Investigating the Role of Zinc and Copper Binding Motifs of Trafficking Sites in the Cyanobacterium Synechocystis PCC 6803

Abstract

Although zinc and copper are required by proteins with very different functions, these metals can be delivered to cellular locations by homologous metal transporters within the same organism, as demonstrated by the cyanobacterial (Synechocystis PCC 6803) zinc exporter ZiaA and thylakoidal copper importer PacS. The N-terminal metal-binding domains of these transporters (ZiaAN and PacSN, respectively) have related ferredoxin folds also found in the metallochaperone Atx1, which delivers copper to PacS, but differ in the residues found in their M/IXCXXC metal-binding motifs. To investigate the role of the nonconserved residues in this region on metal binding, the sequence from ZiaAN has been introduced into Atx1 and PacSN, and the motifs of Atx1 and PacSN swapped. The motif sequence can tune Cu(I) affinity only approximately 3-fold. However, the introduction of the ZiaAN motif (MDCTSC) dramatically increases the Zn(II) affinity of both Atx1 and PacSN by up to 2 orders of magnitude. The Atx1 mutant with the ZiaAN motif crystallizes as a side-to-side homodimer very similar to that found for [Cu(I)2–Atx1]2 (Badarau20726513Biochemistry2010497798). In a crystal structure of the PacSN mutant possessing the ZiaAN motif (PacSNZiaAN), the Asp residue from the metal-binding motif coordinates Zn(II). This demonstrates that the increased Zn(II) affinity of this variant and the high Zn(II) affinity of ZiaAN are due to the ability of the carboxylate to ligate this metal ion. Comparison of the Zn(II) sites in PacSNZiaAN structures provides additional insight into Zn(II) trafficking in cyanobacteria.

Crystallization of Ethylene Plant Hormone Receptor—Screening for Structure

“…In addition to their natural metal cofactor, purified recombinant proteins can bind to other metal ions that can maintain or even stabilize their structure. In this sense, Cu(I) has been replaced by Zn(II) in human and cyanobacterial copper chaperones for structural studies [ 66 , 67 , 68 ]. Thus, in addition to fixing the copper loading state of our purified ETRs, we also used zinc as a replacement in our LCP crystallization trials.…”

Crystal Structure of the Human Copper Chaperone ATOX1 Bound to Zinc Ion

“…However, high-resolution atomic models of Atox1 loaded with Zn ions have not been reported so far. Badarau and colleagues investigated the crosstalk between Zn and Cu in cyanobacteria and obtained the structure of the Cu-chaperone Atx1 from cyanobacterium Synechocystis loaded with Zn ions, gaining insight into the Zn and Cu binding motifs [ 35 , 36 ].…”