The delivery of copper for thylakoid import observed by NMR.
The thylakoid compartments of plant chloroplasts are a vital destination for copper. Copper is needed to form holo-plastocyanin, which must shuttle electrons between photosystems to convert light into biologically useful chemical energy. Copper can bind tightly to proteins, so it has been hypothesized that copper partitions onto ligand-exchange pathways to reach intracellular locations without inflicting damage en route. The copper metallochaperone Atx1 of chloroplast-related cyanobacteria (ScAtx1) engages in bacterial two-hybrid interactions with N-terminal domains of copper-transporting ATPases CtaA (cell import) and PacS (thylakoid import). Here we visualize copper delivery. The N-terminal domain PacS(N) has a ferredoxin-like fold that forms copper-dependent heterodimers with ScAtx1. Removal of copper, by the addition of the cuprous-ion chelator bathocuproine disulfonate, disrupts this heterodimer, as shown from a reduction of the overall tumbling rate of the protein mixture. The NMR spectral changes of the heterodimer versus the separate proteins reveal that loops 1, 3, and 5 (the carboxyl tail) of the ScAtx1 Cu(I) site switch to an apo-like configuration in the heterodimer. NMR data ((2)J(NH) couplings in the imidazole ring of (15)N ScAtx1 His-61) also show that His-61, bound to copper(I) in [Cu(I)ScAtx1](2), is not coordinated to copper in the heterodimer. A model for the PacS(N)/Cu(I)/ScAtx1 complex is presented. Contact with PacS(N) induces change to the ScAtx1 copper-coordination sphere that drives copper release for thylakoid import. These data also elaborate on the mechanism to keep copper(I) out of the ZiaA(N) ATPase zinc sites.
Banci
Lucia
L
Magnetic Resonance Center (CERM) and Department of Chemistry, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Florence, Italy.
Bertini
Ivano
I
Ciofi-Baffoni
Simone
S
Kandias
Nikolaos G
NG
Robinson
Nigel J
NJ
Spyroulias
Georgios A
GA
Su
Xun-Cheng
XC
Tottey
Stephen
S
Vanarotti
Murugendra
M
D001692:Biological Transport; D003300:Copper / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008968:Molecular Conformation; D020524:Thylakoids / Q000737:chemistry* / Q000378:metabolism*
D008024:Ligands; D003300:Copper