the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
20 Jan 2021
20 Jan 2021
The long-standing relationship between Paramagnetic NMR and Iron-Sulfur proteins: the mitoNEET example. An old method for new stories or the other way around?
- 1Consorzio Interuniversitario Risonanze Magnetiche MetalloProteine, Sesto Fiorentino, I-50019, Italy
- 2Department of Pharmacy, University of Patras, Patras, GR-26504, Greece
- 3Magnetic Resonance Center and Department of Chemistry, University of Florence, Sesto Fiorentino, I-50019, Italy
- These authors contributed equally
- 1Consorzio Interuniversitario Risonanze Magnetiche MetalloProteine, Sesto Fiorentino, I-50019, Italy
- 2Department of Pharmacy, University of Patras, Patras, GR-26504, Greece
- 3Magnetic Resonance Center and Department of Chemistry, University of Florence, Sesto Fiorentino, I-50019, Italy
- These authors contributed equally
Abstract. Paramagnetic NMR spectroscopy and iron-sulfur (Fe–S) proteins have maintained a synergic relationship for decades. Indeed, the hyperfine shifts with their temperature dependencies and the relaxation rates of nuclei of cluster-bound residues have been extensively used as a fingerprint of the type and of the oxidation state of the Fe–S cluster within the protein frame. The identification of NMR signals from residues surrounding the metal cofactor is crucial for understanding the structure-function relationship in Fe–S proteins, but it is generally impaired in standard NMR experiments by paramagnetic relaxation enhancement due to the presence of the paramagnetic cluster(s). On the other hand, the availability of systems of different size and stability has, over the years, stimulated NMR spectroscopists to exploit iron-sulfur proteins as paradigmatic cases to develop experiments, models and protocols. Here, the cluster binding properties of human mitoNEET have been investigated by one-dimensional and two-dimensional 1H diamagnetic and paramagnetic NMR, in its oxidized and reduced states. The NMR spectra of both oxidation states of mitoNEET appeared to be significantly different from those reported for previously investigated [Fe2S2]2+/+ proteins. We show how the use of 1D NOE experiments, 13C direct-detected experiments, and the optimization of NMR experiments for paramagnetic systems significantly reduce the blind
sphere of the protein around the paramagnetic cluster. The application of this approach provided a detailed description of the unique electronic properties of mitoNEET, that are responsible for its biological function. Indeed, the NMR properties suggested that the specific electronic structure of the cluster possibly drives the functional properties of different [Fe2S2] proteins.
Francesca Camponeschi et al.
Status: final response (author comments only)
-
RC1: 'Comment on mr-2021-2', Anonymous Referee #1, 22 Jan 2021
In this report Camponeschi et al. present NMR assignments of the dimeric membrane-anchored human CDGSH protein (“mitoNEET”). Each subunit contains a Fe2S2 cluster and the current study aims at investigating its electronic properties in both the oxidized and the reduced states. Due to the paramagnetism of the cluster the resonance assignment requires separate sets of experiments for residues located outside a ~ 10 Å sphere and residues near the cluster. The former involves standard 3D backbone triple-resonance and side-chain experiments. In order to reduce the “blind sphere” around the cluster and observe very fast relaxing resonances 2D 15N-IR-HSQC-AP, developed a couple of years ago by one of the authors, and protonless 13C-detected CON experiments were carried out. Finally, a number of protons from Fe-coordinating residues were assigned using 1D NOE experiments in conjunction with X-ray structure derived distances. Conclusions about the electron distribution within the Fe2S2 cluster were drawn from the envelope of the hyperfine-shifted spectral region, which has some functional implications.
Although I am not an expert for iron-sulfur proteins it appears to me that the system studied here is of high biological relevance (nice literature overview in the Introduction, by the way). Overall, the manuscript is very well-written, except for paragraph 3.2.2 (see below). It describes sound experimental work and comprehensible interpretation of the results. However, it mostly represents an application of established techniques to a well-studied protein. Considering its editorial policy the current paper falls outside the scope of Magnetic Resonance.
Paragraph 3.2.2 (“Paramagnetic experiments on [Fe2S2]-mitoNEET reduced and oxidized”) is to a large extent phrased in a lab-style language and should be rewritten.
Some examples:
Heading: are the experiments paramagnetic or the samples ?
Lines 190, 194, 200: “Spectra were recorded on a Bruker Av600 MHz” (spectrometer ?)
