Studies of copper trafficking proteins from Bacillus subtilis by native mass spectrometry

• Main Author: Kay, Kristine
• Published: University of East Anglia 2016
• Subjects: 669
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.705145

Copper is essential for life, but potentially toxic due to its ability to redox cycle and displace metal cofactors. Therefore, ubiquitous protein networks exist to safely handle and deliver copper. Copper is removed from the cell via an integral membrane P1B-ATPase, characterised by its soluble metal-binding domains (MBDs) which receive copper from Atx1-like copper chaperones in the cytoplasm. The mechanism of copper removal is unknown. Low molecular weight thiol species (LMWT) may play a role in cellular copper trafficking. The metallochaperone (CopZ) and soluble domains of the ATPase (CopAab) from Bacillus subtilis each bind Cu(I) with high affinity/specificity and form higher-order assemblies. Native electrospray ionisation mass spectrometry (ESI-MS) revealed formation of multiple copper-bound species with increasing Cu(I) level; cooperative formation of Cu4(CopZ)2 and Cu6(CopAab)2 was observed. The affinity for Cu(I) of bacillithiol (BSH), the primary LMWT in the B.subtilis cytoplasm, was determined to be b2 = 4.1 x 1017 M-2. ESI-MS revealed reduced intensity of dimeric forms of CopZ and CopAab in the presence of BSH (and other LMWT) due to copper competition; bacillithiolation of CopZ was observed. Higher order copper-bound complexes were observed for metallochaperones from Streptomyces lividans and Saccharomyces cerevisiae, and their decrease in the presence of LMWT. Rapid and reversible copper transfer between CopZ and CopAab was observed with similar rate constants at 25°C for forward (247 ± 2.2 s-1) and reverse (258 ± 2.6 s-1) directions of transfer. The rate constant was independent of concentration, suggesting the rate-limiting step is first-order, and likely to be protein complex formation. Kinetic studies demonstrated that protein complex formation resulted in a 7-fold increase in the rate of copper transfer; a copper-bound CopZ:CopAab complex was observed via ESI-MS. Bidirectional copper transfer between CopZ and CopAab consistent with a regulatory role for the MBDs.