title: Mass spectrometry of B. subtilis CopZ: Cu(i)-binding and interactions with bacillithiol.
abstract: CopZ from Bacillus subtilis is a well-studied member of the highly conserved family of Atx1-like copper chaperones. It was previously shown via solution and crystallographic studies to undergo Cu(i)-mediated dimerisation, where the CopZ dimer can bind between one and four Cu(i) ions. However, these studies could not provide information about the changing distribution of species at increasing Cu(i) levels. To address this, electrospray ionisation mass spectrometry using soft ionisation was applied to CopZ under native conditions. Data revealed folded, monomeric CopZ in apo- and Cu(i)-bound forms, along with Cu(i)-bound dimeric forms of CopZ at higher Cu(i) loading. Cu4(CopZ)2 was the major dimeric species at loadings >1 Cu(i)/CopZ, indicating the cooperative formation of the tetranuclear Cu(i)-bound species. As the principal low molecular weight thiol in B. subtilis, bacillithiol (BSH) may play a role in copper homeostasis. Mass spectrometry showed that increasing BSH led to a reduction in Cu(i)-bound dimeric forms, and the formation of S-bacillithiolated apo-CopZ and BSH adducts of Cu(i)-bound forms of CopZ, where BSH likely acts as a Cu(i) ligand. These data, along with the high affinity of BSH for Cu(i), determined here to be β2(BSH) = ∼4 × 10(17) M(-2), are consistent with a role for BSH alongside CopZ in buffering cellular Cu(i) levels. Here, mass spectrometry provides a high resolution overview of CopZ-Cu(i) speciation that cannot be obtained from less discriminating solution-phase methods, thus illustrating the potential for the wider application of this technique to studies of metal-protein interactions.
authors.affiliation: Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK. n.le-brun@uea.ac.uk.
authors.forename: Kristine L
authors.initials: KL
authors.lastname: Kay
authors.affiliation: School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK.
authors.forename: Chris J
authors.initials: CJ
authors.lastname: Hamilton
authors.affiliation: Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK. n.le-brun@uea.ac.uk.
authors.forename: Nick E
authors.initials: NE
authors.lastname: Le Brun
mesh_terms: D001412:Bacillus subtilis / Q000254:growth & development / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D003300:Copper / Q000378:metabolism*; D003545:Cysteine / Q000031:analogs & derivatives* / Q000378:metabolism; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D001426:Bacterial Proteins; D018832:Molecular Chaperones; C543521:bacillithiol; D003300:Copper; D003545:Cysteine; D005944:Glucosamine