Line 198: “Each experiment consisted from ~ 300k up to ~900k scans.”
Line 202: “…using 16.5 ms and 13.7 ms as acquisition and a t1max delay....”
Line 209: “…2048 scans each fid were collected…”
Line 211: “…the IPAP approach was used for homodecoupling…” Does that mean virtual decoupling of 1JC’Ca ?
Further minor issues:
According to the Material and Methods section 15N relaxation experiments (R1, R2, hetNOE) were performed for the diamagnetic part of the protein. The purpose and result of these experiments is not reported at any point in the manuscript.
Proton 1D spectra were recorded with a spectral width of 320 ppm, much wider than required for spectral range observed here. Were any specialized wide-band pulses employed that would be able to excite a ~ 200-kHz region ?
It is mentioned that the CON experiment was optimized for paramagnetic systems (section 4.2.3). Which modifications were applied ? Simply shorter magnetization transfer periods ? An INEPT delay of 8 ms is specified in the experimental section, which is shorter than 1/(2*1JC’Ca). Is that sufficient to incorporate the IPAP module ?
-
AC1: 'Reply on RC1', Lucia Banci, 25 Jan 2021
The manuscript describes the NMR characterization of the first coordination sphere of mitoNEET in its two oxidation states. Albeit a few NMR studies are available for the protein, the data reported here provide an advancement with respect to the current knowledge on the protein. Hence, to some extent, we agree with the reviewer that the manuscript, with very minor revisions, could be suitable also for other journals and for a more general audience.
However, we believe that the mitoNEET case is a very nice example of how a protocol based on the combination of various experimental approaches tailored to paramagnetic systems spanning from the more recent IR-HSQC-AP to the “ancient” 1D NOEs, could provide insights into the knowledge of a challenging system of high biological interest.
Tailored NMR approaches have recently contributed to advance the knowledge for many different cases in FeS proteins, see for example the cases of Ciapin (Proc Natl Acad Sci U S A. 2013 Apr 30;110(18):7136-41. doi: 10.1073/pnas.1302378110), Glrx5 (Proc Natl Acad Sci U S A. 2014 Apr 29;111(17):6203-8. doi: 10.1073/pnas.1400102111), ISCA2 (J Am Chem Soc. 2014 Nov 19;136(46):16240-50. doi: 10.1021/ja507822j, J Am Chem Soc. 2017 Jan 18;139(2):719-730. doi: 10.1021/jacs.6b09567), NUBP1 (J Am Chem Soc. 2020 Jun 17;142(24):10794-10805. doi: 10.1021/jacs.0c02266), IBA57 (J Am Chem Soc. 2018 Oct 31;140(43):14401-14412. doi: 10.1021/jacs.8b09061), LIAS (J Mol Biol. 2019 Nov 8;431(22):4514-4522. doi: 10.1016/j.jmb.2019.08.018) and also on mitoNEET (J Am Chem Soc. 2017 Jul 19;139(28):9479-9482. doi: 10.1021/jacs.7b05003). These studies were mainly performed in the frame of FeS proteins biogenesis and interactomics, and only sparsely discussed the NMR aspects from a methodological point of view. We thought that MR could be the suitable medium to emphasize how: - the combination of classical and tailored experiments circumvents the loss of information in the proximity of metal centers; - the continuous advancement in NMR methodology contributes to the understanding of FeS proteins, as overviewed in the first part of the article and outlined in the title.
We still believe this. We are, of course, ready to revise the manuscript according to reviewer suggestions but this might not affect the opinion of the reviewer on the appropriateness of the manuscript for MR.
-
AC1: 'Reply on RC1', Lucia Banci, 25 Jan 2021
-
RC2: 'Comment on mr-2021-2', Anonymous Referee #2, 15 Feb 2021
In this study, Camponeschi et al use NMR to characterize mitoNEET, a mitochondrial Fe2S2 protein. By using 1D NOE experiments, 13C direct-detected experiments, and the optimization of NMR experiments for paramagnetic systems, the authors show significantly reduction of the “blind” sphere of the protein around the paramagnetic cluster, thus allowing the detection of residues possibly involved in the biological function of mitoNEET. The study has significant implications in the fields of paramagnetic NMR and FeS proteins. Some revisions are recommended.
