Springer Semin. protein A (SpA),7 for instance, binds immunoglobulin G Fc fragment, and it interacts with certain Fab fragments, thus characterizing SpA as a B-cell superantigen (2, 3). The Fc binding capacity of SpA has also been found to counteract the innate immune defenses of the host by interfering with the activation of the classical pathway of the complement system (4). More recently a group of small excreted proteins has been discovered that also aid in evading complement-mediated bacterial clearance (5-8). MT-7716 hydrochloride The discovery of new evasion molecules, and understanding the molecular basis of the mode of action of these molecules, not only leads to a better knowledge of their role in the pathophysiology of bacterial infections but is also the first step in their possible exploitation as anti-inflammatory disease therapeutics. In addition to SpA, a second staphylococcal immunoglobulin-binding protein, Sbi, has been identified (9) that occurs in many strains (including methicillin-sensitive and -resistant strains). Sbi is a 436-amino acid protein that contains one functional immunoglobulin-binding domain and a second predicted immunoglobulin-binding motif, both with sequence similarity to the five immunoglobulin-binding repeats (E, A, B, C, and D) of SpA (see Fig. 1and in and domain in and and gene constructs were amplified by PCR using strain Mu50 genomic DNA as a template. The following oligonucleotide primers were used for strains BL21(DE3), BL21(DE3)-Star, or Rosetta. Freshly transformed cells were grown in a shaker at 37 C in Luria Bertani broth (LB), containing ampicillin, until they reached an extinction of 0.6 at 600 nm. Isopropyl for 15 min and the supernatant filtered through a 0.45-= 1.5 ?, covering the momentum transfer range 0.013 < < 0.45 ?-1 (= 4sin(where 2is the scattering angle). Prior to data collection, dynamic light scattering analysis was used to ensure the monodispersity of the protein samples. To check for radiation damage, two successive 2-min exposures taken on the same sample were compared; no radiation effects were observed. The data were processed using standard procedures and extrapolated to zero solute concentration using the program package PRIMUS (15). The forward scattering < 1.3/model of Sbi was constructed using the program DAMMIN (18), which represents the protein by an assembly of densely packed beads. Simulated annealing was employed to build a compact interconnected configuration of beads inside a sphere with the diameter is the number of experimental points; is a scaling factor, and model (bead model of Sbi-E determined by DAMMIN (18). The four major globular domains are depicted with roman numerals. A homology model of Sbi domains I and II (shown in and representations) is superimposed onto the SAXS structure. Serum Affinity Pulldown Assay Purified Rabbit polyclonal to Caspase 10 Sbi-I, Sbi-II, Sbi-III-IV, Sbi-III, and Sbi-IV were each covalently coupled to a 1-ml NHS-activated Sepharose high performance column (Amersham Biosciences) according to the manufacturers instructions. After equilibration with phosphate-buffered saline, 5 ml of MT-7716 hydrochloride human serum (Cambrex) and serum from calf, cow, chicken, goat, horse, mouse, rabbit, or sheep (Sigma) was applied to the column. Columns were washed with phosphate-buffered saline, and bound proteins were eluted with a 0-1 m NaCl gradient over 10 ml. The binding of serum proteins by bait proteins Sbi-III-IV, Sbi-III, and Sbi-IV was analyzed using SDS-PAGE, using immobilized Sbi-I as a negative control. Size exclusion chromatography, with a Superdex-200 gel filtration column (Amersham Biosciences), was used for further purification of the bound fragments. Trypsinized protein fragments from excised gel slices MT-7716 hydrochloride were analyzed by MALDI-TOF. Protein-Lynx software was used for protein identification. Surface Plasmon Resonance (SPR) Complement C3/C3 derivative binding experiments were performed on a BIACore 3000 Instrument (BIACore, Life Sciences), which has four flow cells. Each experiment was run in 10 mm HEPES, MT-7716 hydrochloride pH 7.2, 3 mm EDTA, 0.01% surfactant P20 with either physiological salt (150 mm NaCl) or half-physiological salt (75 mm NaCl) at a flow rate of 20 as described previously (26, 27). C3a was obtained from Calbiochem. All complement proteins used as analytes in SPR experiments were exchanged into the SPR running buffer, and separated from any minor oligomeric species formed during storage, by FPLC gel filtration on Superose 6 (Superdex 200 for C3dg and C4Bdg) within 24 h of use. Complement Activity Assays The Wielisa total complement.