Anopheles gambiae odorant binding protein crystal complex with the synthetic repellent DEET: implications for structure-based design of novel mosquito repellents.

Insect odorant binding proteins (OBPs) are the first components of the olfactory system to encounter and bind attractant and repellent odors emanating from various sources for presentation to olfactory receptors, which trigger relevant signal transduction cascades culminating in specific physiological and behavioral responses. For disease vectors, particularly hematophagous mosquitoes, repellents represent important defenses against parasitic diseases because they effect a reduction in the rate of contact between the vectors and humans. OBPs are targets for structure-based rational approaches for the discovery of new repellent or other olfaction inhibitory compounds with desirable features. Thus, a study was conducted to characterize the high resolution crystal structure of an OBP of Anopheles gambiae, the African malaria mosquito vector, in complex with N,N-diethyl-m-toluamide (DEET), one of the most effective repellents that has been in worldwide use for six decades. We found that DEET binds at the edge of a long hydrophobic tunnel by exploiting numerous non-polar interactions and one hydrogen bond, which is perceived to be critical for DEET's recognition. Based on the experimentally determined affinity of AgamOBP1 for DEET (K (d) of 31.3 µΜ) and our structural data, we modeled the interactions for this protein with 29 promising leads reported in the literature to have significant repellent activities, and carried out fluorescence binding studies with four highly ranked ligands. Our experimental results confirmed the modeling predictions indicating that structure-based modeling could facilitate the design of novel repellents with enhanced binding affinity and selectivity.

New inhibitors of glycogen phosphorylase as potential antidiabetic agents.

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.

X-ray sequence ambiguities of Sclerotium rolfsii lectin resolved by mass spectrometry.

X-ray crystallography, although a powerful technique for determining the three-dimensional structure of proteins, poses inherent problems in assigning the primary structure in residues Asp/Asn and Glu/Gln since these cannot be distinguished decisively in the electron density maps. In our recently published X-ray crystal structure of the Sclerotium rolfsii lectin (SRL) at 1.1 A resolution, amino acid sequence was initially deduced from the electron density map and residues Asp/Asn and Glu/Gln were assigned by considering their hydrogen bonding potential within their structural neighborhood. Attempts to verify the sequence by Edman sequencing were not successful as the N terminus of the protein was blocked. Mass spectrometry was applied to verify and resolve the ambiguities in the SRL X-ray crystal structure deduced sequence. From the Matrix assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI TOF-MS) and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis of tryptic and chymotryptic peptides of SRL, we could confirm and correct the sequence at five locations with respect to Asp/Asn and Glu/Gln. Analysis data also confirmed the positions of Leu/Ile, Gln/Lys residues and the sequence covering 118 of the total 141 residues accounting to 83.68% of the earlier deduced sequence of SRL.

Crystal structure of human Charcot-Leyden crystal protein, an eosinophil lysophospholipase, identifies it as a new member of the carbohydrate-binding family of galectins.

BACKGROUND: The Charcot-Leyden crystal (CLC) protein is a major autocrystallizing constituent of human eosinophils and basophils, comprising approximately 10% of the total cellular protein in these granulocytes. Identification of the distinctive hexagonal bipyramidal crystals of CLC protein in body fluids and secretions has long been considered a hallmark of eosinophil-associated allergic inflammation. Although CLC protein possesses lysophospholipase activity, its role(s) in eosinophil or basophil function or associated inflammatory responses has remained speculative. RESULTS: The crystal structure of the CLC protein has been determined at 1.8 A resolution using X-ray crystallography. The overall structural fold of CLC protein is highly similar to that of galectins -1 and -2, members of an animal lectin family formerly classified as S-type or S-Lac (soluble lactose-binding) lectins. This is the first structure of an eosinophil protein to be determined and the highest resolution structure so far determined for any member of the galectin family. CONCLUSIONS: The CLC protein structure possesses a carbohydrate-recognition domain comprising most, but not all, of the carbohydrate-binding residues that are conserved among the galectins. The protein exhibits specific (albeit weak) carbohydrate-binding activity for simple saccharides including N-acetyl-D-glucosamine and lactose. Despite CLC protein having no significant sequence or structural similarities to other lysophospholipase catalytic triad has also been identified within the CLC structure, making it a unique dual-function polypeptide. These structural findings suggest a potential intracellular and/or extracellular role(s) for the galectin-associated activities of CLC protein in eosinophil and basophil function in allergic diseases and inflammation.