In details:
1. I have some general questions about the mitoNEET protein I hope the authors can help answer.
a) If mitoNEET can repair Fe-S proteins by donating its own Fe2S2 cluster, how does it reacquire the Fe2S2 cluster? Can the authors comment on the source of its Fe2S2 cluster?b) The redox states of mitoNEET are crucial for its function and stability. How are the redox states of mitoNEET regulated in cells?
2. Some experimental details are needed.
a) For M9 media growth, how much (15NH4)2SO4 and 13C-glucose were supplemented?
b) What kind of anaerobic environment was used?
c) Does the phosphate buffer contain any NaCl?
d) I assume there were additional steps to remove the extra K4Fe(CN)6 or sodium dithionite?2) What's the Fe2S2: protein ratio 'as purified'? It would be helpful to include UV data to show the load of Fe2S2 on the protein in both redox states.
3) The authors purified the protein in an anaerobic environment, I assume it's because the Fe2S2 is susceptible to oxidative damage. Would addition of 10mM K4Fe(CN)6 to the protein solution damage the Fe2S2 cluster?
4) Is the purified mitoNEET protein a homodimer as shown in Fig. 1A?
5) In Fig. 1A, can the authors highlight the residues that are affected by different redox states?
6) Fig 1B, how were the chemical shift differences between two redox states calculated?
7) It's intriguing to me that the redox state change would mainly affect the regions involved in inter-subunit contacts. Do the authors have any hypothesis why?
8) There is no mention of Fig. 1C in the text. The author might add some.
9) Can the authors provide some explanations why no hyperfine shifted signals were observed for the reduced [Fe2S2]+-bound form of mitoNEET?
10) The authors should provide the data showing the broadening of signal B collected in D2O.
11) The authors might want to highlight the additional residues assigned by 15N-IR-HSQC-AP in the structure of mitoNEET.
12) The labels in Fig.3 are too small to read, the authors might want to improve that.
- AC2: 'Reply on RC2', Lucia Banci, 22 Feb 2021
-
EC1: 'Comment on mr-2021-2', Gottfried Otting, 23 Feb 2021
Dear Professor Banci,
Thank you for the careful response to the comments made by reviewer 2. Some of the detailed comments made by reviewer 1, however, have not been addressed. Any revised version will have to do this.
I am somewhat bewildered by the long list of references for FeS proteins compiled in the response to reviewer 1, where the techniques used in your present work have been applied. Given this background, what exactly is the novelty reported in your article apart from the assignments of some difficult-to-assign resonances of a specific paramagnetic protein using established techniques? Also the title of your article is quite ambiguous in this regard.
The website of Magn. Reson. specifically states “Routine applications of established techniques and minor technical advances are considered to be outside its scope.”
To discuss the NMR aspects from a methodological point of view could be valuable, but there is relatively little of this in the present version of the manuscript beyond a description of the parameters used to set up various experiments. Have the parameters simply been copied from previously published work or have they been adapted specifically to the protein of this work? The discussion and conclusion sections of your manuscript do not discuss the paramagnetic NMR techniques at all. In fact, these sections seem to indicate that observing signals of different linewidths in the 1D 1H NMR experiments was sufficient to draw the conclusions there are. This is not in line with the gist of the letter accompanying the submission and, if the salient information really only comes from the straightforward 1D 1H NMR spectra, the assignments made would be inconsequential and, I am afraid, Magn. Reson. not the right forum for this work. I encourage submission of a revised version only if these concerns can be substantially addressed.
Minor queries:
Line 301: what exactly is reported in red in Table 2?
When referring to distances from the FeS cluster, it is not clear whether the distances refer to the nearest metal ion or the centre of the cluster(s).
Best regards,
Gottfried
Francesca Camponeschi et al.
Data sets
Paramagnetic tailored experiments for the NMR investigation of reduced and oxidized [2Fe-2S]-mitoNEET Francesca Camponeschi, Angelo Gallo, Mario Piccioli, and Lucia Banci https://doi.org/10.5281/zenodo.4442395
Francesca Camponeschi et al.
Viewed
| HTML | XML | Total | BibTeX | EndNote | |
|---|---|---|---|---|---|
| 205 | 56 | 15 | 276 | 5 | 5 |
- HTML: 205
- PDF: 56
- XML: 15
- Total: 276
- BibTeX: 5
- EndNote: 5
Viewed (geographical distribution)
| Country | # | Views | % |
|---|
| Total: | 0 |
| HTML: | 0 |
| PDF: | 0 |
| XML: | 0 |
- 1