Cooperative binding of copper(I) to the metal binding domains in Menkes disease protein.
We have optimised the overexpression and purification of the N-terminal end of the Menkes disease protein expressed in Escherichia coli, containing one, two and six metal binding domains (MBD), respectively. The domain(s) have been characterised using circular dichroism (CD) and fluorescence spectroscopy, and their copper(I) binding properties have been determined. Structure prediction derived from far-UV CD indicates that the secondary structure is similar in the three proteins and dominated by beta-sheet. The tryptophan fluorescence maximum is blue-shifted in the constructs containing two and six MBDs relative to the monomer, suggesting more structurally buried tryptophan(s), compared to the single MBD construct. Copper(I) binding has been studied by equilibrium dialysis under anaerobic conditions. We show that the copper(I) binding to constructs containing two and six domains is cooperative, with Hill coefficients of 1.5 and 4, respectively. The apparent affinities are described by K(0.5), determined to be 65 microM and 19 microM for constructs containing two and six domains, respectively. Our data reveal a unique regulation of Menkes protein upon a change in copper(I) concentration. The regulation does not occur as an 'all-or-none' cooperativity, suggesting that the copper(I) binding domains have a basal low affinity for binding and release of copper(I) at low concentrations but are able to respond to higher copper levels by increasing the affinity, thereby contributing to prevent the copper concentration from reaching toxic levels in the cell.
Jensen
P Y
PY
Department of Analytical and Pharmaceutical Chemistry, Royal Danish School of Pharmacy, Copenhagen, Denmark. pia@carb.nist.gov
Bonander
N
N
Møller
L B
LB
Farver
O
O
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D027682:Cation Transport Proteins; D002412:Cations / Q000737:chemistry; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry*; D000073840:Copper-Transporting ATPases; D004926:Escherichia coli; D005786:Gene Expression Regulation; D006801:Humans; D010957:Plasmids; D011993:Recombinant Fusion Proteins; D013050:Spectrometry, Fluorescence
D002352:Carrier Proteins; D027682:Cation Transport Proteins; D002412:Cations; D011993:Recombinant Fusion Proteins; D003300:Copper; D000251:Adenosine Triphosphatases; C081184:ATP7A protein, human; D000073840:Copper-Transporting ATPases