N1574-GlcNAc intradomain interactions. (A) Part of the crystal structures of the regions of the VWF A2 domain (3ZQK, I1535-N1577) and VWF A1 domain (1AUQ, V1314-S1356) containing the β2-β3 hairpin and α2-α3 loop. (B) Alignment of the VWF A2 and VWF A1 domain (adapted from Zhang et al¹⁷ ) regions containing important glycosylation site and potential hydrophobic interaction sites. Amino acids forming secondary structures in gray and key residues displayed in larger bold font. (C) VWF A2 domain fragments containing mutation(s) to glycosylation site, a candidate hydrophobic interaction partner, or combinations of these were analyzed by SDS-PAGE on a 4% to 12% Bis-Tris gel followed by silver staining after expression and purification to determine changes in MW caused by alteration of the surface glycan structure (the N1515Q/N1574Q/Y1544D variant is labeled as Y1544D/NQ/NQ).