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Functional elucidation of TfuA in peptide backbone thioamidation

Nature Chemical Biology volume 17, pages 585–592 (2021)Cite this article

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Abstract

YcaO enzymes catalyze several post-translational modifications on peptide substrates, including thioamidation, which substitutes an amide oxygen with sulfur. Most predicted thioamide-forming YcaO enzymes are encoded adjacent to TfuA, which when present, is required for thioamidation. While activation of the peptide amide backbone is well established for YcaO enzymes, the function of TfuA has remained enigmatic. Here we characterize the TfuA protein involved in methyl-coenzyme M reductase thioamidation and demonstrate that TfuA catalyzes the hydrolysis of thiocarboxylated ThiS (ThiS-COSH), a proteinaceous sulfur donor, and enhances the affinity of YcaO toward the thioamidation substrate. We also report a crystal structure of a TfuA, which displays a new protein fold. Our structural and mutational analyses of TfuA have uncovered conserved binding interfaces with YcaO and ThiS in addition to revealing a hydrolase-like active site featuring a Ser–Lys catalytic pair.

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Fig. 1: MS and kinetic analysis of TfuA-mediated thioamidation.
Fig. 2: TfuA catalyzes the hydrolysis of ThiS-COSH.
Fig. 3: TfuA and YcaO enhances the activity of each other.
Fig. 4: Crystal structure of MaTfuA.
Fig. 5: Reaction scheme of YcaO–TfuA thioamide synthetase.

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Data availability

We declare that all the data supporting the findings of this study are available within the manuscript, Supplementary dataset or Supplementary Information. Plasmids are available upon request. X-ray crystallographic coordinates were deposited in the PDB under the code 6XP8. Source data are provided for Figs. 1–3 and Extended Data Figs. 6, 8 and 9. Public databases used in the study include Pfam (PF02624, PF07812, PF02597, PF00899, PF13407), UniProt, GenBank (AAB17515, AAB17513, WP_048176273, WP_048175617, WP_048175616, WP_048176081, WP_011023978) and the PDB (1RYJ, 2CU3, 6PEU, 4IRX, 3CNQ, 4PK9). The accession codes are also provided in the manuscript on the first occurrence.

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Acknowledgements

We thank L. Zhu and J. Arrington for assistance with the NMR and nMS experiments, respectively. We thank X. Rui Guo from the Mitchell group for acquiring the high-resolution mass spectral data. This work was supported in part by National Institutes of Health grant nos. GM097142 (to D.A.M.), GM131347 (to S.K.N.) and the Alice Helm Graduate Research Excellence Fellowship in Microbiology (to A.L.). The Bruker UltrafleXtreme MALDI–TOF/TOF mass spectrometer was purchased in part with a grant from the National Institutes of Health (grant no. S10 RR027109 A).

Author information

Author notes

  1. Shi-Hui Dong

    Present address: State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, China

  2. Nilkamal Mahanta

    Present address: Department of Chemistry, Indian Institute of Technology Dharwad, Karnataka, India

Authors and Affiliations

  1. Department of Microbiology, University of Illinois, Urbana, IL, USA

    Andi Liu, Haley N. Penkala & Douglas A. Mitchell

  2. Carl R. Woese Institute for Genomic Biology, University of Illinois, Urbana, IL, USA

    Andi Liu, Yuanyuan Si, Nilkamal Mahanta, Haley N. Penkala, Satish K. Nair & Douglas A. Mitchell

  3. Department of Chemistry, University of Illinois, Urbana, IL, USA

    Yuanyuan Si, Nilkamal Mahanta, Satish K. Nair & Douglas A. Mitchell

  4. Department of Biochemistry, University of Illinois, Urbana, IL, USA

    Shi-Hui Dong & Satish K. Nair

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Contributions

A.L. conducted the bioinformatics, kinetics and binding analyses. Y.S. and A.L. performed mutational and biochemical analyses of TfuA. The crystallographic work was performed by S-.H.D. under supervision by S.K.N. H.N.P. optimized the thiocarboxylate detection assay and assisted in variant generation. N.M. contributed to target selection, cloning, and protein purification. D.A.M. conceived of and supervised the overall project. A.L. wrote the first draft of the manuscript with input from D.A.M. and S.K.N., while all authors reviewed, edited and approved the final version of the manuscript.

