Abstract
Disrupting the interaction between glycogen phosphorylase and the glycogen targeting subunit (G(L)) of protein phosphatase 1 is emerging as a novel target for the treatment of type 2 diabetes. To elucidate the molecular basis of binding, we have determined the crystal structure of liver phosphorylase bound to a G(L)-derived peptide. The structure reveals the C terminus of G(L) binding in a hydrophobically collapsed conformation to the allosteric regulator-binding site at the phosphorylase dimer interface. G(L) mimics interactions that are otherwise employed by the activator AMP. Functional studies show that G(L) binds tighter than AMP and confirm that the C-terminal Tyr-Tyr motif is the major determinant for G(L) binding potency. Our study validates the G(L)-phosphorylase interface as a novel target for small molecule interaction.
Highlights
Diabetes is one of the major public health problems
We show that the glycogen targeting subunit (GL) C-terminal region structurally and functionally mimics AMP binding to human liver glycogen phosphorylase
Using x-ray crystallography we identify the C terminus of GL bound in the allosteric regulator site of human liver glycogen phosphorylase (hlGP)
Summary
Protein Production—Human liver glycogen phosphorylase was prepared as described [7]. Briefly, full-length hlGPa was expressed in insect cells and purified using copper-chelating, anion exchange, and size exclusion chromatography. hlGPb was expressed in Escherichia coli and purified analogously. Crystallization and Structure Determination—Crystals were obtained at 20 °C in 1 ϩ 1 l hanging drops from hlGPa concentrated to 8 mg/ml in 20 mM BES, pH 6.7, 1 mM EDTA, 0.5 mM dithiothreitol, 50 mM glucose, 0.5 mM AMP over a reservoir of 0.1 M Tris, pH 8.5, 7– 8% (w/v) polyethylene glycol 8000. The complex structure was solved using difference Fourier methods with the coordinates of hlGPa-AMP (Protein Data Bank accession code 1FA9) as a template. A biotin-labeled 17-mer GL-peptide, biotin-FPEWPSYLGYEKLGPYY-COOH (GL probe) was synthesized by Interactiva (Ulm, Germany). A one-step phosphorylation reaction by phosphorylase kinase (Sigma) transformed hlGPb into 33P-radiolabeled hlGPa. The incubation reaction contained test compound, 5 g of 33P-hlGPa, 50 pmol of GL probe, 0.4 mg SPA-Beads (streptavidin-SPA beads; Amersham Biosciences, RPNQ 0007) in test buffer
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