Figure 1.
Overall Structure of GrlR with Bound Ligand
(A) Ribbon diagram of monomer. The β-strands and the random coils/turns are depicted in light and dark green colors, respectively. The Triton-X100 molecule is bound in the hydrophobic pore of the eight-stranded β-barrel. The CPK model of the ligand is shown with oxygen and carbon atoms colored in red and grey, respectively.
(B) Ribbon diagram of the dimer. Monomer A is shown in red, monomer B in green. The dimeric interface residues, surface-exposed residues (EDED motif), and the ligand from both monomers are shown in ball and stick representation. This figure was drawn using Molscript and Raster3D [42,43].
Table 1.
Crystallographic Data and Refinement Statistics
Figure 2.
Stereo View of the β-Barrel and the Bound ligand
(A) Stereo view of the Cα trace of GrlR β-barrel shown in green, viewed from the top. The hydrophobic side chains of the residues from the pore region are shown in thick lines. The ball-and-stick representation of the Triton-X100 molecule at the center of the pore is shown. This figure was prepared by using Molscript and Raster3D [42,43].
(B) Simulated annealing Fo-Fc omit map in the pore region of GrlR. The bound triton molecule and all atoms within 2 Å of the triton molecule were omitted prior to refinement. The map contoured at a level of 3σ. This figure was prepared using PyMOL [44].
Figure 3.
The Electrostatic Surface Potential of GrlR Dimer
The surface-exposed (EDED) residues are labeled. It shows a strong negatively charged (red) patch from both EDED motifs of the monomers. The dotted line indicates the dimer interface. Blue represents a positive charge. This figure was prepared using GRASP [20].
Figure 4.
(A) SDS-PAGE gel showing the in vitro pull-down assay to demonstrate the binding of wild-type GrlR and EDED mutants to GrlA. Lane 1, molecular weight marker (MW); lane 2, GST; lane 3, E46A-D47A-E48A-D49A; lane 4, E46A-D47A-E48A; lane 5, wild-type GrlR; lane 6, E46A-D47A; lane 7, E48A; lane 8, D47A. The bands corresponding to GST-GrlA and GrlR were further identified by peptide mass finger printing. The staining was done using coomassie brilliant blue.
(B) SDS-PAGE gel showing the amount of native and mutant GrlR protein used for the in vitro pull-down assay. Lane 1, molecular weight marker; lane 2, E46A-D47A-E48A-D49A; lane 3, wild-type GrlR; lane 4, E46A-D47A-E48A; lane 5, E46A-D47A; lane 6, E48A; lane 7, D47A.
Figure 5.
General Secretion Profile of EHEC EDL933 Harboring pSA10-grlR and Expressing Wild-Type and Mutants of GrlR
Secreted proteins were concentrated from supernatants of bacterial culture grown in DMEM and resolved in 12% SDS-PAGE and stained with coomassie brilliant blue (upper panel). Western blot of EHEC total cell protein against 6His antibody (lower panel).