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Correspondence to Douglas A. Mitchell.

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The authors declare no competing interests.

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Peer review information Nature Chemical Biology thanks Wen Liu, Eugene Mueller and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 TfuA-associated RiPP structures and TfuA sequence analysis.

a, Representative thioamitide structures with thioamide moiety shown in red. b, A sequence similarity network (SSN) of the TfuA protein family PF07812 (n = 2,042 sequences) was generated with protein sequences 100% identical are conflated to a single node, and an alignment score of 60 (BLAST-P expectation value of 10-60) was used as the edge cut-off. Background is shaded based on taxonomy: archaea (orange), Proteobacteria (blue), Actinobacteria (pink), Cyanobacteria (purple), Firmicutes (green), and others (gray). Nodes representing TfuA proteins encoded within ten open-reading frames of a ThiS homolog are black. c, Gene neighborhood diagrams for selected methanogens encoding TfuA near ThiS. NCBI accession identifiers are given for the TfuA proteins.

Extended Data Fig. 2 Correlation analysis of TfuA and ThiS from methanogens with complete genomes.

a, Maximum-likelihood tree of YcaO proteins from methanogens (n = 111 sequences) with clades colored based on taxonomic group: Methanobacteria (purple), Methanococci (orange), Methanopyri (teal), Methanomicrobia (green), and Methanomassiliicoccales (magenta). The absence (open square) or presence (filled square) of tfuA and thiS within each genome is denoted. b, The cooccurrence table calculated for the correlation analysis between TfuA and ThiS. The P-value calculated using Fisher’s exact test is 1.6 × 10-12.

Extended Data Fig. 3 MALDI-TOF-MS analysis of MtThiS-COSH.

a, Top, MS spectrum of MtThiS (m/z 8,001 Da). Bottom, MS spectrum of MtThiS-COSH (thiocarboxylated C-terminus, m/z 8,017 Da). b, The same sample as above was digested with endoproteinase GluC. Shown is the spectral window surrounding the C-terminal peptide: VIRVIYGG.

Extended Data Fig. 4 High-resolution and tandem MS of the MtThiS-COSH C-terminal fragment.

a, High-resolution broadband spectrum of the C-terminal GluC peptide fragment of MtThiS-COSH. b, m/z 892.51 was subjected to CID (collision-induced dissociation) with assigned ions indicated in tabular form. c, Tandem mass spectrum (MS/MS) confirming the location of the +16 Da mass change to the C-terminus. CID also promotes the formal loss of H2S (obsv. Δm/z 33.9884 Da; calc. Δm/z 33.9877 Da) from the parent ion.

Extended Data Fig. 5 High-resolution and tandem MS of the thioamidated McrA peptide.

a, The thioamidated McrA peptide (GG-RLGFYGYDLQD) was characterized by HRMS. b, m/z 1,476.66 was subjected to CID with assigned ions indicated in tabular form. c, MS/MS spectrum confirming the location of the +16 Da mass change to the central Gly residue. Thioamide bond cleavage was not observed under the applied CID conditions.

Extended Data Fig. 6 TfuA catalyzes ThiS-COSH hydrolysis to generate sulfide.

Quantification of sulfide (a) and MtThiS-COSH (b) via fluorescence detection at different time points of the reaction between MtThiS-COSH (150 μM) and MtTfuA of various concentrations. Sulfide concentrations were measured via reaction with 7-amino-4-methylcoumarin followed by fluorescence quantification. ThiS-COSH concentrations were determined using the LRSA-based assay. Data are presented as mean values ± s.d. (n = 3 independent experiments). c, Fluorescence quantification of MtThiS-COSH at different time points after reaction with 50 μM MtYcaO, 5 mM ATP, and McrA peptide of various concentrations. Data are presented as mean values ± s.d. (n = 3 independent experiments).

