PcoB is a defense outer membrane protein that facilitates cellular uptake of copper

Abstract

Copper (Cu) is one of the most abundant trace metals in all organisms, involved in a plethora of cellular processes. Yet elevated concentrations of the element are harmful, and interestingly prokaryotes are more sensitive for environmental Cu stress than humans. Various transport systems are present to maintain intracellular Cu homeostasis, including the prokaryotic plasmid-encoded multiprotein pco operon, which is generally assigned as a defense mechanism against elevated Cu concentrations. Here we structurally and functionally characterize the outer membrane component of the Pco system, PcoB, recovering a 2.2 Å structure, revealing a classical β-barrel architecture. Unexpectedly, we identify a large opening on the extracellular side, linked to a considerably electronegative funnel that becomes narrower towards the periplasm, defining an ion conducting pathway as also supported by metal binding quantification via ICP-MS and MD simulations. However, the structure is partially obstructed towards the periplasmic side, and yet flux is permitted in the presence of a Cu gradient as shown by functional characterization in vitro. Complementary in vivo experiments demonstrated that isolated PcoB confers increased sensitivity towards Cu. Aggregated, our findings indicate that PcoB serves to permit Cu import. Thus, it is possible the Pco system physiologically accumulates Cu in the periplasm as a part of an unorthodox defense mechanism against metal stress. These results point to a previously unrecognized principle of maintaining Cu homeostasis and may as such also assist in the understanding and in efforts towards combatting bacterial infections of Pco-harboring pathogens.

Microfluidic-Derived Detection of Protein-Facilitated Copper Flux Across Lipid Membranes

“…24 In particular, Tris in combination with Zn under prolonged contact may cause unspecific PcoB degradation. 21 We therefore chose 20 mM MOPS with pH adjusted to 7.4 with NaOH, unless mentioned otherwise. 150 mM NaCl or Na 2 SO 4 and 15 mM KCl or K 2 SO 4 were used throughout the experiments.…”