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

• Published in: Journal of biological inorganic chemistry Vol. 15; no. 1; pp. 87 - 98
• Main Authors: Banci, Lucia, Bertini, Ivano, Ciofi-Baffoni, Simone, Poggi, Luisa, Vanarotti, Murugendra, Tottey, Stephen, Waldron, Kevin J, Robinson, Nigel J
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 2010; Springer-Verlag
• Subjects: Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - metabolism; Amino Acid Sequence; Apoenzymes - chemistry; Apoenzymes - genetics; Apoenzymes - metabolism; Bacterial Proteins - metabolism; Biochemistry; Biomedical and Life Sciences; Cation Transport Proteins - metabolism; Copper - metabolism; Copper-transporting ATPases; Life Sciences; Microbiology; Models, Molecular; Molecular Chaperones - metabolism; Molecular Sequence Data; Mutation; Nuclear Magnetic Resonance, Biomolecular; Original Paper; Protein Structure, Tertiary; Solubility; Substrate Specificity; Synechocystis - enzymology; Synechocystis - metabolism; Zinc - metabolism; P-type ATPase; NMR; Zinc
• ISSN: 0949-8257; 1432-1327
• DOI: 10.1007/s00775-009-0568-7

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.