The structure of glycogen phosphorylase b with an alkyldihydropyridine-dicarboxylic acid compound, a novel and potent inhibitor.

BACKGROUND: In muscle and liver, glycogen concentrations are regulated by the reciprocal activities of glycogen phosphorylase (GP) and glycogen synthase. An alkyl-dihydropyridine-dicarboxylic acid has been found to be a potent inhibitor of GP, and as such has potential to contribute to the regulation of glycogen metabolism in the non-insulin-dependent diabetes diseased state. The inhibitor has no structural similarity to the natural regulators of GP. We have carried out structural studies in order to elucidate the mechanism of inhibition. RESULTS: Kinetic studies with rabbit muscle glycogen phosphorylase b (GPb) show that the compound (-)(S)-3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5, 6-tricarboxylate (Bay W1807) has a Ki = 1.6 nM and is a competitive inhibitor with respect to AMP. The structure of the cocrystallised GPb-W1807 complex has been determined at 100K to 2.3 A resolution and refined to an R factor of 0.198 (Rfree = 0.287). W1807 binds at the GPb allosteric effector site, the site which binds AMP, glucose-6-phosphate and a number of other phosphorylated ligands, and induces conformational changes that are characteristic of those observed with the naturally occurring allosteric inhibitor, glucose-6-phosphate. The dihydropyridine-5,6-dicarboxylate groups mimic the phosphate group of ligands that bind to the allosteric site and contact three arginine residues. CONCLUSIONS: 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.

Sulphate activates phosphorylase b by binding to the Ser (P) site.

The notion, in a recent crystallographic study on the R state phosphorylase b (Barford, D. and Johnson, L.N. (1989) Nature 340, 609-616), that sulphate ions activate the enzyme by interacting within 2 A of the phosphorylatable Ser-14, is directly supported by kinetic studies on phosphorylase b', a proteolytic species which lacks the N-terminal 16 residues. The results show that this form of the enzyme is no longer activated by ammonium sulphate.

The refined crystal structure of toxic shock syndrome toxin-1 at 2.07 A resolution.

The pyrogenic toxin toxic shock syndrome toxin-1 from Staphylococcus aureus is a causative agent of the toxic shock syndrome disease. It belongs to a family of proteins known as superantigens that cross-link major histocompatibility class II molecules and T-cell receptors leading to the activation of a substantial number of T cells. The crystal structure of this protein has been refined to 2.07 A with an Rcryst value of 20.4% for 51,240 reflections. The final model contains three molecules in the asymmetric unit with good stereochemistry and a root-mean-square deviation of 0.009 A and 1.63 from ideality for bond lengths and bond angles, respectively. The overall fold is considerably similar to that of other known microbial superantigens (staphylococcal enterotoxins). However, a detailed structural analysis shows that toxic shock syndrome toxin-1 lacks several structural features that affect its specificity for V beta elements of the T-cell receptor and also its recognition by major histocompatibility class II molecules.

Refined crystal structures of native human angiogenin and two active site variants: implications for the unique functional properties of an enzyme involved in neovascularisation during tumour growth.

Human angiogenin (Ang), an unusual member of the pancreatic RNase superfamily, is a potent inducer of angiogenesis in vivo. Its ribonucleolytic activity is weak (10(4) to 10(6)-fold lower than that of bovine RNase A), but nonetheless seems to be essential for biological function. Ang has been implicated in the establishment of a wide range of human tumours and has therefore emerged as an important target for the design of new anti-cancer compounds. We report high-resolution crystal structures for native Ang in two different forms (Pyr1 at 1.8 A and Met-1 at 2.0 A resolution) and for two active-site variants, K40Q and H13A, at 2.0 A resolution. The native structures, together with earlier mutational and biochemical data, provide a basis for understanding the unique functional properties of this molecule. The major structural features that underlie the weakness of angiogenin's RNase activity include: (i) the obstruction of the pyrimidine-binding site by Gln117; (ii) the existence of a hydrogen bond between Thr44 and Thr80 that further suppresses the effectiveness of the pyrimidine site; (iii) the absence of a counterpart for the His119-Asp121 hydrogen bond that potentiates catalysis in RNase A (the corresponding aspartate in Ang, Asp116, has been recruited to stabilise the blockage of the pyrimidine site); and (iv) the absence of any precise structural counterparts for two important purine-binding residues of RNase A. Analysis of the native structures has revealed details of the cell-binding region and nuclear localisation signal of Ang that are critical for angiogenicity. The cell-binding site differs dramatically from the corresponding regions of RNase A and two other homologues, eosinophil-derived neurotoxin and onconase, all of which lack angiogenic activity. Determination of the structures of the catalytically inactive variants K40Q and H13A has now allowed a rigorous assessment of the relationship between the ribonucleolytic and biological activities of Ang. No significant change outside the enzymatic active site was observed in K40Q, establishing that the loss of angiogenic activity for this derivative is directly attributable to disruption of the catalytic apparatus. The H13A structure shows some changes beyond the ribonucleolytic site, but sites involved in cell-binding and nuclear translocation are essentially unaffected by the amino acid replacement.

