Expression, purification and copper-binding studies of the first metal-binding domain of Menkes protein

The cDNA, coding for the first metal-binding domain (MBD1)of Menkes protein , was cloned into the T7-system based vector, pCA. The T7 lysozyme-encoding plasmid, pLysS, is shown to be crucial for expression, suggesting that the protein is toxic to the cells. Adding copper to the growth medium did not affect the plasmid stability. MBD1 is purified in two steps with a typical yield of 12 mg . L-1. Menkes protein, a P-type ATPase, contains a sequence GMXCXSC that is repeat ed six times, at the N-terminus. The paired cysteine residues are involved in metal binding. MBD1 has only two cysteine residues, which can exist as f ree thiol groups (reduced), as a disulphide bond (oxidized) or bound to a m etal ion [e.g. Cu(I)-MBD1]. These three MBD1 forms have been investigated u sing CD. No major spectral change was seen between the different MBD1 forms , indicating that the folding is not changed upon metal binding. A copper-bound MBD1 was also studied by EPR, and the lack of an EPR signal suggests that the oxidation state of copper bound to MBD1 is Cu(I). Cu(PI b inding studies were performed by equilibrium dialysis and revealed a stoich iometry of 1 : I and an apparent K-d = 46 mu M. Oxidized MBD1, however, is not able to bind copper. Different copper complexes were investigated for t heir ability to reconstitute apo-MBD1. Given the same total copper concentr ation CuCl43- was superior to Cu(I)-thiourea (structural analogue of metall othionein) and Cu(I)-glutathione (used at fivefold higher copper concentrat ion) although the latter two were able to partially reconstitute apo-MBD1. Cu(II) was not able to reconstitute apo-MBD1, presumably due to Cu(LI)-indu ced oxidation of the thiol groups. Based on our results, glutathione and/or metallothionein are likely candidates for the in vivo incorporation of cop per to Menkes protein.