Source data

Extended Data Fig. 7 McThiS 1H,15N-HMQC spectra upon MtTfuA titration.

a, An overlay of McThiS 1H,15N-HMQC spectra in the presence (red) and absence (black) of MtTfuA (2 equiv.). Residues with substantial chemical shift perturbations are labeled. b, Zoomed regions of the spectrum showing McThiS residues with the largest chemical shift changes. The MtTfuA: McThiS ratio varies from 0:1 to 6:1 equiv. (MtTfuA concentrations vary from 110 μM to 1.2 mM).

Extended Data Fig. 8 SDS-PAGE analysis of AzoYcaO co-purifying with His-AzoTfuA.

(a) A Coomassie-stained SDS-PAGE gel showing untagged AzoYcaO (BAI72909.1) and His6-AzoTfuA (BAI72908.1) from Azospirillum sp. B510 co-expressed heterologously and co-purified using Ni-NTA affinity chromatography. In-gel trypsin digestion and subsequent MS analysis was performed to confirm the identity of the suspected His6-AzoTfuA (b) and AzoYcaO (c) bands. MALDI-TOF mass spectra of the tryptic peptides are labeled with subscripts corresponding native amino acid sequence covered. The identified tryptic fragments cover ~40% of the total protein sequence for both proteins.

Source data

Extended Data Fig. 9 The presence of ATP alters the outcome of ThiS-COSH hydrolysis.

a, Fluorescence quantification of MtThiS-COSH (initially 200 μM) upon reaction with MtTfuA (3 μM), MtYcaO (3 μM), and McrA peptide (100 μM), in the presence (red circles) or absence (black squares) of ATP (5 mM). Data are presented as mean values ± s.d. (n = 3 independent experiments). An exponential decay model was used to fit the data. b, Fluorescence quantification of sulfide production for MtThiS-COSH control and reactions in panel a at 140 min. Individual data points (n = 3 independent experiments) and mean values (lines) are presented. c, MALDI-TOF mass spectra of the McrA peptide from reactions in panel a at 140 min.

Source data

Extended Data Fig. 10 Structural comparison of MaTfuA and proteins with partial structural similarity.

a, Overall structure of MaTfuA. b, Structure of myo-inositol-binding protein (PDB code: 4IRX) bound to its substrate (yellow stick) shows similarities with the α/β fold of MaTfuA (c) Structure of subtilisin BPN’ in complex with its prodomain (PDB code: 3CNQ) shows similarities with the α/β fold of MaTfuA. d, Close-up view of the superimposition between MaTfuA and 3CNQ shows the putative binding pocket for a peptide substrate. Residues that form the presumptive binding pocket are shown as tan (stick); the prodomain bound by subtilisin BPN’ is shown in blue (stick).

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2, Figs. 1–23 and References.

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Source data

Source Data Fig. 1

Raw data and model fitting statistics for Fig. 1b–e.

Source Data Fig. 2

Chemical shift data for Fig. 2b.

Source Data Fig. 3

Raw data and model fitting statistics for Fig. 3a,b; raw data and representative gel images for Fig. 3c.

Source Data Extended Data Fig. 6

Raw data and representative gel images for Extended Data Fig. 6a–c.

Source Data Extended Data Fig. 8

Uncropped gel image for Extended Data Fig. 8a.

Source Data Extended Data Fig. 9

Raw data and representative gel images for Extended Data Fig. 9a,b.

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Liu, A., Si, Y., Dong, SH. et al. Functional elucidation of TfuA in peptide backbone thioamidation. Nat Chem Biol 17, 585–592 (2021). https://doi.org/10.1038/s41589-021-00771-0

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