Kinetic properties of tetrameric glycogen phosphorylase b in solution and in the crystalline state.

R-state monoclinic P2(1) crystals of phosphorylase have been shown to be catalytically active in the presence of an oligosaccharide primer and glucose-1-phosphate in 0.9 M ammonium sulfate, 10 mM beta-glycerophosphate, 0.5 mM EDTA, and 1 mM dithiothreitol, the medium in which the crystals are grown or equilibrated for crystallographic studies (Barford, D. & Johnson, L.N., 1989, Nature 360, 609-616; Barford, D., Hu, S.-H., & Johnson, L.N., 1991, J. Mol. Biol. 218, 233-260). Kinetic data suggest that the activity of crystalline tetrameric phosphorylase is similar to that determined in solution for the enzyme tetramer. However, large differences were found in the maximal velocities for both oligosaccharide or glucose-1-phosphate substrates between the soluble dimeric and crystalline tetrameric enzyme.

Binding of phosphate and pyrophosphate ions at the active site of human angiogenin as revealed by X-ray crystallography.

Human angiogenin (Ang) is an unusual homolog of bovine pancreatic RNase A that utilizes its ribonucleolytic activity to induce the formation of new blood vessels. The pyrimidine-binding site of Ang was shown previously to be blocked by glutamine 117, indicating that Ang must undergo a conformational change to bind and cleave RNA. The mechanism and nature of this change are not known, and no Ang-inhibitor complexes have been characterized structurally thus far. Here, we report crystal structures for the complexes of Ang with the inhibitors phosphate and pyrophosphate, and the structure of the complex of the superactive Ang variant Q117G with phosphate, all at 2.0 A resolution. Phosphate binds to the catalytic site of both Ang and Q117G in essentially the same manner observed in the RNase A-phosphate complex, forming hydrogen bonds with the side chains of His 13, His 114, and Gln 12, and the main chain of Leu 115; it makes an additional interaction with the Lys 40 ammonium group in the Ang complex. One of the phosphate groups of pyrophosphate occupies a similar position. The other phosphate extends toward Gln 117, and lies within hydrogen-bonding distance from the side-chain amide of this residue as well as the imidazole group of His 13 and the main-chain oxygen of Leu 115. The pyrimidine site remains obstructed in all three complex structures, that is, binding to the catalytic center is not sufficient to trigger the conformational change required for catalytic activity, even in the absence of the Gln 117 side chain. The Ang-pyrophosphate complex structure suggests how nucleoside pyrophosphate inhibitors might bind to Ang; this information may be useful for the design of Ang antagonists as potential anti-angiogenic drugs.

Control of phosphorylase b conformation by a modified cofactor: crystallographic studies on R-state glycogen phosphorylase reconstituted with pyridoxal 5'-diphosphate.

Previous crystallographic studies on glycogen phosphorylase have described the different conformational states of the protein (T and R) that represent the allosteric transition and have shown how the properties of the 5'-phosphate group of the cofactor pyridoxal phosphate are influenced by these conformational states. The present work reports a study on glycogen phosphorylase b (GPb) complexed with a modified cofactor, pyridoxal 5'-diphosphate (PLPP), in place of the natural cofactor. Solution studies (Withers, S.G., Madsen, N.B., & Sykes, B.D., 1982, Biochemistry 21, 6716-6722) have shown that PLPP promotes R-state properties of the enzyme indicating that the cofactor can influence the conformational state of the protein. GPb complexed with pyridoxal 5'-diphosphate (PLPP) has been crystallized in the presence of IMP and ammonium sulfate in the monoclinic R-state crystal form and the structure refined from X-ray data to 2.8 A resolution to a crystallographic R value of 0.21. The global tertiary and quaternary structure in the vicinity of the Ser 14 and the IMP sites are nearly identical to those observed for the R-state GPb-AMP complex. At the catalytic site the second phosphate of PLPP is accommodated with essentially no change in structure from the R-state structure and is involved in interactions with the side chains of two lysine residues (Lys 568 and Lys 574) and the main chain nitrogen of Arg 569. Superposition of the T-state structure shows that were the PLPP to be incorporated into the T-state structure there would be a close contact with the 280s loop (residues 282-285) that would encourage the T to R allosteric transition. The second phosphate of the PLPP occupies a site that is distinct from other dianionic binding sites that have been observed for glucose-1-phosphate and sulfate (in the R state) and for heptulose-2-phosphate (in the T state). The results indicate mobility in the dianion recognition site, and the precise position is dependent on other linkages to the dianion. In the modified cofactor the second phosphate site is constrained by the covalent link to the first phosphate of PLPP. The observed position in the crystal suggests that it is too far from the substrate site to represent a site for catalysis.

