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Analysis of a Soluble (UreD:UreF:UreG)2 Accessory Protein Complex and Its Interactions with Klebsiella aerogenes Urease by Mass Spectrometry

  • Research Article
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Journal of The American Society for Mass Spectrometry

Abstract

Maturation of the nickel-containing urease of Klebsiella aerogenes is facilitated by the UreD, UreF, and UreG accessory proteins along with the UreE metallo-chaperone. A fusion of the maltose binding protein and UreD (MBP-UreD) was co-isolated with UreF and UreG in a soluble complex possessing a (MBP-UreD:UreF:UreG)2 quaternary structure. Within this complex a UreF:UreF interaction was identified by chemical cross-linking of the amino termini of its two UreF protomers, as shown by mass spectrometry of tryptic peptides. A pre-activation complex was formed by the interaction of (MBP-UreD:UreF:UreG)2 and urease. Mass spectrometry of intact protein species revealed a pathway for synthesis of the urease pre-activation complex in which individual hetero-trimer units of the (MBP-UreD:UreF:UreG)2 complex bind to urease. Together, these data provide important new insights into the structures of protein complexes associated with urease activation.

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Acknowledgments

The authors thank Dr. Dan Jones and the Mass Spectrometry and Metabolomics Core facility at Michigan State University for assistance with MALDI-TOF MS, along with Dr. Jones and Dr. Lee Macomber for helpful discussions. The authors also thank Joseph Eschweiler (UM) for aiding in the collection of some of the intact MS data on the urease complexes reported here. This project was funded by the National Institutes of Health awards to R.P.H. (DK045686) and to B.T.R. (GM095832).

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Author notes

  1. Jodi L. Boer

    Present address: Department of Translational Science and Molecular Medicine, Michigan State University College of Human Medicine, Grand Rapids, MI, 49503, USA

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, 48824, USA

    Mark A. Farrugia, Jodi L. Boer & Robert P. Hausinger

  2. Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, 48824, USA

    Robert P. Hausinger

  3. Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA

    Linjie Han, Yueyang Zhong & Brandon T. Ruotolo

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  1. Mark A. Farrugia
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Correspondence to Brandon T. Ruotolo or Robert P. Hausinger.

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Farrugia, M.A., Han, L., Zhong, Y. et al. Analysis of a Soluble (UreD:UreF:UreG)2 Accessory Protein Complex and Its Interactions with Klebsiella aerogenes Urease by Mass Spectrometry. J. Am. Soc. Mass Spectrom. 24, 1328–1337 (2013). https://doi.org/10.1007/s13361-013-0677-y

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