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Monitoring Fe–S cluster occupancy across the E. coli proteome using chemoproteomics

Nature Chemical Biology volume 19, pages 356–366 (2023)Cite this article

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Abstract

Iron–sulfur (Fe–S) clusters are ubiquitous metallocofactors involved in redox chemistry, radical generation and gene regulation. Common methods to monitor Fe–S clusters include spectroscopic analysis of purified proteins and autoradiographic visualization of radiolabeled iron distribution in proteomes. Here, we report a chemoproteomic strategy that monitors changes in the reactivity of Fe–S cysteine ligands to inform on Fe–S cluster occupancy. We highlight the utility of this platform in Escherichia coli by (1) demonstrating global disruptions in Fe–S incorporation in cells cultured under iron-depleted conditions, (2) determining Fe–S client proteins reliant on five scaffold, carrier and chaperone proteins within the Isc Fe–S biogenesis pathway and (3) identifying two previously unannotated Fe–S proteins, TrhP and DppD. In summary, the chemoproteomic strategy described herein is a powerful tool that reports on Fe–S cluster incorporation directly within a native proteome, enabling the interrogation of Fe–S biogenesis pathways and the identification of previously uncharacterized Fe–S proteins.

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Fig. 1: A two-dimensional chemoproteomic platform to monitor Fe–S cluster binding.
Fig. 2: Monitoring reactivity changes for Fe–S-ligating cysteines.
Fig. 3: Interrogating the role of scaffold and carrier proteins within the Isc Fe–S biogenesis pathway.
Fig. 4: Interrogating the role of the chaperone/cochaperone proteins HscA and HscB within the Isc Fe–S biogenesis pathway.
Fig. 5: Mapping coverage of the E. coli Fe–S proteome.
Fig. 6: Predicting Fe–S binding sites from changes in cysteine reactivity.

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Data availability

MS proteomics data for this study have been deposited in ProteomeXchange via the PRIDE partner repository with the dataset identifier PXD026488. Databases used for MS/MS searches against the E. coli proteome were obtained from the UniProtKB at www.uniprot.org. Source data are provided with this paper.

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Acknowledgements

We thank all members of the E.W. laboratory for discussions and feedback. This work was supported by NIH grant R35GM134964 to E.W.

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  1. Department of Chemistry, Boston College, Chestnut Hill, MA, USA

    Daniel W. Bak & Eranthie Weerapana

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Contributions

D.W.B. and E.W. designed the research. D.W.B. performed the research and proteomic analyses. D.W.B. and E.W. analyzed and interpreted the data. D.W.B. and E.W. wrote the paper.

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Correspondence to Daniel W. Bak or Eranthie Weerapana.

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Extended data

Extended Data Fig. 1 A functional catalogue of the E. coli Fe–S proteome.

The 144 members of the E. coli Fe-S proteome categorized by cluster function (see Supplementary Dataset 1). General Fe-S proteins/complexes (blue) are represented as cartoon diagrams displaying the approximate orientation of all (metallo)cofactors, with a list of individual Fe-S family members presented in the associated green boxes. Abbreviations: XDH, xanthine dehydrogenase; DMSO, dimethyl sulfoxide reductase; SDH, succinate dehydrogenase; FRD, fumarate reductase; GltS, glutamate synthase; NiFe, dinuclear nickel-iron center; SAM, S-adenosylmethionine; Moco, molybdenum cofactor.

Extended Data Fig. 2 Cysteine labeling and protein abundance changes in the E. coli Fe-S proteome upon iron-depletion.

a, b, Intracellular (a) iron and (b) zinc concentration determined by ICP-OES for E. coli grown under iron-replete (control) (gray) or iron-depleted (green) conditions. Significance is calculated as *** p < 0.005 (p = 2.2E-05), paired t-test (two-tailed) from n = 3 biological replicates. Error bars represent the standard error of the mean. c, Reductive dimethylation (ReDiMe) proteomic platform employed to measure protein abundance changes upon growth of E. coli under iron-depletion conditions. d, IsoTOP-ABPP proteomic platform employed to measure cysteine labeling changes upon growth of E. coli under iron-depletion conditions. e, Waterfall plot of log2 L/H ratio (RP) changes for all quantified proteins from an iron-depleted E. coli proteome (see Supplementary Dataset 2). Proteins with RP>|0.5| are highlighted in red. f, Gene-ontology analysis of processes enriched in proteins with RP>0.5. Processes involved in iron import and homeostasis are highlighted in red. g, Pie chart analysis of the number of quantified Fe-S proteins with RP<−0.5 (red), RP ~ 0.0 (gray), and RP>0.5 (green).

