Sodium Iodide

GluCl_cryst was expressed from baculovirus-infected Sf9 cells and purified by metal ion affinity chromatography. The Fab complex was isolated by size-exclusion chromatography. The GluCl_cryst-Fab complex was concentrated to 1-2 mg/mL and supplemented with synthetic lipids and ivermectin. Crystallization was performed by hanging drop vapor diffusion at 4°C with a precipitating solution containing 21-23% PEG 400, 50 mM sodium citrate pH 4.5 and 70 mM sodium chloride. Cryoprotection was achieved by soaking crystals in precipitant solution supplemented with 30% PEG 400. Additional complexes were obtained by soaking crystals in cryoprotectant containing L-glutamate, picrotoxin or sodium iodide. Diffraction data were indexed, integrated and scaled and the structure solved by molecular replacement using a GLIC-derived homology model of GluCl_cryst and a Fab homology model as search probes. The molecular replacement phases were used to initiate autobuilding and the resulting model was iteratively improved by cycles of manual adjustment and crystallographic refinement. Function of GluCl was examined by two-electrode voltage clamp experiments and by [³H]-L-glutamate saturation and competition binding assays.

Inhibitor APV (from AIDS reagent program) was dissolved in DMSO by vortex-mixing. The mixture was incubated on ice prior to centrifugation to remove any insoluble material. The inhibitor was mixed with 2.2 mg/ml protein in molar ratio of 5:1 in most cases. The two exceptions were: 3.5 mg/ml of PR_I50V was used with inhibitor-protein ratio of 10:1, and 3.7 mg/ml of PR_L90M. The crystallization trials employed the hanging drop method using equal volumes of enzyme-inhibitor and reservoir solution. PR_WT-APV was crystallized from 0.1M MES, pH 5.6, and 0.6–0.8 M sodium chloride. Crystals of PR_V32I-APV and PR_I50V-APV were grown from 0.1M sodium acetate, pH 5.4, 0.4 M and 1.2 M sodium chloride, respectively. PR_I54M-APV crystals were grown from 0.1M sodium acetate, pH 4.6, and 0.67 M sodium chloride. PR_I54V-APV and PR_I84V-APV crystals were grown from 0.1 M sodium acetate, pH 5.4 and 4.0, respectively, and 0.13 M sodium iodide, and PR_L90M-APV crystals from 0.1 M sodium acetate, pH 4.8 and 0.2 M sodium iodide. Single crystals were mounted on fiber loops with 20 to 30 % (v/v) glycerol as cryoprotectant in the reservoir solution. X-ray diffraction data were collected at the SER-CAT beamline of the Advanced Photon Source, Argonne National Laboratories. Diffraction data were integrated, scaled, and merged using the HKL2000 package [54 ]. PR_WT-APV, PR_V32I-APV and PR_I50V-APV were solved by molecular replacement program Phaser [55 (link)] with the protein atoms of structure 2QCI[32 (link)] as the starting model. The other complexes were solved by MOLREP [56 ], using the protein atoms of 2F8G as the starting model [19 (link)]. The crystal structures were refined using SHELX-97 [57 (link)], except that the lower resolution structure of PR_I84V-APV was refined with REFMAC 5.2 [58 (link)]. The diffraction-data precision indicator (DPI) was used for determining the accuracy in the atomic positions [59 ]. The molecular graphics program COOT was used for map display and model building [60 (link)]. Structural figures were made by PyMol [61 ]. The structures were compared by superimposing their C_α atoms and using HIVAGENT [62 ] to calculate the distance between two atoms. The cut-off distances for different interactions were as described in [30 (link)].