Exploring the role of the various methionine residues in the Escherichia coli CusB adapter protein

The dissemination of resistant pathogenic microbes has become one of the most challenging problems that modern medicine has faced. Developing novel drugs based on new molecular targets that previously were not targeted, is therefore the highest priority in antibiotics research. One approach that has been recently suggested is to inhibit copper transporters in prokaryotic systems. Copper is required for many biological pathways, but sometimes it can harm the cell. Pathogenic systems have a highly sophisticated copper-regulation network; therefore, a better understanding of how this network operates at the molecular level should assist in developing the next generation of antibiotics. The CusB protein is part of the CusCBA periplasmic Cu(I) efflux system in Gram-negative bacteria, and was recently reported to play a key role in the functioning of the whole CusCBA system, in which conformational changes as well as the assembly/disassembly process control the opening of the transporter. More knowledge of the underlying mechanism is needed to attain a full understanding of CusB functioning, which is associated with targeting specific and crucial residues in CusB. Here, we combine in-vitro structural measurements, which use EPR spectroscopy and UV-Vis measurements, with cell experiments to explore the role of the various methionine residues in CusB. We targeted two methionine residues (M227 and M241) that are essential for the proper functioning of CusB.


Addition of Cu(I) ion to protein solution
Cu(I) (tetrakis (acetonitrile) copper(I) hexafluorophosphate (Sigma-Aldrich, St. Louis, MO, USA)) was added to the protein solution under nitrogen gas to maintain inert anaerobic conditions. No Cu(II) EPR signal was observed at any time. A Cu(I):CusB ratio of 3:1 was used for all EPR measurements.

Electron Paramagnetic Resonance (EPR) spectroscopy
A constant-time four-pulse double electron-electron resonance (DEER) experiment with pulse sequence π/2(νobs)-τ1-π(νobs)-t′−π(νpump)-(τ1 + τ2 − t′)-π(νobs)-τ2(νobs)-τ2-echo was performed at (50 ± 0.5 K) on a Q-band Elexsys E580 equipped with a 2-mm probe head; bandwidth, 220 MHz. A two-step phase cycle was applied to the first pulse. The echo was measured as a function of t′, and τ2 was kept constant to eliminate relaxation effects. The pump pulse frequency was set to the maximum of the EPR spectrum and the observer pulse frequency was set 60 MHz higher than that of the pump pulse. The observer π/2 and π pulses, as well as the π pump pulse, had durations of 40 ns; the dwell time was 20 ns. The observer frequency was 33.82 GHz. The power of the 40-ns π-pulse was 20.0 mW. The parameter τ1 was set to 200 ns, and τ2 was set to 1200 ns. The repetition time was set to 12 ms, and 30 shots per point were applied. The samples were measured in 1.6-mm quartz capillary tubes (Wilmad-Labglass, Vineland, NJ, USA). The data were analyzed with the DeerAnalysis 2016 program and Tikhonov regularization (4,5). The regularization parameter in the L curve was optimized by examining the fit of the time-domain data and was found to be between 30 and 40. The modulation depth for the four-spin system was about 15%.

Determining the Cu(I) dissociation constant
BCA (Bicinchoninic acid disodium salt hydrate; Sigma-Aldrich, St. Louis, MO, USA) and Cu(I) tetrakis (acetonitrile) copper(I) hexafluorophosphate (Sigma-Aldrich) solution was titrated with CusB and their UV-VIS spectra were recorded with a Chirascan spectrometer (Applied Photophysics, Surrey, UK) at RT. Measurements were carried out in a cell with a 1-mm optical path length. The Cu(I) concentration was 100 µM and the BCA concentration was 220 µM to ensure the absence of free Cu(I) from the solution. Spectra were recorded from 200 to 800 nm with a step size and bandwidth of 0.5 nm. The spectra were baselined according to the absorption value at 800 nm, which is zero.
To correlate between the concentration of BCA-Cu(I) and its absorbance at 562 nm, UV-Vis spectra were recorded for 200 µM BCA titrated with Cu(I)(6) (Fig B).  Tables A-C present the three repetitions for KD values calculated for ΔCusB-mutants

