Solution Structures of a Cyanobacterial Metallochaperone: INSIGHT INTO AN ATYPICAL COPPER-BINDING MOTIF *

The Atx1 copper metallochaperone from Synechocystis PCC 6803, ScAtx1, interacts with two P(1)-type copper ATPases to supply copper proteins within intracellular compartments, avoiding ATPases for other metals en route. Here we report NMR-derived solution structures for ScAtx1. The monomeric apo form has a betaalphabetabetaalpha fold with backbone motions largely restricted to loop 1 containing Cys-12 and Cys-15. The tumbling rate of Cu(I)ScAtx1 (0.1-0.8 mm) implies dimers. Experimental restraints are satisfied by symmetrical dimers with Cys-12 or His-61, but not Cys-15, invading the copper site of the opposing subunit. A full sequence of copper ligands from the cell surface to thylakoid compartments is proposed, considering in vitro homodimer liganding to mimic in vivo liganding in ScAtx1-ATPase heterodimers. A monomeric high resolution structure for Cu(I)ScAtx1, with Cys-12, Cys-15, and His-61 as ligands, is calculated without violations despite the rotational correlation time. (2)J(NH) couplings in the imidazole ring of His-61 establish coordination of N(epsilon2) to copper. His-61 is analogous to Lys-65 in eukaryotic metallochaperones, stabilizing Cu(I)S(2) complexes but by binding Cu(I) rather than compensating charge. Cys-Cys-His ligand sets are an emergent theme in some copper metallochaperones, although not in related Atx1, CopZ, or Hah1. Surface charge (Glu-13) close to the metal-binding site of ScAtx1 is likely to support interaction with complementary surfaces of copper-transporting ATPases (PacS-Arg-11 and CtaA-Lys-14) but to discourage interaction with zinc ATPase ZiaA and so inhibit aberrant formation of copper-ZiaA complexes

Surplus Zinc is Handled by Zym1 Metallothionein and Zhf Endoplasmic Reticulum Transporter in Schizosaccharomyces pombe

Homeostatic mechanisms prevent the accumulation of free zinc in the cytoplasm, raising questions regarding where surplus zinc is stored and how it is delivered to and from these stores. A genetic screen for zinc hypersensitivity in Schizosaccharomyces pombe identified a missense mutation truncating Zhf, an endoplasmic reticulum transporter. These cells were ~5-fold more zinc-sensitive than other independent mutants. The targeted disruption of zhf prevented growth on low zinc medium and caused hypersensitivity to elevated zinc/cobalt but resistance to cadmium. The exposure to elevated zinc but not copper also promotes the accumulation of transcripts encoding a metallothionein designated Zym1. The Sty1 pathway is required for maximal zym1 expression but is not obligatory for zinc perception. The targeted disruption of zym1 impaired cadmium tolerance but only slightly impaired zinc tolerance, whereas zym1 overexpression substantially rescued zinc hypersensitivity of zhf cells. Four equivalents of zinc were displaced from Zym1 by up to 12 equivalents of p-(hydroxymercuri)phenylsulphonate. Zym1 thiols react rapidly with 5,5'-dithiobis-(2-nitrobenzoic acid) compared with bacterial zinc metallothionein (6.8 and 0.2 × 104 s1, respectively). Zym1 is unlike known fungal metallothioneins that are induced by and sequester copper but not zinc. Less zinc but normal cadmium was accumulated by zym1, consistent with zinc sequestration by Zym1 in vivo