PONE-D-19-25528

Investigating the ferric ion binding site of magnetite biomineralisation protein Mms6

PLOS ONE

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #2: Yes

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Reviewer #1: Re: Investigating the ferric ion binding site of magnetite biomineralisation protein Mms6

The presented work an in-depth investigation on the mode of iron ion binding by a protein aiding magnetite crystals formation in magnetotactic bacteria. The study is well planned and conducted and is consistent with previous works on the same protein. In particular, the authors have identified three amino acid residues involved in Fe3+ binding, and demonstrated that those are different from the residues binding Fe2+ ions.

The article could be accepted for publication as is, however I would comment on a couple of points:

1. The Monte-Carlo simulation would have been more sensible if the 2+/3+ metal ions were included in the structure. Although I understand, there might be some limitations in the force field.

2. On Fig. 3c, dotted line could be rather interpreted as that there’s a metal binding to the mutant, but it doesn’t lead to nucleation (for whatever reasons). I would appreciate authors’ comment on that.

Minor and technical comments:

1. Ln. 67: “…nucleate the formation…” is a tautology.

Reviewer #2: This manuscript from Staniland and co-workers describes studies of iron binding by the Mms6 protein from Magnetospirillum magneticum AMB-1. The approach taken used two assays to assess iron binding capability, which are very different in their means of sensing bound iron. One uses only ferric-citrate as a chelated ligand, while the other uses a combination of ferric/ferrous citrate. One is a measurement with proteins in solution and the other measures binding to proteins on a surface. In solution the SUMO-fused proteins are reported here to be monomers and dimers. The non-fusion protein has been shown as a multimer, and contrary to the SUMO-fused proteins, it has been refolded from a denatured state. Although the authors did not investigate the structure of the protein in this study when it is attached to a surface, it is reasonably likely that its multimeric state remains. The interaction between adjacent C-terminal domains or adsorption to the surface could profoundly influence the structure of an intrinsically disordered polypeptide and alter the means by which the protein binds iron compared with the SUMO-fused monomers. As the authors did not measure the affinity for iron in either assay, they do not evidence to support the assumption that the two C-terminal domains have the same structure or that the same iron-binding configuration of residues is evaluated by each assay. Bringing the results from these two assays together to make a single conclusion regarding the binding site of Mms6 for iron is not justified.

Other aspects of the submission are also of concern. First, the procedure for attaching protein to the surface for nanoplasmonic sensing involved a few minutes of flow until a stable plasmonic signal was reached. The authors state that this short period resulted in saturation of the sensor by both proteins, but provide no evidence to support this statement, which is contrary to published results that show it takes about an hour for the reaction between gold and a thiol group to reach saturation (Bard et al. 2018 Photochemistry and Photobiology 94 (6) 1109-1115). Regarding the results from the plasmonic sensing assay the authors note the slight reduction of signal for Mms6MM over time to level of at about 70% of the wild type protein signal and propose, without experimental support, this lower level of binding is the binding of ferrous ions only, or weaker unspecific general binding. The authors also noted a large wavelength shift when the system was returned to water, which they interpreted as being due to the precipitation of iron oxide on the surface of the gold. The lack of shift for the triple mutant protein was interpreted as being the result of the mutant being unable to bind ferric ions. Again there is no experimental support given to this interpretation. At the very least a protein that does not bind iron should have been used as a control condition. It is also not stated if this assay was performed more than once and how the results varied between tests, if done.

Figure 2 is unnecessary as this information including the chemistry involved is published as a figure in an open-source publication that has been cited by the authors

The manuscript also contains typographical errors that need to be corrected.

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Reviewer #1: No

Reviewer #2: No

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Investigating the ferric ion binding site of magnetite biomineralisation protein Mms6

PONE-D-19-25528R1

Dear Dr. Staniland,

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With kind regards,

Eugene A. Permyakov, Ph.D., Dr.Sci.

Academic Editor

PLOS ONE

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