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 plasmidencoded 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 2.0 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 inductively coupled plasma mass spectrometry and molecular dynamics (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 demonstrate 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 Ping Li and Niloofar Nayeri contributed equally.

The intracellular helical bundle of human glucose transporter GLUT4 is important for complex formation with ASPL

“…Far dot western blotting was carried out as previously described with minor modifications [ 23 ]. Briefly, 1 μL of ASPL‐C and PcoC (copper transporter expressed in E. coli [ 24 ], as a control) containing 100 pmol protein were spotted onto nitrocellulose membranes, before blocking in 2% fish gelatin and 1% BSA for 40 min. GLUT4 and AQP7 (as a control) lysates were prepared as described above and applied to membrane at room temperature for 40 min before washing twice with PBS supplemented with 0.1% DM.…”

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.…”

“…The coverslip with the double film consisting of protein–agarose gel covered with lipids was placed inside a Sykes–Moore chamber and covered with 400 μL of swelling buffer; in order to avoid interference between buffers and metal ions, many commonly used buffers had to be avoided . In particular, Tris in combination with Zn under prolonged contact may cause unspecific PcoB degradation . We therefore chose 20 mM MOPS with pH adjusted to 7.4 with NaOH, unless mentioned otherwise.…”

“…Specifically, we selected the outer membrane protein PcoB from Escherichia coli that serves as a Cu-specific porin in a number of bacteria. 5 , 19 − 21 To follow the changes in the Cu 2+ concentrations, we employed a membrane-impermeable Zn sensor FluoZin-3. 8 In a complex with Zn 2+ ions, FluoZin-3 exhibits strong and stable fluorescence, with 494 nm excitation and 516 nm emission maxima.…”

“…Building on our previous successful experiences with reconstitution of membrane proteins into artificial vesicles, we employed the method to study copper flux in real time. − As a proof of concept, we designed a setup combining a membrane protein metal-ion channel reconstituted into GUVs with a fluorescent sensor to detect Cu 2+ flux. Specifically, we selected the outer membrane protein PcoB from Escherichia coli that serves as a Cu-specific porin in a number of bacteria. ,− To follow the changes in the Cu 2+ concentrations, we employed a membrane-impermeable Zn sensor FluoZin-3 . In a complex with Zn 2+ ions, FluoZin-3 exhibits strong and stable fluorescence, with 494 nm excitation and 516 nm emission maxima.…”