Article Figures & Data
Figures
- FIG. 1.
Structure of S. aureus pI 258 CadC. (A) Ribbon diagram of the CadC dimer with secondary structural units (N-α1-α2-α3-β1-α4-α5-β2-β3-α6-C). All figures were created with MolScript (17), Raster3D (19), and PyMOL (9). The two zinc ions are shown as purple spheres. (B) To illustrate the location of the type 2 metal binding sites, the dimer was rotated 90° relative to the orientation above. (C) Sequence alignment of CadC with SmtB and ArsR. The residues that comprise the metal binding sites are shown in red. The numbers are for the CadC type 1 and type 2 metal binding sites. Each site is composed of two residues from one monomer (no “prime”) and two residues from the other monomer (designated “prime”). The CadC secondary-structure elements are indicated above the sequence (cylinders, α-helices; rectangle, β-strands).
- FIG. 2.
Location of the type 1 metal binding sites. (A) The type 1 metal binding site is located at the interface of α3 and α4 from one monomer (magenta) and α1′ from the other monomer (green). Residues at the dimer interface are labeled. (B) The vicinity of the type 1 metal binding site. The 2Fo-Fc simulated annealing omit electron density map at the 1σ level is shown for residues 58 to 61 in helix α4 and residues 11 to 13 from the other monomer. A hydrogen bond is formed between the hydroxyl of Tyr-12 and the thiol of Cys-58. (C) Molecules 1, 2, and 4 of SeMet CadC structure can be traced up to Cys-7. The 2Fo-Fc simulated annealing omit electron density map at the 1σ level is shown for residues 7 to 10 of molecule 2. (D) The N termini of molecules 2 and 4 of the SeMet CadC structure are superimposed on molecule 1, showing that, without metal ions bound to the type 1 site, Cys-7 can assume multiple orientations. The side chains of Cys-7, Cys-58′, and Cys-60′ are shown.
- FIG. 3.
Location of the type 2 metal binding sites. The two type 2 metal binding sites in each monomer are formed at the interface of the α6 and α6′ helices in each dimer. The asymmetry in the distribution of zinc is shown for the two dimers in the asymmetric unit. (A) Dimera has zinc (purple spheres) bound at both ends of the dimer interface via side chain ligands arranged tetrahedrally (donated by the side chains of Asp-101 and His-103 of one monomer [green] and His-114 and Glu-117 of the other monomer [magenta]). (B) Dimerb has only a single bound zinc, because the second type 2 site is more disordered, and Glu117 is not visible in the structure.
- FIG. 4.
Comparison of a filled and an empty type 2 metal binding site. (A and C) Close-ups of the type 2 metal binding sites (shown is the 2Fo-Fc simulated annealing omit electron density map contoured at the 1σ level) with and without zinc (purple sphere), respectively. (B) Superposition of the empty (in dimerb) and filled (in dimera) zinc sites shows how the backbone and side chain atoms of the histidine and aspartate pairs differ. His-114 is the last residue that can be seen in dimerb. The filled site is shown as a ball-and-stick diagram, and the empty site as bonds.
- FIG. 5.
Comparison of the structures of CadC and SmtB. Stereo view of superposition of the CadC (magenta) and SmtB (gray) monomers. CadC and SmtB were overlapped using only helices 2, 3, and 6 (RMSD = 1.25 Å). The positions of Ala-71 of CadC and Ser-74 of SmtB are shown.
- FIG. 6.
Model for the evolutionary history of type 1 and type 2 metal binding sites. The type 1 metal binding sites in ArsR are proposed to be the most ancient, because each site requires only two residues (Cys-32 and Cys-34) from a single subunit of the homodimer. In CadC, the equivalents of these two cysteine residues, Cys-58 and Cys-60, form more-complicated type 1 sites with residues Cys-7′ and Cys-11′ from the other subunit. CadC also has type 2 sites composed of residues Asp-101 and His-103 from one subunit and residues His-114′ and Glu-117′ from the other subunit. The equivalent type 2 sites in SmtB are composed of residues Asp-104 and His-106 from one subunit and His-117′ and Glu-120′ from the other subunit. SmtB also has residue Cys-14, which corresponds to either CadC residue Cys-7 or CadC residue Cys-11, and residue Cys-61′, which corresponds to CadC residue Cys-58′ and may represent a degenerate type 1 site.
Tables
- TABLE 1.
Data collection
Measurement Value for crystala Native CadC SeMet CadC Space group P41 P43 Cell dimensions (Å) a = b = 116.5; c = 41.8 a = b = 90.2; c = 147.8 Beamline 14-BM-C 14-ID-B Wavelength (Å)b 0.9000 0.9795, 0.9793, 0.9563 Resolution (Å) 1.9 2.5, 2.5, 2.5 No. of unique reflections 40,691 40,682 40,488 40,784 Completeness (%) 95.6 (99.6)c 98.8, 98.7, 98.8 I/σ 28.0 (8.0) 26.3, 28.0, 26.0 Redundancy 3.5 (3.4) 4.0, 4.0, 4.0 R sym d 0.045 (0.225) 0.047, 0.044, 0.050 ↵ a Mean figure of merit (before/after density modification), 0.35/0.76.
↵ b Data in the SeMet CadC column were collected at three different wavelengths: peak, inflection point, and high-energy remote.
↵ c Last shell value is in parentheses.
↵ d R sym = Σhkl(Σi|Ihkl,i − <Ihkl>|)/Σhkl,i<Ihkl>, where Ihkl,i is the intensity of an individual measurement of the reflection with Miller indices h, k, and l, and <Ihkl> is the mean intensity of that reflection.
- TABLE 2.
Refinement statistics
Measurement Value Resolution (Å) 50-1.9 (1.95-1.9) I/σ cutoff 0 No. of residues 404 No. of ions 3 No. of protein atoms 3,152 No. of water molecules 470 R/R free (%)a 20.5/26.1 (27.8/32.9) RMSDs Bond (Å) 0.019 Angle (°) 1.77 Bonded B-factors (Å2), main chain/side chain 1.26/3.39 ↵ a Last shell value is in parentheses.
