The homoeostasis of metal ions in cells is the result of the contribution of several cellular pathways that involve transient, often weak, protein-protein interactions. Metal transfer typically implies the formation of adducts where the metal itself acts as a bridge between proteins, by co-ordinating residues of both interacting partners. In the present study we address the interaction between the human copper(I)-chaperone HAH1 (human ATX1 homologue) and a metal-binding domain in one of its partners, namely the P-type copper-transporting ATPase, ATP7A (ATPase, Cu+ transporting, alpha polypeptide). The adduct was structurally characterized in solution, in the presence of copper(I), and through X-ray crystallography, upon replacing copper(I) with cadmium(II). Further insight was obtained through molecular modelling techniques and site-directed mutagenesis. It was found that the interaction involves a relatively small interface (less than 1000 A(2), 1 A=0.1 nm) with a low fraction of non-polar atoms. These observations provide a possible explanation for the low affinity of the two apoproteins. It appears that electrostatics is important in selecting which domain of the ATPase is able to form detectable amounts of the metal-mediated adduct with HAH1.

Copper metallochaperones supply copper to cupro-proteins through copper-mediated protein-protein-interactions and it has been hypothesized that metallochaperones thereby inhibit copper from causing damage en route. Evidence is presented in support of this latter role for cyanobacterial metallochaperone, Atx1. In cyanobacteria Atx1 contributes towards the supply of copper to plastocyanin inside thylakoids but it is shown here that in copper-replete medium, copper can reach plastocyanin without Atx1. Unlike metallochaperone- independent copper-supply to superoxide dismutase in eukaryotes, glutathione is not essential for Atx1- independent supply to plastocyanin: Double mutants missing atx1 and gshB (encoding glutathione synthetase) accumulate the same number of atoms of copper per cell in the plastocyanin pool as wild type. Critically, atx1gshB are hypersensitive to elevated copper relative to wild type cells and also relative to gshB single mutants with evidence that hyp ersensitivity arises due to the mislocation of copper to sites for other metals including iron and zinc. The zinc site on the amino-terminal domain (ZiaA N) of the P 1-type zinctransporting ATPase is especially similar to the copper site of the Atx1 target PacS N, and ZiaA N will bind Cu(I) more tightly than zinc. An NMR model of a substituted-ZiaA N-Cu(I)-Atx1 heterodimer has been generated making it possible to visualize a juxtaposition of residues surrounding the ZiaA N zinc site, including Asp 18, which normally repulse Atx1. Equivalent repulsion between bacterial copper metallochaperones and the amino-terminal regions of P 1-type ATPases for metals other than Cu(I) is conserved, again consistent with a role for copper metallochaperones to withhold copper from binding sites for other metals

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