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Affinity gradients drive copper to cellular destinations

Abstract

Copper is an essential trace element for eukaryotes and most prokaryotes1. However, intracellular free copper must be strictly limited because of its toxic side effects. Complex systems for copper trafficking evolved to satisfy cellular requirements while minimizing toxicity2. The factors driving the copper transfer between protein partners along cellular copper routes are, however, not fully rationalized. Until now, inconsistent, scattered and incomparable data on the copper-binding affinities of copper proteins have been reported. Here we determine, through a unified electrospray ionization mass spectrometry (ESI-MS)-based strategy, in an environment that mimics the cellular redox milieu, the apparent Cu(I)-binding affinities for a representative set of intracellular copper proteins involved in enzymatic redox catalysis, in copper trafficking to and within various cellular compartments, and in copper storage. The resulting thermodynamic data show that copper is drawn to the enzymes that require it by passing from one copper protein site to another, exploiting gradients of increasing copper-binding affinity. This result complements the finding that fast copper-transfer pathways require metal-mediated protein–protein interactions and therefore protein–protein specific recognition3. Together with Cu,Zn-SOD1, metallothioneins have the highest affinity for copper(I), and may play special roles in the regulation of cellular copper distribution; however, for kinetic reasons they cannot demetallate copper enzymes. Our study provides the thermodynamic basis for the kinetic processes that lead to the distribution of cellular copper.

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Figure 1: Determination of the relative Cu(I)-binding affinity of GSH and DETC.
Figure 2: Demetallation of Cu 1 Sco1 and the Cu A site of CcO by apo-MT-2 as followed by ESI-MS.
Figure 3: Free-energy gradients of cellular Cu(I) delivery pathways.

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Acknowledgements

This work was supported by grants from the Estonian Science Foundation project 7191, the Estonian Ministry of Education and Research (grant SF0140055s08), by the SPINE-II-COMPLEXES contract LSHG-CT-2006-031220, by the FIRB PROTEOMICA MIUR contract RBRN07BMCT and by a World Federation of Scientists scholarship to K.Z. We thank K. Saar for preparative work with rat mitochondrial fractions and for measuring the enzymatic activity of CcO, and C. Massagni for preparing the CCS expression plasmid.

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L.B., I.B., S.C.-B., K.Z., P.P. designed the research; K.Z., T.K. and P.P. performed the research. All authors analysed the data and contributed to the writing of the paper.

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Correspondence to Ivano Bertini or Peep Palumaa.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures S1-S18 with legends. Figure S1 is a simple schematic summarizing the main findings in the paper. Figures S2-18 report ESI-MS spectra performed on the copper proteins, the fittings of the ESI-MS data to obtain the apparent dissociation constants and kinetics data on demetallation processes of copper enzymes. (PDF 3420 kb)

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Banci, L., Bertini, I., Ciofi-Baffoni, S. et al. Affinity gradients drive copper to cellular destinations. Nature 465, 645–648 (2010). https://doi.org/10.1038/nature09018

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