Structure-Function Analyses of the ATX1 Metallochaperone

Abstract

Saccharomyces cerevisiae Atx1p represents a member of the family of metallochaperone molecules that escort copper to distinct intracellular targets. Atx1p specifically delivers copper to the Ccc2p copper transporter in the Golgi. Additionally, when overproduced, Atx1p substitutes for superoxide dismutase 1 in preventing oxidative damage; however the mechanistic overlap between these functions is unresolved. The crystal structure of Atx1p has been solved recently. By examining a surface electrostatic potential distribution, multiple conserved lysines are revealed on one face of Atx1p. An additional conserved lysine (Lys 65 ) lies in close proximity to the metal binding site. Through site-directed mutagenesis, residues in the metal binding region including Lys 65 were found to be necessary for both copper delivery to Ccc2p and for Atx1p antioxidant activity. Copper trafficking to Ccc2p also relied on the lysine-rich face of Atx1p. Surprisingly however, elimination of these lysines did not inhibit the antioxidant activity of Atx1p. We provide evidence that Atx1p does not suppress oxidative damage by a metallochaperone mechanism but may directly consume superoxide. Purified Cu-Atx1p reacts noncatalytically with superoxide anion in vitro. We conclude that the copper-trafficking and antioxidant functions of Atx1p arise from chemically and structurally distinct attributes of this metallochaperone.

Yeast Cox17 Solution Structure and Copper(I) Binding

“…4b, meaningful differences between apo and CuCox17 occur where the rmsd between the two representative structures is greater than the sum of the rmsds of the two ensembles of structures. There are slight differences for coordinating residue Cys 26 and for Met 58 , which is close to the modeled Cu(I) ion (Fig. 4a).…”

“…This residue, which is essential for function (58), has been proposed to modulate copper transfer (50,52). In the CuCox17 structure, conserved residue Lys 30 is proximal to the copper-binding site and could counterbalance the negative charge because of the presence of three cysteines.…”

Binding of Copper(I) by the Wilson Disease Protein and Its Copper Chaperone

“…Consideration of the electrostatic properties of each domain furnishes part of the answer. Interactions between Atox1 or Atx1 and their target domains involve complementary electrostatic surfaces (15,16,37,48). In particular, positively charged residues on the chaperone surface interact with negatively charged residues on the target domain.…”

Characterization of the Interaction between the Wilson and Menkes Disease Proteins and the Cytoplasmic Copper Chaperone, HAH1p

“…Recent yeast two-hybrid analyses of the yeast HAH1 homologue, ATX1, showed that mutation of the corresponding residues, Lys 65 3 Glu and Lys 61 3 Glu ϩ Lys 62 3 Glu, as well as other compound lysine to glutamate mutations, abolish interaction between the copper chaperon and CCC2p, the WD/ MNK homologue (35). As the x-ray crystal structures for ATX1p (36) and the fourth metal-binding domain of MNKp (MNK4) have recently been reported (37), we attempted to reconcile these differences by modeling the WDp/MNKp -HAH1p interaction.…”