NMR structural analysis of the soluble domain of ZiaA-ATPase and the basis of selective interactions with copper metallochaperone Atx1

A Cu(I) metallochaperone, Atx1, interacts with the amino-terminal domain of a Cu(I)-transporting ATPase, PacS_N, but not with a domain of related Zn-transporting ATPase, ZiaA_N in Synechocystis PCC 6803. This is thought to prevent ZiaA_N from acquiring Cu(I), which it binds more tightly than Zn. Solution structures of Atx1, PacS_N, and the heterodimer were previously described. Here we report solution structural studies of the ZiaA_N soluble domain. Apo-ZiaA_N has a typical ferredoxin-like fold followed by an atypical 34 residues of unstructured polypeptide containing a His₇ motif. ZiaA_N competes with the metallochromic indicator 4-(2-pyridylazo)resorcinol for 1 equiv of Zn, which can be displaced by thiol-modifying p-mercuriphenylsulfonic acid, establishing that a high-affinity site involves thiols of the CXXC motif within the ferredoxin-like fold. A single equivalent of Zn affects nuclear magnetic resonance signals arising from the CXXC motif as well as all seven His residues. The presence of NMR-line broadening in both sites implies that Zn₁-ZiaA_N undergoes exchange phenomena, consistent with CXXC-bound Zn coincidentally sampling various His ligands. These Zn-dependent dynamic changes could either aid metal transfer or alter intramolecular interactions. No formation of Atx1–Cu(I)–ZiaA_N heterodimers was observed, and in the presence of equimolar ZiaA_N and PacS_N, only Atx1–Cu(I)–PacS_N complexes were detected. Residues flanking the CXXC motif of PacS_N (R₁₃-ASS₂₀) differ in charge and bulk from those of ZiaA_N (D₁₈-KLK₂₅) and make contacts in the Atx1–Cu(I)–PacS_N complex. Crucially, swapping these residues flanking the CXXC motifs of ZiaA_N and PacS_N reciprocally swaps partner choice by Atx1. These few residues of the two ATPases have diverged during evolution to bias Atx1 interactions in favor of PacS_N rather than ZiaA_N.