Structural Analysis of LasR Ligand Complexes

Recombinant 6xHis-LasR LBD and 6xHis-LasR LBD T75V/Y93F/A127W proteins bound to 3OC₁₂HSL, mBTL, BB0020, and BB0126 were expressed in E. coli BL21 (DE3) cells (Invitrogen). The strains were supplied with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and 100 μM of test compound and grown for 4 h at 25°C. The cells were harvested via centrifugation at 16,100 × g for 15 min. LasR complexes were purified as previously described for LasR LBD:3OC₁₂HSL using Ni-NTA affinity columns (Qiagen) followed by size exclusion chromatography (GE Healthcare)^{38 (link)}. 6xHis-LasR LBD and 6xHis-LasR LBD T75V/Y93F/A127W proteins complexed with mBTL, BB0020, and BB0126 were crystallized by the hanging drop diffusion method. Diffraction data were processed using the HKL-300 software package^{63 (link)}. The structures were solved using Phaser in Phenix by molecular replacement, with the structure of LasR LBD:3OC₁₂HSL used as the search model^{39 (link), 64 (link), 65 (link)}. Model building was performed using Coot^{66 (link)} and further refinement was accomplished using Phenix^{64 (link)}.