Circular Dichroism (CD) measurements
In order to verify that CusB mutants have no effect on the secondary structure of the protein, we used Circular dichroism (CD) measurements. CD measurements were conducted using a Chirascan spectrometer (Applied Photophysics). Measurements were performed in a 1-cm optical path length cell, and the spectra were recorded from 270 to 190 nm with a step size and a bandwidth of 0.5 nm. The CD signal was averaged for 10 s every 2 nm, 3 scans per sample.  The CD spectra reveal that upon mutation the secondary structures of the protein is affected by less than 3%.

Western Blot
In order to compare the growth rate for the wt-CusB and its various mutants their protein expression level must be equivalent. WT-CusB and the various mutants were grown during 16 hours in LB medium. Pellets were suspended in SDS sample buffer, separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane using a transfer apparatus according to themanufacturer's protocols (Bio-Rad). After incubation with 3% BSA in TBST (10 mMTris

Generation of a PCR product from the functional cassette flanked with homology arms
The oligonucleotides were suspended in double distilled water at a final concentration of 10 µM.
The PCR reaction was conducted following Table D.
To target the chromosome at the site of choice, it is necessary to incorporate short homology regions into the functional cassette carrying the selectable marker "Sm". This is done by designing two oligonucleotides for use in PCR amplification. Each oligonucleotide consists of two parts: 1. The first part is the homology region (see   1 µL of Red/ET Recombination protein expression plasmid pRed/ET was added to cell pellets.
Cells were transferred into chilled 1 mm electroporation cuvettes.
Cells were transferred into 1 mL LB medium incubated at 30 C for 70 min, shaking at 1000 rpm.
tetracycline (3 µg/mL) + kanamycin (15 μg/ml), and C. kanamycin (15 μg/ml) as control experiments. Plates were incubated at 30 C overnight wrapped in tinfoil. No colonies were found for plates B and C; however, 9 colonies were found on plate A.

Disruption of a chromosomal DNA fragment by the FRT-flanked PGK-gb2-neo cassette:
Colonies were picked from tetracycline plates and control plates and inoculated in a microfuge tube containing 1 mL LB medium + 3 µg/mL tetracycline.  ∆CusB cells were streaked out on petri dishes to obtain single colonies, which were then analyzed by PCR using: 1) Primer A and primer 2 (kit): 5'-CGAGACTAGTGAGACGTGCTAC-3'.

3) Primer A and primer B.
PCR profile: Initial denaturation step 5 min at 98 ºC; thirty cycles: 30 sec at 98 ºC, 30 sec at 57.5 ºC, 120 sec at 72 ºC; final elongation step 10 min at 72 ºC. (See Fig G lanes 1-3 accordingly.) ∆CusB cells, recombinant CusB cells, and BL21 cells (endogenic CusB) were streaked out on a petri dish to obtain single colonies, which were then analyzed by PCR using:   Each experiment was repeated five times under identical conditions. The presented results are for M9 medium unless indicated otherwise.

Growth rate experiments in rich medium
To determine the effect of the medium on ∆CusB-mutant cell growth, LB broth was used as a rich medium for comparison of the growth rates of native E. coli cells, and of ∆CusB and ∆CusBmutants. This set of experiments was also conducted to observe the growth-rate dependence on the concentrations of Cu(II).

Cell viability
Cells were dyed using the L7007 LIVE/DEAD Bacterial Viability kit (Molecular Probes, Oregon, USA); cells were grown in M9 medium. Images were acquired on a Leica SP8 confocal microscope, running LASX acquisition software. The magnification used was a 63 X 1.4 NA objective. Cells were counted with ImageJ software, with a script fully described below. Error calculations were based on three repetitions (Standard deviation), with each experiment scanned at nine different tilesall together 27 repetitions.