The ammonium sulfate activation of phosphorylase b.

The ammonium sulfate activation of phosphorylase b has been studied. Ammonium sulfate, when present in high concentrations, induces properties of phosphorylase a in phosphorylase b, such as an enhanced affinity for AMP, a reversal of the glucose-6-P inhibition and enzyme tetramerization. The data are consistent with the interpretation that sulfates bind to the Ser-14 site and the sulfate-protein interactions at this site are responsible for activation of phosphorylase b.

The binding of C5-alkynyl and alkylfurano[2,3-d]pyrimidine glucopyranonucleosides to glycogen phosphorylase b: synthesis, biochemical and biological assessment.

C5-alkynyl and alkylfurano[2,3-d]pyrimidine glucopyranonucleosides have been synthesized and studied as inhibitors of glycogen phosphorylase b (GPb). Kinetic experiments have shown that most of these compounds were low micromolar inhibitors of the enzyme. The best inhibitor was 1-(ß-D-glucopyranosyl)-5-ethynyluracil (K(i)=4.7 µM). Crystallographic analysis of these compounds in complex with GPb revealed that inhibitors with a long C5-alkynyl group exploited interactions with ß-pocket of the active site and induced significant conformational changes of the 280s loop compared to GPb in complex with compounds with a short C5-alkynyl group. The results highlight the importance in the length of the aliphatic groups used to enhance inhibitory potency for the exploitation of the hydrophobic ß-pocket. The best of the inhibitors had also a moderate effect on glycogenolysis in the cellular lever with an IC(50) value of 291.4 µM.

Inhibition of rabbit muscle glycogen phosphorylase by D-gluconohydroximo-1,5-lactone-N-phenylurethane.

The effect of the beta-glycosidase inhibitor D-gluconohydroximo-1,5-lactone-N-phenylurethane (PUG) on the kinetic and ultracentrifugation properties of glycogen phosphorylase has been studied. Recent crystallographic work at 2.4 A resolution [D. Barford et al. (1988) Biochemistry 27, 6733-6741] has shown that PUG binds in the catalytic site of phosphorylase b crystals with its gluconohydroximolactone moiety occupying a position similar to that observed for other glucosyl compounds and the N-phenylurethane side chain fitting into an adjacent cavity with little conformational change in the enzyme. In solution, PUG was shown to be a potent inhibitor of phosphorylase b, directly competitive with alpha-D-glucopyranose 1-phosphate (glucose-1-P) (Ki = 0.40 mM) and noncompetitive with respect to glycogen and AMP. When PUG was tested for synergistic inhibition in the presence of caffeine, the Dixon plots of reciprocal velocity versus PUG concentration at different fixed caffeine concentrations provided intersecting lines with interaction constant (alpha) values of 0.95-1.38, indicating that the binding of one inhibitor is not significantly affected by the binding of the other. For glycogen phosphorolysis, PUG was noncompetitive with respect to phosphate, suggesting that it can bind to the central enzyme-AMP-glycogen-phosphate complex. PUG was shown to inhibit phosphorylase alpha (without AMP) activity (Ki = 0.43 mM) in a manner similar to that of the b form. However, in the presence of AMP, PUG exhibited complex kinetics, acting as a noncompetitive inhibitor with respect to glucose-1-P, while a twofold decrease of PUG binding to the enzyme-AMP-glycogen complex was observed. Ultracentrifugation experiments demonstrated that PUG does not cause any significant dissociation of phosphorylase alpha tetramer. Furthermore the dimerization of phosphorylase alpha by glucose is completely prevented in the presence of PUG. These observations are consistent with PUG binding to both the R and the T conformations of phosphorylase.