Source data

Extended Data Fig. 3 Cysteine reactivity under iron-depletion is independent of lysate preparation and IA-alkyne labeling conditions.

a, Two-dimensional proteomic dataset for the E. coli proteome grown under iron-replete (control) conditions (see Supplementary Dataset 3). All quantified cysteine residues are plotted in the main graph (annotated Fe-S cluster cysteine ligands - green circles, non-annotated cysteine residues - light gray small circles). Inset to right: cysteine residues with no protein abundance data (annotated Fe-S cluster cysteine ligands - green circles, non-annotated cysteine residues - light gray small circles). Inset below: proteins with no cysteine reactivity data (annotated Fe-S protein - red circles, non-annotated proteins - light gray small). b,c, Comparison plot of cysteine reactivity ratios (iron-depleted versus iron-replete) for quantified Fe-S ligands (green circles) from lysates IA-alkyne labeled under standard aerobic (x-axis) versus (b) anaerobic or (c) reducing conditions (y-axis) (see Supplementary Dataset 2).

Extended Data Fig. 4 Cysteine reactivity changes for the ligands of the two Fe-S clusters of biotin synthase.

a, Rc values for Fe-S cysteine ligands on the Fe-S protein, BioB. b, Crystal structure of E. coli BioB with clusters and cysteine residues highlighted (Insets: AdoMet radical [4Fe-4S] cluster – green and auxiliary [4Fe-4S] cluster – orange).

Extended Data Fig. 5 suf operon expression in E. coli isc genetic deletion strains.

a, Structural organization of the E. coli isc and suf operons. b, Proposed model of suf operon function and regulation by the IscU client protein and transcription factor, IscR. c, Protein abundance changes for scaffold complex proteins from the suf operon across all experimental growth conditions and deletion strains.

Extended Data Fig. 6 Reactivity profile of cysteine residues involved in Fe-S ligation.

a, Proteomic workflow for measuring the cysteine reactivity of Fe-S cluster ligands in an iron-depleted E. coli proteome labeled with 100 µM IA-Light or 10 µM IA-Heavy. b, Cysteine reactivity curve for all quantified cysteine residues (light gray) from an iron-depleted E. coli proteome (see Supplementary Dataset 9). Fe-S cluster cysteine ligands are highlighted (green circles). c,d Violin plot of L/H ratios for unique groups of (c) functional cysteine residues, including Fe-S ligands (red), active site residues (blue), zinc ligands (purple), disulfides (green), and iron ligands (yellow) and (d) Fe-S ligands from Fe-S client proteins (red) and Fe-S scaffold proteins (yellow). The median R value for each functional group of cysteine residues is displayed as a dashed line, while the average R values (red) and number of unique values (black) are indicated for each group.

Extended Data Fig. 7 Biochemical characterization of the putative Fe-S protein DppD.

a, Organization of the dpp and opp operons. b, the protein domain structure of the nucleotide-binding subunits, DppD/F and OppD/F. Conserved cysteine residues in the C-terminal extension (orange) are shown as red lines. c, AlphaFold structural model of DppD (AF-P0AAG0-F1). The four conserved c-terminal cysteine residues are highlighted. d, Representative SDS-PAGE analysis of purified E. coli DppD (experiment independently repeated twice). e, Visible color of purified E. coli DppD protein. f, ICP-OES analysis of the iron content of purified E. coli DppD from n = 3 experimental replicates. Error bars represent the standard error of the mean. g, UV-visible absorbance spectrum of as-isolated E. coli DppD.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–3 and Tables 1 and 2.

Reporting Summary

Supplementary Data

MS-based proteomic data.

Source data

Source Data Fig. 1

UV-visible spectroscopy data.

Source Data Fig. 1

Unprocessed SDS–PAGE gels.

Source Data Fig. 6

ICP-OES, UV-visible spectroscopy and EPR spectroscopy data.

Source Data Fig. 6

Unprocessed SDS–PAGE gels.

Source Data Extended Data Fig./Table 2

ICP-OES data.

Source Data Extended Data Fig. 7

ICP-OES and UV-visible spectroscopy data.

Source Data Extended Data Fig. 7

Unprocessed SDS–PAGE gels.

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Bak, D.W., Weerapana, E. Monitoring Fe–S cluster occupancy across the E. coli proteome using chemoproteomics. Nat Chem Biol 19, 356–366 (2023). https://doi.org/10.1038/s41589-022-01227-9

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