1. Isolation of a Common Receptor for Coxsackie B Viruses and Adenoviruses 2 and 5
2. Development of optimized vectors for gene therapy
3. Coxsackievirus and Adenovirus Receptor Amino-Terminal Immunoglobulin V-Related Domain Binds Adenovirus Type 2 and Fiber Knob from Adenovirus Type 12
4. The knob fiber protein (Ad12 knob) and the NH 2 -terminal fragment (residues 22 to 125) of the cellular receptor (CAR D1) were expressed in E. coli and purified as described previously (3). Purified proteins were proteolysed separately with trypsin (10 mg/ml). The 1:3 (trimeric knob: CAR D1) complex was formed at room temperature and purified by anion exchange chromatography. Crystals of Ad12 knob were grown at room temperature with the sitting drop vapor diffusion method from an Ad12 knob solution of 20 mg/ml suspended over a reservoir of 26% polyethylene glycol (PEG) 3350. Showers of small poorly ordered crystals grew over the course of a week and were used to seed a 10-μl drop containing equal volumes of Ad12 knob and 26% PEG 3350 over a reservoir of 26% PEG 3350. Typically crystals grew overnight as rhombohedral plates (0.5 mm by 0.5 mm by 0.2 mm). They were flash cooled at 99 K with 50% PEG 3350 as a cryoprotectant. Crystals of the complex were grown at room temperature with the sitting drop vapor diffusion method from 0.9 M ammonium sulfate in 100 mM MES (pH 6.2). Mercury was introduced into the Ad12 knob–CAR D1 complex by soaking a single CAR D1-knob complex crystal in 10 mM thimerosal for 6 hours. Crystals were flash cooled at 99 K with 50% ethylene glycol as a cryoprotectant. Data were processed with the HKL Program Suite (22). The structure of Ad12 knob was solved by molecular replacement (23) with a monomer of Ad5 knob [Protein Data Bank (PDB) accession number 1KNB.PDB] as a search model. Six monomers were placed and their positions were refined with rigid body refinement. Simulated annealing protocols in CNS (24) with the use of tight NCS restraints were punctuated by rounds of model building. The refinement statistics are shown in Table 1. The structure of the Ad12 knob–CAR D1 complex was determined with a combination of single isomorphous replacement (SIR) solvent flattening and molecular replacement. The refined structure of the Ad12 knob monomer was used as a search model in molecular replacement. A single clear solution was found corresponding to a monomer in the asymmetric unit such that the biological three-fold axis was coincident with the crystallographic axis. The heavy atom position was determined by visual inspection of a difference map with model phases and its position was refined with MLPHARE (23). Phase combination with the use of the Ad12 knob structure and the experimental SIR phases followed by solvent flattening with the program DM (23) resulted in a map with a mean figure of merit (FOM) of 0.74. The structure was refined with CNS punctuated by rounds of model building.
5. Crystal structure of the receptor-binding domain of adenovirus type 5 fiberprotein at 1.7 Å resolution