Probing determinants of cyclopiazonic acid sensitivity of bacterial Ca2+-ATPases

Authors

  • Aljona Kotšubei,

    1. Centre for Membrane Pumps in Cells and Disease – PUMPKIN, Aarhus University, Denmark
    2. Department of Molecular Biology and Genetics, Aarhus University, Denmark
    3. Department of Gene Technology, Tallinn University of Technology, Estonia
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  • Manuela Gorgel,

    1. Centre for Membrane Pumps in Cells and Disease – PUMPKIN, Aarhus University, Denmark
    2. Department of Molecular Biology and Genetics, Aarhus University, Denmark
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  • Jens P. Morth,

    1. Centre for Membrane Pumps in Cells and Disease – PUMPKIN, Aarhus University, Denmark
    Current affiliation:
    1. Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo, Blindern, Oslo, Norway
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  • Poul Nissen,

    1. Centre for Membrane Pumps in Cells and Disease – PUMPKIN, Aarhus University, Denmark
    2. Department of Molecular Biology and Genetics, Aarhus University, Denmark
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  • Jacob L. Andersen

    Corresponding author
    1. Centre for Membrane Pumps in Cells and Disease – PUMPKIN, Aarhus University, Denmark
    2. Department of Molecular Biology and Genetics, Aarhus University, Denmark
    • Correspondence

      J. L. Andersen, Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10c, DK-8000 Aarhus C, Denmark

      Fax: +45 8612 3178

      Tel: +45 8715 4936

      E-mail: jla@mb.au.dk

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Abstract

Cyclopiazonic acid (CPA) is a specific and potent inhibitor of the sarcoplasmic reticulum Ca2+-ATPase 1a (SERCA1a). Despite high sequence similarity to SERCA1a, Listeria monocytogenes Ca2+-ATPase 1 (LMCA1) is not inhibited by CPA. To test whether a CPA binding site could be created while maintaining the functionality of the ATPase we targeted four amino acid positions in LMCA1 for mutational studies based on a multiple sequence alignment of SERCA-like Ca2+-ATPases and structural analysis of the CPA site. The identification of CPA-sensitive gain-of-function mutants pinpointed key determinants of the CPA binding site. The importance of these determinants was further underscored by the characterization of the CPA sensitivity of two additional bacterial Ca2+-ATPases from Lactococcus lactis and Bacillus cereus. The CPA sensitivity was predicted from their sequence compared with the LMCA1 results, and this was experimentally confirmed. Interestingly, a cluster of Lactococcus bacteria applied in the production of fermented cheese display Ca2+-ATPases that are predictably CPA insensitive and may originate from their coexistence with CPA-producing Penicillum and Aspergillus fungi in the cheese. The differences between bacterial and mammalian binding pockets encompassing the CPA site suggest that CPA derivatives that are specific for bacteria or other pathogens can be developed.

Database

LMCA1 (EC: 3.6.3.8), SERCA1a (EC: 3.6.3.8)

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