title: Probing determinants of cyclopiazonic acid sensitivity of bacterial Ca2+-ATPases.
abstract: Cyclopiazonic acid (CPA) is a specific and potent inhibitor of the sarcoplasmic reticulum Ca(2+)-ATPase 1a (SERCA1a). Despite high sequence similarity to SERCA1a, Listeria monocytogenes Ca(2+)-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 Ca(2+)-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 Ca(2+)-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 Ca(2+)-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.
authors.affiliation: Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Aarhus University, Denmark; Department of Molecular Biology and Genetics, Aarhus University, Denmark; Department of Gene Technology, Tallinn University of Technology, Estonia.
authors.forename: Aljona
authors.initials: A
authors.lastname: Kotšubei
authors.forename: Manuela
authors.initials: M
authors.lastname: Gorgel
authors.forename: Jens P
authors.initials: JP
authors.lastname: Morth
authors.forename: Poul
authors.initials: P
authors.lastname: Nissen
authors.forename: Jacob L
authors.initials: JL
authors.lastname: Andersen
mesh_terms: D001409:Bacillus cereus / Q000187:drug effects / Q000201:enzymology; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007211:Indoles / Q000494:pharmacology*; D013294:Lactococcus lactis / Q000187:drug effects / Q000201:enzymology; D008089:Listeria monocytogenes / Q000187:drug effects* / Q000201:enzymology; D008826:Microbial Sensitivity Tests; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics*; D011487:Protein Conformation; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D007211:Indoles; D050505:Mutant Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; C000543:cyclopiazonic acid
keywords: Ca2+-ATPase; LMCA1; Listeria monocytogenes; SERCA; cyclopiazonic acid