Structural mechanism for glycogen phosphorylase control by phosphorylation and AMP

Abstract

The crystal structures of activated R state glycogen phosphorylase a (mGP a) and R and T state glycogen phosphorylase b (GP b) complexed with AMP have been solved at 2.9 Å, 2.9 Å and 2.2 Å resolution, respectively. The structure of R state GP a is nearly identical to the structure of sulphate-activated R state GP b, except in the region of Ser14, where there is a covalently attached phosphate group in GP a and a non-covalently attached sulphate group in GP b. The contacts made by the N-terminal tail residues in R state GP a at the subunit interface of the functionally active dimer are similar to those observed previously for T state GP a. The quaternary and tertiary structural changes on the T to R transition allow these interactions to be relayed to the catalytic site in R state GP a. The transition from the T state GP b structure to the R state GP a structure results in a change in the N-terminal residues from a poorly ordered extended structure that makes intrasubunit contacts to an ordered coiled conformation that makes intersubunit contacts. The distance between Arg10, the first residue to be located from the N terminus, in R state GP a and T state GP b is 50 Å. One of the important subunit-subunit interactions in the dimer molecule involves contacts between the helix α2 and the cap′ (residues 35′ to 45′ that form a loop between the 1st and 2nd α helices, α1′ and α2′ of the other subunit. The prime denotes residues from the other subunit). The interactions made by the N-terminal residues induce structural changes at the cap′/α2 helix interface that lead to the creation of a high-affinity AMP site. The tertiary structural changes at the cap (shifts 1.2 to 2.1 Å for residues 35 to 45) are partially compensated by the quaternary structural change so that the overall shifts in these residues after the combined tertiary and quaternary changes are between 0.5 and 1.3 Å. AMP binds to R state GP b with at least 100-fold greater affinity and exhibits four additional hydrogen bonds, stronger ionic interactions and more extensive van der Waals' interactions with 116 Å 2 greater solvent accessible surface area buried compared with AMP bound to T state GP b. A hydrogen bond observed in the R state complex between Asn44′ and the N-1 of the adenine moiety of AMP provides a possible explanation for the differences in affinity between AMP and IMP, and the different allosteric properties of the two nucleotides. The observed correlation between tertiary and quaternary conformational changes form the basis for a structural explanation for allosteric control by phosphorylation and by AMP.