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Protein-folding location can regulate manganese-binding versus copper- or zinc-binding

Abstract

Metals are needed by at least one-quarter of all proteins1,2. Although metallochaperones3,4,5,6,7,8 insert the correct metal into some proteins, they have not been found for the vast majority, and the view is that most metalloproteins acquire their metals directly from cellular pools. However, some metals form more stable complexes with proteins than do others. For instance, as described in the Irving–Williams series9, Cu2+ and Zn2+ typically form more stable complexes than Mn2+. Thus it is unclear what cellular mechanisms manage metal acquisition by most nascent proteins. To investigate this question, we identified the most abundant Cu2+-protein, CucA (Cu2+-cupin A), and the most abundant Mn2+-protein, MncA (Mn2+-cupin A), in the periplasm of the cyanobacterium Synechocystis PCC 6803. Each of these newly identified proteins binds its respective metal via identical ligands within a cupin fold. Consistent with the Irving–Williams series, MncA only binds Mn2+ after folding in solutions containing at least a 104 times molar excess of Mn2+ over Cu2+ or Zn2+. However once MncA has bound Mn2+, the metal does not exchange with Cu2+. MncA and CucA have signal peptides for different export pathways into the periplasm, Tat and Sec respectively. Export by the Tat pathway allows MncA to fold in the cytoplasm, which contains only tightly bound copper or Zn2+ (refs 10–12) but micromolar Mn2+ (ref. 13). In contrast, CucA folds in the periplasm to acquire Cu2+. These results reveal a mechanism whereby the compartment in which a protein folds overrides its binding preference to control its metal content. They explain why the cytoplasm must contain only tightly bound and buffered copper and Zn2+.

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Figure 1: CucA is the principal periplasmic Cu 2+ -protein.
Figure 2: MncA is the principal periplasmic Mn 2+ -protein.
Figure 3: MncA and CucA have similar metal-sites.
Figure 4: MncA prefers Cu2+ but entraps Mn2+.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The crystallographic coordinates of MncA have been deposited in the Protein Data Bank under accession number 2VQA.

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Acknowledgements

This work was supported by the BBSRC PMS committee (BBS/B/02576 and BB/E001688/1). B.R. was supported by J. Gitlin and the Children's Discovery Institute. M.J.B. is a Royal Society University Research Fellow. J. Kotz provided advice on the draft manuscript. We thank T. Palmer, B. Ize and G. Buchanan for donating E. coli BL21 tat deletion strains and for advice.

Author Contributions S.T. and K.J.W. contributed equally, doing most of the laboratory work and playing a part in planning and data interpretation. S.J.F. and M.J.B. collected and interpreted the X-ray diffraction data. C.D. and K.S. collected and interpreted the EPR data. B.R. did the metal exchange experiment. J.G. did the mass fingerprinting, C.B. generated the algorithm for principal component analysis and T.R.C. did the confocal microscopy. N.J.R. managed the programme, and had overall responsibility for data interpretation and writing the manuscript.

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Correspondence to Nigel J. Robinson.

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This file contains Supplementary Tables 1 and 2, Supplementary Figures S1-S16 and a Supplementary Discussion with Supplementary References (PDF 2980 kb)

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Tottey, S., Waldron, K., Firbank, S. et al. Protein-folding location can regulate manganese-binding versus copper- or zinc-binding. Nature 455, 1138–1142 (2008). https://doi.org/10.1038/nature07340

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