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. 2012 Aug 28;51(34):6767-75.
doi: 10.1021/bi300596a. Epub 2012 Aug 17.

N-terminal region of CusB is sufficient for metal binding and metal transfer with the metallochaperone CusF

Tiffany D Mealman  1 , Mowei Zhou, Trisiani Affandi, Kelly N Chacón, Mariana E Aranguren, Ninian J Blackburn, Vicki H Wysocki, Megan M McEvoy
Affiliations

N-terminal region of CusB is sufficient for metal binding and metal transfer with the metallochaperone CusF

Tiffany D Mealman et al. Biochemistry. .

Abstract

Gram-negative bacteria, such as Escherichia coli, utilize efflux resistance systems in order to expel toxins from their cells. Heavy-metal resistance is mediated by resistance nodulation cell division (RND)-based efflux pumps composed of a tripartite complex that includes an RND-transporter, an outer-membrane factor (OMF), and a membrane fusion protein (MFP) that spans the periplasmic space. MFPs are necessary for complex assembly and have been hypothesized to play an active role in substrate efflux. Crystal structures of MFPs are available, however incomplete, as large portions of the apparently disordered N- and C-termini are unresolved. Such is the case for CusB, the MFP of the E. coli Cu(I)/Ag(I) efflux pump CusCFBA. In this work, we have investigated the structure and function of the N-terminal region of CusB, which includes the metal-binding site and is missing from previously determined crystal structures. Results from mass spectrometry and X-ray absorption spectroscopy show that the isolated N-terminal 61 residues (CusB-NT) bind metal in a 1:1 stoichiometry with a coordination site composed of M21, M36, and M38, consistent with full-length CusB. NMR spectra show that CusB-NT is mostly disordered in the apo state; however, some slight structure is adopted upon metal binding. Much of the intact protein's function is maintained in this fragment as CusB-NT binds metal in vivo and in vitro, and metal is transferred between the metallochaperone CusF and CusB-NT in vitro. Functional analysis in vivo shows that full-length CusB is necessary in an intact polypeptide for full metal resistance, though CusB-NT alone can contribute partial metal resistance. These findings reinforce the theory that the role of CusB is not only to bind metal but also to play an active role in efflux.

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Figures

Figure 1
Figure 1
Schematic of the periplasmic Cu(I)/Ag(I) efflux pump, CusCFBA: trimeric RND-transporter CusA (orange), trimeric OMF CusC (pink), MFP CusB (green) and metallochaperone CusF (blue).
Figure 2
Figure 2
Overlay of NMR 1H-15N correlation (HSQC) spectra of apo-CusB-NT (red) and Ag(I)-CusB-NT (black).
Figure 3
Figure 3
NanoESI mass spectra of 40 μM apo-CusB-NT (top) and 40 μM Ag(I)-CusB-NT (bottom). Peaks corresponding to apo-CusB-NT and Ag(I)-CusB-NT are labeled with open and filled circles, respectively, along with their corresponding charge states.
Figure 4
Figure 4
X-ray absorption spectroscopy of CusB-NT. Main panel shows the Fourier transform with the EXAFS plotted in the top insert. Black traces are experimental data and red traces are simulated data. The bottom insert shows a comparison of CusB-NT (blue) with the full-length protein (red). The Cu-S(Met) bond length determined from simulation is 2.29 Å for CusB-NT.
Figure 5
Figure 5
NanoESI mass spectra of (a) apo-CusB-NT at 40 μM, (b) Ag(I)-CusF at 40 μM, (c) 1:1 mixture of apo-CusB-NT and Ag(I)-CusF, both at 40 μM, and (d) 1:1 mixture of Apo-CusB-NT-M36I and Ag(I)-CusF. Apo-CusB-NT and Ag(I)-CusB-NT are labeled with open and filled circles respectively. Apo-CusF and Ag(I)-CusF are represented with open and filled triangles respectively. Apo-CusB-NT-M36I is labeled with open squares. Relative abundances of the apo vs. Ag(I)-bound proteins after deconvolution are shown as bar graphs in the column on the right for corresponding mass spectra on the left. The asterisk at m/z 2125 in (c) corresponds to the Ag(I)/CusF/CusB complex.
Figure 6
Figure 6
Functional analysis of E. coli cells expressing different CusB constructs grown on copper-containing LB-agar plates. Copper sulfate concentrations in the media are given on the x-axis and relative growth is scored on the y-axis.
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