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 His 7 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 Zn 1 -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 (R 13 -ASS 20 ) differ in charge and bulk from those of ZiaA N (D 18 -KLK 25 ) 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.