Tying Up a Loose End: On the Role of the C‐Terminal CCHHRAG Fragment of the Metalloregulator CueR

Figures

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  Figure 1. Structural models of the C‐terminal metal binding region of WT‐CueR and ▵7C‐CueR. One monomer of the protein homodimer is shown in cyan and the other in grey. Cysteine residues are shown in purple (with sulfur atoms in yellow). The structural model (including the C‐terminal fragment which is disordered in the crystal structure) was prepared in PyMOL on the basis of the AgI‐bound form of CueR (PDB: 1Q06). [8] The complete amino acid sequences of WT‐CueR and ▵7C‐CueR are included in the Supporting Information (Figure S1).

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  Figure 2. Deletion of the seven C‐terminal residues significantly affects the stability of the AgI binding site of CueR, but the overall fold is conserved. Panels A–B: CD spectra of WT (A) and Δ7C‐CueR (B) at increasing AgI : protein monomer ratios. The upper figures are ellipticity traces at representative wavelength values selected near the maxima and minima of the CD curves. The solid lines in panel B represent the fit of data, see the text. c WT‐CueR=c Δ7C‐CueR=20 μM. Panels C–D: Deconvoluted ESI‐MS data for WT‐CueR (C), and Δ7C‐CueR (D) with increasing amounts of AgI. c WT‐CueR=c Δ7C‐CueR=20 μM.

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  Figure 3. Metal site structure of WT‐ and Δ7C‐CueR probed by 111Ag PAC spectroscopy (spectra for WT‐CueR are from Ref. [18]). Measured and fitted A 2 G 2(t) (left) and Fourier transformed (right) data and fit for the WT‐ and Δ7C‐CueR proteins recorded at −196 and 1 °C, pH=8.0, c protein=11 μM, c AgI=5.5 μM, w/w%sucrose=55 %.

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  Figure 4. Possible interactions of the metal ion binding loop (residues 112–120) with the seven C‐terminal amino acid residues. A: Structural data from the AgI‐CueR (activator) complex with DNA (PDB code 4WLW, in all other published structures the entire C‐terminal seven AA fragment is disordered). B: Backbone trace of the CuI‐CueR‐DNA complex with the last seven amino acid residues included (the last five AAs were added as an alpha‐helical extension, see Figure S6) at t=0 of an MD simulation, see the Supporting Movie. The structure at t=0 (transparent) is superimposed on the final (t=1 μs) structure (opaque) from the MD simulation (Supporting Movie), and the movement of the C‐terminus is indicated by the black arrow. C: At t=1 μs, in one of the two dimers, the C‐terminus has established contacts (between Arg133 and Asp119 and Glu124) with residues near the metal binding Cys120. D: Amino acid sequence alignment of CueR, CupR, GolS and HmrR from different bacteria. Cysteines, acidic (D/E) and basic (R/K) residues are highlighted in yellow, red and blue, respectively.