The molecular basis for Pompe disease revealed by the structure of human acid a-glucosidase

Derrick Deming; Karen Lee; Tracey McSherry; Ronnie R. Wei; Tim Edmunds; Scott C. Garman

Proteolytic treatment prior to crystallization mimics proteolytic maturation in the late endosome 124 and lysosome. Electron density is missing for loops 116-122, 199-205, and 782-792. 125 Presumably, the flexible loops in the intact protein limited the resolution of the intact GAA 126 crystals. After cleavage of the three loops, the remaining fragments remain associated by non- Active site and ligand binding 136 The active site of GAA is an acidic pocket at the base of a conical funnel lined by hydrophobic 137 residues in the (β/α) 8 barrel catalytic domain (Fig. 1, Fig. 2, and Fig. 2 supplement A) . The To better understand substrate recognition in GAA, we determined complexes of the enzyme 155 with isomaltose and maltotriose (di-and tri-saccharide fragments of glycogen) at 2.0 and 1.9Å 156 resolution, respectively. The structures show glucose present in the active site with no clear proximity of active site residues D282, M519, and R600, β-linked substituents beyond a 161 hydroxyl are sterically blocked, and thus we conclude that the β-glucose appears in the active 162 site after cleavage of α-linked substrate and subsequent ring opening of the α-glucose product. 163 Residues D404, D616, and H674 confer glucose substrate specificity by hydrogen bonding to 164 hydroxyls on the ligand (Fig. 2) . Hydrophobic residues W376, L405, I441, W481, W516, M519, 165 W613 and F649 contribute to active site architecture and substrate binding. We also determined the complex of GAA bound to 1-deoxynojirimycin (DNJ) (Fig. 2) shallow cleft in the N1 domain 34Å away from the active site ( Fig. 3 and Fig. 3 supplement A) . 176 In the second site, the non-reducing ends of isomaltose and maltotriose bind identically, with 177 hydrogen bonds between the glucose hydroxyls and the side chain of D91 and the main chain per side chain atom possessing less than 2 Å 2 , between 2-9 Å 2 , and above 9 were considered 249 buried, partially buried or exposed respectively. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/212837 doi: bioRxiv preprint . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/212837 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/212837 doi: bioRxiv preprint