Crystal structures of ribonuclease A complexes with 5'-diphosphoadenosine 3'-phosphate and 5'-diphosphoadenosine 2'-phosphate at 1.7 A resolution.

High-resolution (1.7 A) crystal structures have been determined for bovine pancreatic ribonuclease A (RNase A) complexed with 5'-diphosphoadenosine 3'-phosphate (ppA-3'-p) and 5'-diphosphoadenosine 2'-phosphate (ppA-2'-p), as well as for a native structure refined to 2.0 A. These nucleotide phosphates are the two most potent inhibitors of RNase A reported so far, with Ki values of 240 and 520 nM, respectively. The binding modes and conformations of ppA-3'-p and ppA-2'-p were found to differ markedly from those anticipated on the basis of earlier structures of RNase A complexes. The key difference is that the 5'-beta-phosphate rather than the 5'-alpha-phosphate of each inhibitor occupies the P1 phosphate binding site. As a consequence, the ribose moieties of the two nucleotides are shifted by approximately 2 A compared to the positions of their counterparts in earlier complexes, and the adenine rings are rotated into unusual syn conformations. Thus, the six-membered and five-membered rings of both adenines are reversed with respect to the others but nonetheless engage in extensive interactions with the residues that form the B2 purine binding site of RNase A. Despite the close structural similarity of the two inhibitors, the puckers of their furanose rings are different: C2'-endo and C3'-endo, respectively. Moreover, their 5'-alpha-phosphates and 3'(2')-monophosphates interact with largely different sets of RNase residues. The results of this crystallographic study emphasize the difficulties inherent in qualitative modeling of protein-inhibitor interactions and the compelling reasons for high-resolution structural studies in which quantitative design of improved inhibitors was enabled. The structures presented here provide a promising starting point for the rational design of tight-binding RNase inhibitors, which may be used as therapeutic agents in restraining the ribonucleolytic activities of RNase homologues such as angiogenin, eosinophil-derived neurotoxin, and eosinophil cationic protein.

Toward rational design of ribonuclease inhibitors: high-resolution crystal structure of a ribonuclease A complex with a potent 3',5'-pyrophosphate-linked dinucleotide inhibitor.

The crystal structure of ribonuclease A (RNase A) in complex with pdUppA-3'-p [5'-phospho-2'-deoxyuridine-3'-pyrophosphate (P'-->5') adenosine 3'-phosphate] has been determined at 1.7 A resolution. This dinucleotide is the most potent low molecular weight inhibitor of RNase A reported to date (K(i) = 27 nM) and is also effective against two major nonpancreatic RNases: eosinophil-derived neurotoxin and RNase-4; in all cases, tight binding in large part derives from the unusual 3',5'-pyrophosphate internucleotide linkage [Russo, N., and Shapiro, R. (1999) J. Biol. Chem. 274, 14902-14908]. The design of pdUppA-3'-p was based on the crystal structure of RNase A complexed with 5'-diphosphoadenosine 3'-phosphate (ppA-3'-p) [Leonidas, D. D., Shapiro, R., Irons, L. I., Russo, N., and Acharya, K. R. (1997) Biochemistry 36, 5578-5588]. The adenosine of pdUppA-3'-p adopts an atypical syn conformation not observed for standard adenosine nucleotides bound to RNase A. This conformation, which allows extensive interactions with Asn 67, Gln 69, Asn 71, and His 119, is associated with the placement of the 5'-beta-phosphate of the adenylate, rather than alpha-phosphate, at the site where substrate phosphodiester bond cleavage occurs. The contacts of the deoxyuridine 5'-phosphate portion of pdUppA-3'-p appear to be responsible for the 9-fold increased affinity of this compound as compared to ppA-3'-p: the uracil base binds to Thr 45 in the same manner as previous pyrimidine inhibitors, and the terminal 5'-phosphate is positioned to form medium-range Coulombic interactions with Lys 66. The full potential benefit of these added interactions is not realized because of compensatory losses of hydrogen bonds of Lys 7 and Gln 11 with the terminal 3'-phosphate and the adenylate 5'-alpha-phosphate, which were not predicted by modeling. The results reported here have important implications for the design of improved inhibitors of RNase A and for the development of therapeutic agents to control the activities of RNase homologues such as eosinophil-derived neurotoxin and angiogenin that have roles in human pathologies.

The binding of D-gluconohydroximo-1,5-lactone to glycogen phosphorylase. Kinetic, ultracentrifugation and crystallographic studies.

