ATP and autophosphorylation driven conformational changes of HipA kinase revealed by ion mobility and crosslinking mass spectrometry.
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.
Center for Translational Medicine, Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xianning West Road 28, Xi'an, Shaanxi, 710049, China.|Unit for Biological Mass Spectrometry and Proteomics, Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Ghent University, K.L. Ledeganckstraat 35, 9000, Ghent, Belgium.
Yurong
Y
Wen
Biomolecular & Analytical Mass Spectrometry Group and UA-VITO Center for Proteomics (CFP-CEPROMA), University of Antwerp, Groenenborgerlaan 171, 2020, Antwerp, Belgium.
Frank
F
Sobott
Unit for Biological Mass Spectrometry and Proteomics, Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Ghent University, K.L. Ledeganckstraat 35, 9000, Ghent, Belgium. Bart.Devreese@Ugent.be.
Bart
B
Devreese
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D020592:Shewanella / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C070740:hipB protein, E coli; C070741:hipA protein, E coli; D000255:Adenosine Triphosphate; D011494:Protein Kinases
Chemical crosslinking; HipAB; Ion mobility; Mass spectrometry; Toxin-antitoxin system