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

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Abstract

A Cu(I) metallochaperone, Atx1, interacts with the amino-terminal domain of a Cu(I)-transporting ATPase, PacSN, but not with a domain of related Zn-transporting ATPase, ZiaAN in Synechocystis PCC 6803. This is thought to prevent ZiaAN from acquiring Cu(I), which it binds more tightly than Zn. Solution structures of Atx1, PacSN, and the heterodimer were previously described. Here we report solution structural studies of the ZiaAN soluble domain. Apo-ZiaAN has a typical ferredoxin-like fold followed by an atypical 34 residues of unstructured polypeptide containing a His7 motif. ZiaAN 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-ZiaAN 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)–ZiaAN heterodimers was observed, and in the presence of equimolar ZiaAN and PacSN, only Atx1–Cu(I)–PacSN complexes were detected. Residues flanking the CXXC motif of PacSN (R13-ASS20) differ in charge and bulk from those of ZiaAN (D18-KLK25) and make contacts in the Atx1–Cu(I)–PacSN complex. Crucially, swapping these residues flanking the CXXC motifs of ZiaAN and PacSN reciprocally swaps partner choice by Atx1. These few residues of the two ATPases have diverged during evolution to bias Atx1 interactions in favor of PacSN rather than ZiaAN.

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Abbreviations

HSQC:

Heteronuclear single quantum coherence

NMR:

Nuclear magnetic resonance

NOE:

Nuclear Overhauser effect

NOESY:

Nuclear Overhauser effect spectroscopy

PAR:

4-(2-Pyridylazo)resorcinol

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Acknowledgments

This work was supported by research grants from the BBSRC (BB/E001688/1) and from the European Commission (“European Network of Research Infrastructures for Providing Access and Technological Advancements in Bio-NMR” contract no. 026145, SPINE II contract no. LSHG-CT-2006-031220 “From Receptor to Gene: Structures of Complexes from Signaling Pathways Linking Immunology, Neurobiology and Cancer,” and Marie Curie host fellowships for early stage research training no. MEST-CT-2004-504391 “NMR in Inorganic Structural Biology”).

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Authors and Affiliations

  1. Magnetic Resonance Center, University of Florence, Via L. Sacconi, 6, 50019, Sesto Fiorentino, Italy

    Lucia Banci, Ivano Bertini, Simone Ciofi-Baffoni, Luisa Poggi & Murugendra Vanarotti

  2. Institute for Cell and Molecular Biosciences, Medical School, University of Newcastle, NE2 4HH, Newcastle, UK

    Stephen Tottey, Kevin J. Waldron & Nigel J. Robinson

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  1. Lucia Banci
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Correspondence to Ivano Bertini or Nigel J. Robinson.

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This article will be printed in the upcoming Journal of Biological Inorganic Chemistry special issue CELL BIOLOGY OF COPPER.

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Banci, L., Bertini, I., Ciofi-Baffoni, S. et al. NMR structural analysis of the soluble domain of ZiaA-ATPase and the basis of selective interactions with copper metallochaperone Atx1. J Biol Inorg Chem 15, 87–98 (2010). https://doi.org/10.1007/s00775-009-0568-7

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