Crystallographic Structure of Human β-Hexosaminidase A: Interpretation of Tay-Sachs Mutations and Loss of GM2 Ganglioside Hydrolysis

Abstract

Lysosomal beta-hexosaminidase A (Hex A) is essential for the degradation of GM2 gangliosides in the central and peripheral nervous system. Accumulation of GM2 leads to severely debilitating neurodegeneration associated with Tay-Sachs disease (TSD), Sandoff disease (SD) and AB variant. Here, we present the X-ray crystallographic structure of Hex A to 2.8 A resolution and the structure of Hex A in complex with NAG-thiazoline, (NGT) to 3.25 A resolution. NGT, a mechanism-based inhibitor, has been shown to act as a chemical chaperone that, to some extent, prevents misfolding of a Hex A mutant associated with adult onset Tay Sachs disease and, as a result, increases the residual activity of Hex A to a level above the critical threshold for disease. The crystal structure of Hex A reveals an alphabeta heterodimer, with each subunit having a functional active site. Only the alpha-subunit active site can hydrolyze GM2 gangliosides due to a flexible loop structure that is removed post-translationally from beta, and to the presence of alphaAsn423 and alphaArg424. The loop structure is involved in binding the GM2 activator protein, while alphaArg424 is critical for binding the carboxylate group of the N-acetyl-neuraminic acid residue of GM2. The beta-subunit lacks these key residues and has betaAsp452 and betaLeu453 in their place; the beta-subunit therefore cleaves only neutral substrates efficiently. Mutations in the alpha-subunit, associated with TSD, and those in the beta-subunit, associated with SD are discussed. The effect of NGT binding in the active site of a mutant Hex A and its effect on protein function is discussed.

Crystal Structure of β-Hexosaminidase B in Complex with Pyrimethamine, a Potential Pharmacological Chaperone

“…PYR was found bound in the active site in slightly different conformations in the β1 and β2 molecules of the asymmetric unit ( Figure 5). The pyrimidine ring occupies a space similar to that of NGT 6 and forms hydrogen bonding interactions with several residues of the pocket (Table 2, Figures 4 and 6). In order to accommodate the p-chlorophenyl ring, Tyr450 moves by approximately 10° around χ 1 relative to its conformation observed in the native structure.…”

“…The pyrimidine ring superimposes onto the NGT molecule and forms hydrogen bonds with some of the same residues from the active site, as well as stacking against Trp489β2 (PYR) and Trp460β1(NGT). 6 …”

“…Introduction of a side chain at this position will cause a serious clash and would likely disrupt the α-helix. 6 Substitutions at Pro504 and Arg505 can also destabilize the protein by interfering with the backbone conformation at Pro504 and by a loss of hydrogen-bond and salt-bridge interactions at Arg505. Since most of the destabilizing mutations are not in the immediate vicinity of the active site, they will not interfere with PYR binding, making the small molecule suitable for EET.…”

“…4,5 The substrate binding environment also ensures that a β-configuration is retained by directing the position of the incoming nucleophilic water molecule. A detailed description of the catalytic mechanism can be found in the paper by Lemieux et al 6 Genetic defects in either gene encoding the subunits of HexA can result in the accumulation of G M2 ganglioside in neural tissues and two of three lysosomal storage diseases collectively known as G M2 gangliosidosis. Tay-Sachs disease (defects in the α subunit) and Sandhoff disease (defects in the β subunit) are the most "common" and best studied of this family of diseases.…”

Identification and characterization of mature β-hexosaminidases associated with human placenta lysosomal membrane

“…β-Hexosaminidase (Hex, EC 3.2.1.52) is an acidic glycohydrolase that catalyses the cleavage of terminal β-N-acetylglucosamine or β-N-acetylgalactosamine from a broad range of glycoconjugated substrates into lysosomes [12,13]. However, the presence of Hex has also been described in cytosol [14] and associated to PM of different cell populations, such as ascidia and Drosophila melanogaster sperm [15][16][17], human erythrocytes [18], laeukaemic cells [19], lymphocytes and monocytes purified from peripheral blood of multiple sclerosis patients [20].…”