Abstract
The human copper chaperone HAH1 transports copper to the Menkes and Wilson proteins, which are copper-translocating P-type ATPases located in the trans-Golgi apparatus and believed to provide copper for important enzymes such as ceruloplasmin, tyrosinase, and peptidylglycine monooxygenase. Although a substantial amount of structural data exist for HAH1 and its yeast and bacterial homologues, details of the copper coordination remain unclear and suggest the presence of two protein-derived cysteine ligands and a third exogenous thiol ligand. Here we report the preparation and reconstitution of HAH1 with Cu(I) using a protocol that minimizes the use of thiol reagents believed to be the source of the third ligand. We show by x-ray absorption spectroscopy that this reconstitution protocol generates an occupied Cu(I) binding site with linear biscysteinate coordination geometry, as evidenced by (i) an intense edge absorption centered at 8982.5 eV, with energy and intensity identical to the rigorously linear two-coordinate model complex bis-2,3,5,6-tetramethylbenzene thiolate Cu(I) and (ii) an EXAFS spectrum that could be fit to two Cu-S interactions at 2.16 A, a distance typical of digonal Cu(I) coordination. Binding of exogenous ligands (GSH, dithiothreitol, and tris-(2-carboxyethyl)-phosphine) to the Cu(I) was investigated. When GSH or dithiothreitol was added to the chaperone during the reconstitution procedure, the resulting Cu(I)- HAH1 remained two-coordinate, whereas the addition of the phosphine during reconstitution elicited a three-coordinate species. When the exogenous ligands were titrated into the Cu(I)-HAH1, all formed three-coordinate adducts but with differing affinities. Thus, GSH and dithiothreitol showed weaker binding, with estimated KD values in the range 10-25 mm, whereas tris-(2-carboxyethyl)-phosphine showed stronger affinity, with a KD value of <5 mm. The implications of these findings for mechanisms of copper transport are discussed.
Highlights
The human copper chaperone HAH1 transports copper to the Menkes and Wilson proteins, which are copper-translocating P-type ATPases located in the transGolgi apparatus and believed to provide copper for important enzymes such as ceruloplasmin, tyrosinase, and peptidylglycine monooxygenase
We show by x-ray absorption spectroscopy that this reconstitution protocol generates an occupied Cu(I) binding site with linear biscysteinate coordination geometry, as evidenced by (i) an intense edge absorption centered at 8982.5 eV, with energy and intensity identical to the rigorously linear twocoordinate model complex bis-2,3,5,6-tetramethylbenzene thiolate Cu(I) and (ii) an EXAFS spectrum that could be fit to two Cu–S interactions at 2.16 Å, a distance typical of digonal Cu(I) coordination
Cu(I)-bound chaperone ever be prepared free from ligation by an exogenous third ligand, and if so, what is the structure? (ii) What is the role of binding of a third ligand in chaperone function? In the present study, we used biochemical and X-ray absorption spectroscopy (XAS) studies to show that a linear two-coordinate Cu(I) center free from ligation by any exogenous ligand can be prepared in HAH1
Summary
Cloning and Isolation of HAH1—The clone for HAH1 was obtained from the expressed sequence tag data bank (accession number AA703181). 8 ml of ϳ100 M protein was reconstituted to a final copper concentration of ϳ50 – 80 M. For XAS experiments, the protein was concentrated using an Ultrafree centrifugation system In the first set of experiments, GSH, DTT, and TCEP were added in a 10-fold excess (typically 1000 – 1500 M) to the apoprotein and incubated for 1 h before adding copper. In the second set of experiments, the three ligands were each titrated into the protein after metal reconstitution, with ratios of 1:1, 5:1, and 25:1 reductant/protein. The nickel K fluorescence, which is generated by the nickel filter and is always present in the single channel analyzer window, was removed by collecting data on a blank sample under identical conditions of detector/Soller slit geometry and subtracting this as a background file from each data set.
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