter from Phanerochaete chrysporium. The manuscript will be of interest to those involved in the study of purine transporters (substrate specificity and transport efficiency) and in general in protein structure and function analysis. The work was performed applying cutting-edge techniques that supports the findings and conclusions stated. However, there are minor details that deserve attention.

1.- The IC50 value depends on substrate concentration used, therefore, how the concentration of radiolabeled substrate used (at least 10 times lower than the Km value) affected the Ki determination?

2.- In the same context, please explain in the text the meaning of Km/i and include a table as supplementary material with the values of IC50 obtained and indicate the value of Km used.

3.- Why do not try to use an AI software such as Alphafold to generate the 3D model of the transporter?

4.- Please include the stereochemical evaluation of the final model used for structural and docking studies.

5.- If the protein is a transporter, why did not perform the MD simulation using a membrane instead of an aqueous system?

6.- In would be desirable to generate the corresponding 3D models at least from the most interesting mutants to perform docking studies to explain the experimental data and not only with the native protein structure.

Reviewer #3: In this work, the authors describe a structure-function study of the purine transporter PhZ from Phanerochaete chrysosporium, initially focused on 4 conserved motifs of the NAT family, that unveiled a set of amino acid residues critical for transport activity and substrate specificity.

General comments:

This work utilizes a PhZ 3D model constructed by homology modeling using the crystal structure of UapA, further refined by molecular dynamics. Given the recent advances in 3D structure prediction by AlphaFold it would be interesting to include some data on this model, and compare it with the model used in this work. If the authors have already tested it, they could include some information in the manuscript. If not, they could retrieve the predicted structure by AlphaFold2 at https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb.

It is important that the authors include statistical analysis of the data to determine if, in fact, there are significant differences in some of the presented results, as sometimes stated in the manuscript.

Abstract

The authors should state here why the A418 residue is pivotal and its location in the protein.

Introduction

Line 48: Please clarify if AzgA subfamily lacks homologs in animals or structural homologs

Line 84: Papakostas and colleagues (2013), the authors should include the reference number

Line 87-90: please include here what were the main findings of this study, as they are important for the work presented here.

Line 108: The reference regarding the post-translational regulation of the PhZ transporter is missing.

Materials and Methods

Line 177: Why weren’t the Km values calculated directly with the labeled substrate at different concentrations? Why did the authors prefer to use a fixed concentration of labelled hypoxanthine and various concentrations of unlabelled hypoxanthine to determine the Ki?

Line 186: What was the specific activity of the labelled hypoxanthine?

Results

Line 246: The authors should include the accession of all the sequences used in the alignment in the supplementary data.

Lines 259-262: where are located motifs 1 and 3? This will help the reader understand why this study discarded these motifs.

Figure 1 B) This image could be improved as several mutations overlap. Instead of showing the complete alignment, repeating the previous image, the authors could use a simplified scheme where the position of each amino acid residue is reported regarding the PhZ protein, evidencing the effect of each mutation.

Line 305: Please clarify here the name of the strain and the main purine transporters deleted.

Line 305: The authors state that the GFP did not affect the transporter activity, as the same growth phenotypes were observed in previous work. However, did the authors test the kinetic parameters of hypoxantine transport without the C- terminus GFP fusion? This method is more accurate to determine if the GFP tag affects protein activity.

Line 319: Instead of “ less than that of the wild-type” consider “ is less than that of cells expressing the wild-type”

line 330: include the growth phenotypes of this strain as mentioned for the previous strains.

line 360: modify the efficiency is not very clear, as it can be an increase or decrease in transport activity. In this case the phenotype suggests an increase in transport capacity.

line 361: please state the hypoxanthine concentration used to determine the initial uptake rate, and why this concentration was chosen.

line 368: Statistical analysis should be applied to this data to evaluate which are significantly different.

line 385 (figure 4): no SD are visible in the data concerning the uptakes of the L214M mutant. Is this correct?

line 389: The authors state that this mutant has lower transport capacity, but they only report the assay where they tested one concentration. The Vmax of the transporter was not determined to evaluate transporter capacity.

lines 394-397: This depends on the concentration of the unlabelled substrate. What were the conditions tested?

Line 415 (Fig 5) I consider it not easy to visualize the degree of inhibition of each substrate for the different mutants. I believe it would be easier if the data were presented differently, first for the WT and then for each mutant as follows: first, the V of hypoxanthine uptake w/o any inhibitor (100%), then with unlabelled hypoxanthine, and then the corresponding V % for all the tested inhibitors.

Also, statistical analysis should be presented here.

Discussion:

Line 616: Please explain how an increase in the intrinsic stability of the mutant protein would decrease the hypoxanthine uptake rate.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Mutational analysis of Phanerochaete chrysosporium´s purine transporter

PONE-D-24-30056R1

Dear Dr. M. Sanguinetti

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