Acquisition of ionic copper by the bacterial outer membrane protein OprC through a novel binding site.

Satya Prathyusha Bhamidimarri,Sandra Soderholm,Arnaud Baslé,Dirk Bumann,Tessa R Young,Muralidharan Shanmugam,Bert Van Den Berg,Ann M Stock

doi:10.1371/journal.pbio.3001446

Satya Prathyusha Bhamidimarri, Sandra Soderholm + Show 6 more

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https://doi.org/10.1371/journal.pbio.3001446

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Abstract

Copper, while toxic in excess, is an essential micronutrient in all kingdoms of life due to its essential role in the structure and function of many proteins. Proteins mediating ionic copper import have been characterised in detail for eukaryotes, but much less so for prokaryotes. In particular, it is still unclear whether and how gram-negative bacteria acquire ionic copper. Here, we show that Pseudomonas aeruginosa OprC is an outer membrane, TonB-dependent transporter that is conserved in many Proteobacteria and which mediates acquisition of both reduced and oxidised ionic copper via an unprecedented CxxxM-HxM metal binding site. Crystal structures of wild-type and mutant OprC variants with silver and copper suggest that acquisition of Cu(I) occurs via a surface-exposed "methionine track" leading towards the principal metal binding site. Together with whole-cell copper quantitation and quantitative proteomics in a murine lung infection model, our data identify OprC as an abundant component of bacterial copper biology that may enable copper acquisition under a wide range of conditions.

Highlights

Metals fulfil cellular functions that cannot be met by organic molecules and are indispensable for the biochemistry of life in all organisms
The CxxxM-HxM configuration, which coordinates the copper in a tetrahedral manner (Fig 1G and 1H), is highly unusual and has, to our knowledge, not been observed before in copper homeostasis proteins
Our data suggest that the TonB-dependent transporters (TBDTs) OprC binds both Cu(I) and Cu(II) very tightly at an unusual CxxxM-HxM binding site that becomes solvent excluded upon metal binding, kinetically trapping the metal and precluding determination of metal binding affinities

Summary

Introduction

Metals fulfil cellular functions that cannot be met by organic molecules and are indispensable for the biochemistry of life in all organisms. Copper is the third-most abundant transition metal in biological systems after iron and zinc. It has key roles as structural component of proteins or catalytic cofactor for enzymes [1], most notably associated with the biology of oxygen and in electron transfer. An excess of copper can be deleterious due to its ability to catalyse production of hydroxyl radicals [2,3]. Excessive copper may disrupt protein structure by interaction with the polypeptide backbone, or via replacement of native metal cofactors from proteins, abolishing enzymatic activities via mismetallation [1,4,5]. Cellular copper levels and availability must be tightly controlled.

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