FIG 6.
GCDCA effects on HBGA binding. (A) Superposition of GII.12 P domain B-trisaccharide complex (light gray) (PDB accession number 3R6K) and GII.1 P domain apo (black) (PDB accession number 4ROX) structures onto the GII.1 P domain GCDCA (cyan and orange) complex structure. The GII.1 apo structure loop B (residues 293 to 301) was previously disordered, whereas in the GII.1 GCDCA complex, this loop was stabilized (clear electron density), and the His299 side chain was repositioned. Also, GII.1 loop A (residues 370 to 377) in the GII.1 GCDCA complex was positioned similarly to the equivalent loop in the GII.12 B-trisaccharide complex structure. Importantly, the GII.1 Trp371 and Asp375 side chains were almost identical to the equivalent side-chain residues in the GII.12 B-trisaccharide complex. In this position, together with the other fucose binding residues (i.e., Cys345, Arg346, and Gly438), the GII.1 Asp375 side chain could now interact with the fucose of the HBGAs at this pocket. (B) GII.1 VLPs or GII.10 VLPs were preincubated with 2-fold serially diluted GCDCA (starting dilution of 25 μM). Microtiter plates were coated with PGM, and after washing and blocking, the VLP-GCDCA mixture was added to the wells at final concentrations of 5.0 μg/ml for GII.1 VLPs and 0.5 μg/ml for GII.10. GII.1 and GII.10 VLPs in PBS without GCDCA are shown as a reference. The GII.1 GCDCA-treated VLPs bound to PGM in a dose-dependent manner, while the GII.10 GCDCA-treated VLPs bound at all dilutions. A lower concentration of GII.10 VLPs was required for binding to PGM than for the GII.1 VLPs.