Allosteric Regulation of Glycogen Phosphorylase by Order/Disorder Transition of the 250′ and 280s Loops

Allostery is a fundamental mechanism of protein activation,
yet
the precise dynamic changes that underlie functional regulation of
allosteric enzymes, such as glycogen phosphorylase (GlyP), remain
poorly understood. Despite being the first allosteric enzyme described,
its structural regulation is still a challenging problem: the key
regulatory loops of the GlyP active site (250′ and 280s) are
weakly stable and often missing density or have large b-factors in
structural models. This led to the longstanding hypothesis that GlyP
regulation is achieved through gating of the active site by (dis)order
transitions, as first proposed by Barford and Johnson. However, testing
this requires a quantitative measurement of weakly stable local structure
which, to date, has been technically challenging in such a large protein.
Hydrogen–deuterium-exchange mass spectrometry (HDX-MS) is a
powerful tool for studying protein dynamics, and millisecond HDX-MS
has the ability to measure site-localized stability differences in
weakly stable structures, making it particularly valuable for investigating
allosteric regulation in GlyP. Here, we used millisecond HDX-MS to
measure the local structural perturbations of glycogen phosphorylase
b (GlyPb), the phosphorylated active form (GlyPa), and the inhibited
glucose-6 phosphate complex (GlyPb:G6P) at near-amino acid resolution.
Our results support the Barford and Johnson hypothesis for GlyP regulation
by providing insight into the dynamic changes of the key regulatory
loops.