Iron is a ligand of SecA-like metal-binding domains in vivo

Tamar Cranford-Smith, Mohammed Jamshad, Mark Jeeves, Rachael Chandler, Jack Yule, Ashley Robinson, Farhana Alam, Karl Dunne, Edwin Aponte Angarita, Mashael Alanazi, Cailean Carter, Ian Henderson, Janet Lovett, Peter Winn, Tim Knowles, Damon Huber

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The ATPase SecA is an essential component of the bacterial Sec machinery, which transports proteins across the cytoplasmic membrane. Most SecA proteins contain a long C-terminal tail (CTT). In Escherichia coli, the CTT contains a structurally flexible linker domain and a small metal-binding domain (MBD). The MBD coordinates zinc via a conserved cysteine-containing motif and binds to SecB and ribosomes. In this study, we screened a high-density transposon library for mutants that affect the susceptibility of E. coli to sodium azide, which inhibits SecA-mediated translocation. Results from sequencing this library suggested that mutations removing the CTT make E. coli less susceptible to sodium azide at subinhibitory concentrations. Copurification experiments suggested that the MBD binds to iron and that azide disrupts iron binding. Azide also disrupted binding of SecA to membranes. Two other E. coli proteins that contain SecA-like MBDs, YecA and YchJ, also copurified with iron, and NMR spectroscopy experiments indicated that YecA binds iron via its MBD. Competition experiments and equilibrium binding measurements indicated that the SecA MBD binds preferentially to iron and that a conserved serine is required for this specificity. Finally, structural modeling suggested a plausible model for the octahedral coordination of iron. Taken together, our results suggest that SecA-like MBDs likely bind to iron in vivo.

Original languageEnglish
Pages (from-to)7516-7528
Number of pages13
JournalJournal of Biological Chemistry
Issue number21
Early online date2 Apr 2020
Publication statusPublished - 22 May 2020

Bibliographical note

Funding Information:
This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) Midlands Integrated Integrative Biosciences Training Partnership (MIBTP) (to T. C.-S.), the Jouf University (to M. A.), BBSRC Grant BB/L019434/1 (to D. H. and M. J.), BBSRC Grant BB/P009840/1 (to T. K.), the Ministerio de Ciencia, Tecnología e Innovación del govierno de Colombia and the British Council (to E. H. A. A.), and a Royal Society University Research Fellowship and the Wellcome Trust for the Q-band EPR spectrometer Grant 099149/Z/12/Z (to J. E. L.). The authors declare that they have no conflicts of interest with the contents of this article.

Funding Information:
Acknowledgments—We thank J. Cole, J. Green, A. Peacock, O. Daub-ney, and D. Collison for advice and assistance. We thank Drs. C. Stark, S. Baker, M. Thompson, H. El Mkami, and A. Shah for technical assistance and members of the Henderson, Lund, and Grainger labs for insightful discussions. NMR work was supported by Wellcome Trust Grant 099185/Z/12/Z, and we thank HWB-NMR at the University of Birmingham for providing open access to their Wellcome Trust funded 900 MHz spectrometer.

Publisher Copyright:
© 2020 Cranford-Smith et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.

Copyright 2020 Elsevier B.V., All rights reserved.


  • SecA
  • Sec
  • protein translocation
  • protein transport
  • metal binding
  • iron
  • zinc
  • NMR
  • EPR


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