Analytical and Bioanalytical Chemistry

, Volume 408, Issue 21, pp 5925–5933

ATP and autophosphorylation driven conformational changes of HipA kinase revealed by ion mobility and crosslinking mass spectrometry

Research Paper

DOI: 10.1007/s00216-016-9709-3

Cite this article as:
Wen, Y., Sobott, F. & Devreese, B. Anal Bioanal Chem (2016) 408: 5925. doi:10.1007/s00216-016-9709-3


Toxin-antitoxin systems are genetic modules involved in a broad range of bacterial cellular processes including persistence, multidrug resistance and tolerance, biofilm formation, and pathogenesis. In type II toxin-antitoxin systems, both the toxin and antitoxin are proteins. In the prototypic Escherichia coli HipA-HipB module, the antitoxin HipB forms a complex with the protein kinase HipA and sequesters it in the nucleoid. HipA is then no longer able to phosphorylate glutamyl-tRNA-synthetase and this prevents the initiation of the forthcoming stringent response. Here we investigated the assembly of the Shewanella oneidensis MR-1 HipA-HipB complex using native electrospray ion mobility-mass spectrometry and chemical crosslinking combined with mass spectrometry. We revealed that the HipA autophosphorylation was accompanied by a large conformational change, and confirmed structural evidence that S. oneidensis MR-1 HipA-HipB assembly was distinct from the prototypic E. coli HipA-HipB complex.

Graphical abstract

Ion mobility mass spectrometry shows a two phase transition from unstructured HipA to a compact folded phosphorylated protein


Toxin-antitoxin system HipAB Ion mobility Chemical crosslinking Mass spectrometry 

Supplementary material

216_2016_9709_MOESM1_ESM.pdf (177 kb)
ESM 1(PDF 177 kb)

Funding information

Funder NameGrant NumberFunding Note
Belgian Federal Goverment Science Policy-Interuniversity attraction poles
  • IAP 7/44
National Natural Science Foundation of China
  • NO.31500051

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.Center for Translational Medicine, Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and TechnologyXi’an Jiaotong UniversityXi’anChina
  2. 2.Unit for Biological Mass Spectrometry and Proteomics, Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE)Ghent UniversityGhentBelgium
  3. 3.Biomolecular & Analytical Mass Spectrometry Group and UA-VITO Center for Proteomics (CFP-CEPROMA)University of AntwerpAntwerpBelgium

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