Metallochaperones and Metal-Transporting ATPases: A Comparative Analysis of Sequences and Structures

Abstract

A comparative structural genomic analysis of a new class of metal-trafficking proteins can provide insights into the intracellular chemistry of reactive cofactors such as copper and zinc. Starting from the sequences of the metallochaperone Atx1 and from the first soluble domain of the copper-transporting ATPase Ccc2, both from yeast, a search on the available genomes was performed using a homology criterion and a metal-binding motif xЈ-xЉ-C-xٞ-x٣-C. By limiting ourselves to 20% identity with any of the proteins found, several soluble copper-transport proteins were identified, as well as soluble domains of membrane-bound ATPases. Structural models were calculated using high-resolution solution structures as templates, and the models were validated using statistical and energy criteria. Residue conservation and substitution have been interpreted and discussed in terms of structure-function relationship. The potential energy surfaces have been analyzed in terms of protein-protein interactions. We find that metallochaperones and their physiological partner ATPases from several phylogenetic kingdoms recognize one another, via an interplay of electrostatics, hydrogen bonding, and hydrophobic interactions, in a manner that precisely orients the metal-binding side chains for rapid metal transfer between otherwise tight binding sites. Finally, other putative metal-transport proteins are mentioned that have low homology and/or a different metal-binding consensus motif and that appear to use similar structures for recognition and transfer. This analysis highlights the wealth and the complexity of the field.

Structure of human Wilson protein domains 5 and 6 and their interplay with domain 4 and the copper chaperone HAH1 in copper uptake

“…4; see also the supporting information). The three negatively charged residues of Ccc2a are conserved in WLN4, but only two are conserved in WLN2, WLN5, and WLN6 (16). As HAH1 interacts with WLN4 and WLN2 but not with WLN5-6, we may conclude that electrostatic charges are important but not unique factors in the protein-protein interactions.…”

“…The longest linker between WLN4 and WLN5 provides natural separation of N-WLNP into two parts: WLN1-4 and WLN5-6. Such a feature is not found in the bacterial and yeast copper-transporting ATPases, which have only one or two metal-binding domains (16). This topological organization suggests that the two parts can be treated independently from a structural point of view and that the different linker lengths connecting these domains is likely to be important for copper trafficking and regulation of WLNP in response to copper binding.…”

“…The structure of N-WLNP has yet to be determined; however, the NMR structures of several single metal-binding domains from the highly homologous human copper-transporting ATPase ATP7A, Menkes protein, have been solved (13)(14)(15). Sequence alignments and homology models on the six metal-binding domains of WLNP demonstrate that the Ϸ70 aa domains of N-WLNP are likely to be folded very similarly into ferredoxin-like units (16). This same fold is also found in both NMR and x-ray structures of the yeast Atx1 and human HAH1 metallochaperones (17)(18)(19)(20), the soluble cytosolic proteins that deliver copper to the copper-transporting P-type ATPases (21).…”

Conserved residues modulate copper release in human copper chaperone Atox1

“…1A). Structural work has demonstrated that Cu chaperones and target metal-binding domains from various organisms possess the same fold and coordinate the metal via two surfaceexposed cysteines in the MXCXXC motif (13)(14)(15)(16)(17)(18)(19)(20)(21). Although the methionine in the first position of the MXCXXC motif is conserved in all organisms, it is not directly involved in metal ligation (13).…”

“…Human Atox1 is a 68-residue protein that, like the metalbinding domains in ATP7A and ATP7B, has a ferredoxin-like ␣/␤-fold and a single MXCXXC copper-binding motif (12,13) (Fig. 1A).…”