Allosteric activation and inhibition of glycogen phosphorylase share common transient structural features

It remains a major challenge to ascertain the specific structurally dynamic changes that underpin protein functional switching. There is a growing need to complement structural models with the ability to determine the dynamic structural changes that occur as these proteins are regulated and function. The archetypal allosteric enzyme, glycogen phosphorylase is one of the most studied proteins and is a clinical target of much interest to treat type II diabetes and metastatic cancers. However, a lack of understanding of its complex regulation, mediated by dynamic structural changes, hinder its exploitation as a drug target. Here, we precisely locate dynamic structural changes upon allosteric activation and inhibition of glycogen phosphorylase, by developing a time-resolved non-equilibrium millisecond hydrogen/deuterium-exchange mass spectrometry (HDX-MS) approach. We resolved obligate transient changes in localized structure that are absent when directly comparing active/inactive states of the enzyme and show that they are common to allosteric activation by AMP and inhibition by caffeine, operating at different sites. This indicates that opposing allosteric regulation by inhibitor and activator ligands is mediated by pathways that intersect at a common structurally dynamic motif. This approach has broad application to determine the structural kinetic mechanisms by which proteins are regulated.