Glycogen phosphorylase revisited: extending the resolution of the R- and T-state structures of the free enzyme and in complex with allosteric activators

Abstract

The crystal structures of free T-state and R-state glycogen phosphorylase (GP) and of R-state GP in complex with the allosteric activators IMP and AMP are reported at improved resolution. GP is a validated pharmaceutical target for the development of antihyperglycaemic agents, and the reported structures may have a significant impact on structure-based drug-design efforts. Comparisons with previously reported structures at lower resolution reveal the detailed conformation of important structural features in the allosteric transition of GP from the T-state to the R-state. The conformation of the N-terminal segment (residues 7–17), the position of which was not located in previous T-state structures, was revealed to form an α-helix (now termed α0). The conformation of this segment (which contains Ser14, phosphorylation of which leads to the activation of GP) is significantly different between the T-state and the R-state, pointing in opposite directions. In the T-state it is packed between helices α4 and α16 (residues 104–115 and 497–508, respectively), while in the R-state it is packed against helix α1 (residues 22′–38′) and towards the loop connecting helices α4′ and α5′ of the neighbouring subunit. The allosteric binding site where AMP and IMP bind is formed by the ordering of a loop (residues 313–326) which is disordered in the free structure, and adopts a conformation dictated mainly by the type of nucleotide that binds at this site.

Validated liquid chromatography‐mass spectrometry method for the quantification of glycogenolysis phosphorylase inhibitor in mouse tissues – 5‐isopropyl‐4‐(2‐chlorophenyl)‐1‐ethyl‐1,4‐dihydro‐6‐methyl‐2,3,5‐pyridinetricarboxylic acid ester disodium salt hydrate

“…Beyond their therapeutic potential, PYG inhibitors may also serve as powerful experimental tools in basic studies focused on the biochemistry of glycogen metabolism and regulation of glucostasis. A perfect example of such interplay is the role of liver PYG inhibitors in the discovery of novel antihyperglycemic drugs in type 2 diabetes [7] and the subsequent experimental utilization of these compounds which allowed a precise determination of the relationship between the covalent modification of PYG, and the synthesis/degradation of glycogen [8,9].…”

Structural Basis of Sequential and Concerted Cooperativity

“…The structure includes two domains, the larger N-terminal being responsible for the intersubunit interface. The intersubunit interface is isologous and is provided by two contact regions formed by α-helices 7 (the “tower” helices; residues 262–276 in rabbit muscle GP; see Figure 4 ) of each subunit, which contact each other in antiparallel directions, and the cap region of each subunit (residues 35–46) contacting the α-helix 2 (residues 47–78) of the opposite subunit [ 38 , 39 , 40 ].…”

Transient Structural Dynamics of Glycogen Phosphorylase from Nonequilibrium Hydrogen/Deuterium-Exchange Mass Spectrometry

“…GlyP catalyzes the first executed energetic step of carbohydrate metabolism from glycogen stores and, as such, is one of the most highly regulated enzymes known, with at least six allosteric sites for natural ligands and drugs. GlyP has been intensively studied since the 1930s, thus there is abundance of structural and kinetic data on this protein system, − with detailed studies on the activation mechanism and stabilization of R/T-states (active/inactive states) by allosteric ligands and phosphorylation . GlyP is, therefore, an ideal system to challenge our ability to discern dynamic structural mechanisms of allosteric regulation as it is alternately activated by adenosine monophosphate (AMP) at the “nucleotide site” and inhibited by caffeine (CFF) at the “inhibitor site”, , while also being constitutively active by Ser 14 phosphorylation .…”