UC San Diego

Lafora Disease (LD), a type of progressive myoclonus epilepsy, is a fatal autosomal recessive disorder. The hallmark of LD is the accumulation of insoluble polyglucosan deposits known as Lafora bodies in nerve, heart, liver, skin and skeletal muscle cells. Half of all LD cases are caused by mutations in the gene that encodes the dual specificity phosphatase, laforin. Laforin contains two domains: a carbohydrate-binding module (CBM) and a phosphatase domain. Together, they allow laforin to bind and dephosphorylate complex carbohydrates, an activity conserved from humans to plants. However, laforin has yet to be crystallized successfully, and the dynamic behavior of the two domains remains unknown. Due to the unavailability of laforin for study, we probed solvent accessibility of the plant functional equivalent of laforin, starch-excess 4 (SEX4), utilizing enhanced peptide amide hydrogen/deuterium (H/D) exchange mass spectrometry (DXMS) in order to elucidate the structural dynamics of these proteins. We also explored changes in H/ D exchange of the SEX4 protein upon the binding of amylopectin, a glucose polymer and known substrate of both laforin and SEX4. Employing DXMS, we observed substrate- induced exchange decrease in regions of the CBM with conserved binding residues and in the phosphatase active site pocket. Exchange decrease was also observed in a loop of the CBM not implicated in carbohydrate binding, indicating a site undergoing substrate-induced allosteric changes or yet unidentified carbohydrate-binding residues. The lack of exchange rate changes in all other regions of SEX4 indicates that the phosphatase domain of SEX4 undergoes little conformational changes upon substrate binding, and likely retains an open and active conformation. The functional homology of SEX4 with laforin makes it highly likely our results hold true for laforin