Ivory, D. P. | bioRxiv Science

bioRxiv2023

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.

biophysics↗

bioRxiv2022

Extensive substrate recognition by the streptococcal antibody-degrading enzymes IdeS and EndoS

Enzymatic cleavage of IgG antibodies is a common strategy used by pathogenic bacteria to ablate immune effector function. The Streptococcus pyogenes bacterium secretes the protease IdeS and the glycosidase EndoS, which specifically catalyse cleavage and deglycosylation of human IgG, respectively. IdeS has received clinical approval for kidney transplantation in hypersensitised individuals, while EndoS has found application in engineering antibody glycosylation. Here, we present crystal structures of both enzymes in complex with their IgG1 Fc substrate, which was achieved using Fc engineering to disfavour preferential Fc crystallisation. The IdeS protease displays extensive Fc recognition and encases the antibody hinge. Conversely, the glycan hydrolase domain in EndoS traps the Fc glycan in a flipped-out conformation, while additional recognition of the Fc peptide is driven by the so-called carbohydrate binding module. Understanding the molecular basis of antibody recognition by bacterial enzymes will facilitate the development of next-generation enzymes for clinical and biotechnological use.

immunology↗