Combined kinetic, ultracentrifugation and X-ray-crystallographic studies have characterized the effect of the beta-glucosidase inhibitor gluconohydroximo-1,5-lactone on the catalytic and structural properties of glycogen phosphorylase. In the direction of glycogen synthesis, gluconohydroximo-1,5-lactone was found to competitively inhibit both the b (Ki 0.92 mM) and the alpha form of the enzyme (Ki 0.76 mM) with respect to glucose 1-phosphate in synergism with caffeine. In the direction of glycogen breakdown, gluconohydroximo-1,5-lactone was found to inhibit phosphorylase b in a non-competitive mode with respect to phosphate, and no synergism with caffeine could be demonstrated. Ultracentrifugation and crystallization experiments demonstrated that gluconohydroximo-1,5-lactone was able to induce dissociation of tetrameric phosphorylase alpha and stabilization of the dimeric T-state conformation. A crystallographic binding study with 100 mM-gluconohydroximo-1,5-lactone at 0.24 nm (2.4 A) resolution showed a major peak at the catalytic site, and no significant conformational changes were observed. Analysis of the electron-density map indicated that the ligand adopts a chair conformation. The results are discussed with reference to the ability of the catalytic site of the enzyme to distinguish between two or more conformations of the glucopyranose ring.

Structural basis for the recognition of carbohydrates by human galectin-7.

Knowledge about carbohydrate recognition domains of galectins, formerly known as S-type animal lectins, is important in understanding their role(s) in cell-cell interactions. Here we report the crystal structure of human galectin-7 (hGal-7), in free form and in the presence of galactose, galactosamine, lactose, and N-acetyl-lactosamine at high resolution. This is the first structure of a galectin determined in both free and carbohydrate-bound forms. The structure shows a fold similar to that of the prototype galectins -1 and -2, but has greater similarity to a related galectin molecule, Gal-10. Even though the carbohydrate-binding residues are conserved, there are significant changes in this pocket due to shortening of a loop structure. The monomeric hGal-7 molecule exists as a dimer in the crystals, but adopts a packing arrangement considerably different from that of Gal-1 and Gal-2, which has implications for carbohydrate recognition.

Selective recognition of mannose by the human eosinophil Charcot-Leyden crystal protein (galectin-10): a crystallographic study at 1.8 A resolution.

The role(s) of the eosinophil Charcot-Leyden crystal (CLC) protein in eosinophil or basophil function or associated inflammatory processes is yet to be established. Although the CLC protein has been reported to exhibit weak lysophospholipase activity, it shows virtually no sequence homology to any known member of this family of enzymes. The X-ray crystal structure of the CLC protein is very similar to the structure of the galectins, members of a beta-galactoside-specific animal lectin family, including a partially conserved galectin carbohydrate recognition domain (CRD). In the absence of any known natural carbohydrate ligand for this protein, the functional role of the CLC protein (galectin-10) has remained speculative. Here we describe structural studies on the carbohydrate binding properties of the CLC protein and report the first structure of a carbohydrate in complex with the protein. Interestingly, the CLC protein demonstrates no affinity for beta-galactosides and binds mannose in a manner very different from those of other related galectins that have been shown to bind lactosamine. The partial conservation of residues involved in carbohydrate binding led to significant changes in the topology and chemical nature of the CRD, and has implications for carbohydrate recognition by the CLC protein in vivo and its functional role in the biology of inflammation.

Sulphate-activated phosphorylase b: the pH-dependence of catalytic activity.

The pH-dependence of sulphate-activated phosphorylase b has been studied in the direction of glycogen synthesis. The bell-shaped curve of the pH-dependence of the catalytic constant for the AMP-activated enzyme showed pK values of 6.1 and 7.3, but the curve for the enzyme activated by 0.9 M ammonium sulphate showed a drop of activity on the acid side at much higher pH values. Its bell was centred at pH 7.8 but it was too narrow to be characterized by only two pK values. The narrowness of the curve could be explained by positive co-operativity, but not its unusually steep acid side. We suggest that the fall on the acid side is due to more than one hydronation (addition of H+). The points can be fitted by a curve with two de-activating hydronations and a de-activating dehydronation having identical titration pK values of 7.5, and hence molecular values of 7.0, 7.5 and 8.0. If both 0.9 M ammonium sulphate and 5 mM AMP are added, the bell is as broad as with AMP alone, but is somewhat raised in pH optimum. The results are discussed in the light of new structural data from crystallographic studies on binary complexes of the enzyme.