High affinity binding of Ca2؉ to ␣-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 Ca 2؉ 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 Ca 2؉ . 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 ␣-helical and ␤- ␣-Lactalbumin (LA)1 is the regulatory protein of lactose synthase (1-3), modulating the affinity of the catalytic component, UDP-galactose ␤-N-acetylglucosaminide ␤1,4-galactosyltransferase I, for acceptor substrates through a reversible proteinprotein interaction (4). LA is also secreted into milk and aggregates of a partially folded form of the human protein have been found to have selective apoptotic effects on tumor cells (5). LA is homologous with the c-type lysozymes (LZs) (6) and provides an example of extreme functional divergence in homologous proteins with closely similar structures (7-9). LA binds calcium strongly and specifically but the LZs include subgroups that bind Ca 2ϩ at a site corresponding to that in LA and others ("conventional" LZs) that lack a Ca 2ϩ -binding site (10). The K d app for Ca 2ϩ binding to apo-LA under physiological conditions is of the order of 10 Ϫ7 M (11-13). The Ca 2ϩ ion in LA has a structural role, being required for folding and native disulfide bond formation in the reduced denatured protein (14, 15). At ambient temperature and low ionic strength, apo-LA undergoes a transconformation to a molten globule (MG) state, which lacks a fixed tertiary structure but retains much of the native secondary structure. The MG is also stabilized by mildly disruptive treatments such as low pH or intermediate concentrations of denaturants (16,17); it is also identical to a kinetic MG that is highly populated during the refolding of chemically denatured LA (18 -20).Crystal structures of holo-LA from various species in different crystal forms and metal complexes (7,8,(21)(22)(23)(24)(25)(26) are similar to those of the homologous LZs, with two lobes of structure separated by a cleft (Fig. 1A). The larger ␣-helical lobe is formed by the amino-and carboxyl-terminal sections of the polypeptide chain (residues 1-34 and 86 -123) while the smaller lobe, which encompasses a small three-stranded antiparallel ␤-sheet, a small 3 10 helix and some irregular structure, is formed by the central section of the polypeptide chain (residues 35-85). We refer to the lobes as "subdomains" since they lack properties normally expected of true domains such as sequence contiguity and distinct functional properties and origins. The high affinity Ca 2ϩ -binding site is l...

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.

The increasing demand for the development of efficient biocatalysts is a consequence of their broad industrial applications. Typical difficulties that are encountered during their exploitation in a variety of processes are interconnected with factors such as temperature, pH, product inhibitors etc. To eliminate these, research has been directed towards the identification of new enzymes that would comply with the required standards. To this end, the recently discovered glucuronoyl esterases (GEs) are an enigmatic family within the carbohydrate esterase (CE) family. Structures of the thermophilic StGE2 esterase from Myceliophthora thermophila (synonym Sporotrichum thermophile), a member of the CE15 family, and its S213A mutant were determined at 1.55 and 1.9 Å resolution, respectively. The first crystal structure of the S213A mutant in complex with a substrate analogue, methyl 4-O-methyl-β-D-glucopyranuronate, was determined at 2.35 Å resolution. All of the three-dimensional protein structures have an α/β-hydrolase fold with a three-layer αβα-sandwich architecture and a Rossmann topology and comprise one molecule per asymmetric unit. These are the first crystal structures of a thermophilic GE both in an unliganded form and bound to a substrate analogue, thus unravelling the organization of the catalytic triad residues and their neighbours lining the active site. The knowledge derived offers novel insights into the key structural elements that drive the hydrolysis of glucuronic acid esters.

The high affinity of W1807 for GP appears to arise from the numerous nonpolar interactions made between the ligand and the protein. Its potency as an inhibitor results from the induced conformational changes that lock the enzyme in a conformation known as the T' state. Allosteric enzymes, such as GP, offer a new strategy for structure-based drug design in which the allosteric site can be exploited. The results reported here may have important implications in the design of new therapeutic compounds.

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 ␤-Dglucopyranose, 2-(␤-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) M, 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-methyloxadiazole 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. Keywords: type 2 diabetes; glycogen phosphorylase; ␤-D-glucopyranosyl analogs; inhibition; X-ray crystallographyGlycogen phosphorylase (GP), because of its central role in glycogen metabolism, has been exploited as a potential target for structure-based design of potent inhibitors, that may be relevant to the control of blood glucose concentrations in type 2 diabetes (Aiston et al. 2001(Aiston et al. , 2003Latsis et al. 2002). Several regulatory binding sites, identified in GP, have been used as molecular targets (for review, see Oikonomakos 2002). These are the allosteric site that binds the activator AMP and the inhibitor glucose-6-P, the catalytic site that binds substrates glucose-1-P and glycogen, and the inhibitor Article published online ahead of print. Article and publication date are at