Lysosomal beta-hexosaminidase A (Hex A) is essential for the degradation of G(M2) 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 angstrom resolution and the structure of Hex A in complex with NAG-thiazoline, (NGT) to 3.25 angstrom 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 alpha beta heterodimer, with each subunit having a functional active site: Only the a-subunit active site can hydrolyze G(M2) gangliosides due to a flexible loop structure that is removed post-translationally from beta, and to the presence of alpha Asn423 and alpha Arg424. The loop structure is involved in binding the G(M2) activator protein, while aArg424 is critical for binding the carboxylate group of the N-acetyl-neuraminic acid residue of G(M2). The beta-subunit lacks these key residues and has beta Asp452 and beta Leu453 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. (c) 2006 Published by Elsevier Ltd.