High affinity binding of Ca²⁺to α-lactalbumin (LA) stabilizes the native structure and is required for the efficient generation of native protein with correct disulfide bonds from the reduced denatured state. A progressive increase in affinity of LA conformers for Ca²⁺ as they develop increasingly native structures can account for the tendency of the apo form to assume a molten globule state and the large acceleration of folding by Ca²⁺. To investigate the effect of calcium on structure of bovine LA, x-ray structures have been determined for crystals of the apo and holo forms at 2.2-Å resolution. In both crystal forms, which were grown at high ionic strength, the protein is in a similar global native conformation consisting of α-helical and β-subdomains separated by a cleft. Even though alternative cations and Ca²⁺ liganding solvent molecules are absent, removal of Ca²⁺ has only minor effects on the structure of the metal-binding site and a structural change was observed in the cleft on the opposite face of the molecule adjoining Tyr¹⁰³ of the helical lobe and Gln⁵⁴ of the β-lobe. Changes include increased separation of the lobes, loss of a buried solvent molecule near the Ca²⁺-binding site, and the replacement of inter- and intra-lobe H-bonds of Tyr¹⁰³ by interactions with new immobilized water molecules. The more open cleft structure in the apo protein appears to be an effect of calcium binding transmitted via a change in orientation of helix H3 relative to the β-lobe to the inter-lobe interface. Calcium is well known to promote the folding of LA. The results from the comparison of apo and holo structures of LA provide high resolution structural evidence that the acceleration of folding by Ca²⁺ is mediated by an effect on interactions between the two subdomains.

The protein glycogen phosphorylase has been linked to type 2 diabetes, indicating the importance of this target to human health. Hence, the search for potent and selective inhibitors of this enzyme, which may lead to antihyperglycaemic drugs, has received particular attention. Glycogen phosphorylase is a typical allosteric protein with five different ligand binding sites, thus offering multiple opportunities for modulation of enzyme activity. The present survey is focused on recent new molecules, potential inhibitors of the enzyme. The biological activity can be modified by these molecules through direct binding, allosteric effects or other structural changes. Progress in our understanding of the mechanism of action of these inhibitors has been made by the determination of high-resolution enzyme inhibitor structures (both muscle and liver). The knowledge of the three-dimensional structures of protein-ligand complexes allows analysis of how the ligands interact with the target and has the potential to facilitate structure-based drug design. In this review, the synthesis, structure determination and computational studies of the most recent inhibitors of glycogen phosphorylase at the different binding sites are presented and analyzed.

In an attempt to identify leads that would enable the design of inhibitors with enhanced affinity for glycogen phosphorylase (GP), that might control hyperglycaemia in type 2 diabetes, three new analogs of β‐D‐glucopyranose, 2‐(β‐D‐glucopyranosyl)‐5‐methyl‐1, 3, 4‐oxadiazole, ‐benzothiazole, and ‐benzimidazole were assessed for their potency to inhibit GPb activity. The compounds showed competitive inhibition (with respect to substrate Glc‐1‐P) with K_i values of 145.2 (±11.6), 76 (±4.8), and 8.6 (±0.7) μM, respectively. In order to establish the mechanism of this inhibition, crystallographic studies were carried out and the structures of GPb in complex with the three analogs were determined at high resolution (GPb‐methyl‐oxadiazole complex, 1.92 Å; GPb‐benzothiazole, 2.10 Å; GPb‐benzimidazole, 1.93 Å). The complex structures revealed that the inhibitors can be accommodated in the catalytic site of T‐state GPb with very little change of the tertiary structure, and provide a rationalization for understanding variations in potency of the inhibitors. In addition, benzimidazole bound at the new allosteric inhibitor or indole binding site, located at the subunit interface, in the region of the central cavity, and also at a novel binding site, located at the protein surface, far removed (∼ 32 Å) from the other binding sites, that is mostly dominated by the nonpolar groups of Phe202, Tyr203, Val221, and Phe252.