The structural basis for specificity of substrate and recruitment peptides for cyclin-dependent kinases.

Progression through the eukaryotic cell cycle is driven by the orderly activation of cyclin-dependent kinases (CDKs). For activity, CDKs require association with a cyclin and phosphorylation by a separate protein kinase at a conserved threonine residue (T160 in CDK2). Here we present the structure of a complex consisting of phosphorylated CDK2 and cyclin A together with an optimal peptide substrate, HHASPRK. This structure provides an explanation for the specificity of CDK2 towards the proline that follows the phosphorylatable serine of the substrate peptide, and the requirement for the basic residue in the P+3 position of the substrate. We also present the structure of phosphorylated CDK2 plus cyclin A3 in complex with residues 658-668 from the CDK2 substrate p107. These residues include the RXL motif required to target p107 to cyclins. This structure explains the specificity of the RXL motif for cyclins.

Bleeding per vaginam is associated with funisitis in women with preterm prelabour rupture of the fetal membranes.

OBJECTIVE: To evaluate the risk of funisitis among women with preterm prelabour rupture of the membranes (PPROM) and subsequent bleeding per vaginam. DESIGN: Prospective cohort study. SETTING: A University Hospital in the USA. POPULATION: A total of 157 women with PPROM, divided into those with bleeding per vaginam during the hospital admission (n = 46) and those without bleeding per vaginam (n = 111). METHODS: Pathologist blinded to bleeding status assessed placental pathology for funisitis. MAIN OUTCOME MEASURES: Funisitis. RESULTS: Women with bleeding per vaginam were more likely to have funisitis (67.4% versus 36%, P < 0.001) compared with those without bleeding. Logistic regression demonstrated that bleeding per vaginam predicted funisitis after controlling for gestational age at admission, latency period and gestational age at delivery. CONCLUSIONS: Among women with PPROM, those with bleeding per vaginam are more likely to have funisitis than those without bleeding per vaginam.

Cryopreservation of buffy-coat-derived platelet concentrates in dimethyl sulfoxide and platelet additive solution.

Platelets prepared in plasma can be frozen in 6% dimethyl sulfoxide (Me(2)SO) and stored for extended periods at -80°C. The aim of this study was to reduce the plasma present in the cryopreserved product, by substituting plasma with platelet additive solution (PAS; SSP+), whilst maintaining in vitro platelet quality. Buffy coat-derived pooled leukoreduced platelet concentrates were frozen in a mixture of SSP+, plasma and 6% Me(2)SO. The platelets were concentrated, to avoid post-thaw washing, and frozen at -80°C. The cryopreserved platelet units (n=9) were rapidly thawed at 37°C, reconstituted in 50% SSP+/plasma and stored at 22°C. Platelet recovery and quality were examined 1 and 24h post-thaw and compared to the pre-freeze samples. Upon thawing, platelet recovery ranged from 60% to 80%. However, there were differences between frozen and liquid-stored platelets, including a reduction in aggregation in response to ADP and collagen; increased CD62P expression; decreased viability; increased apoptosis and some loss of mitochondrial membrane integrity. Some recovery of these parameters was detected at 24h post-thaw, indicating an extended shelf-life may be possible. The data suggests that freezing platelets in 6% Me(2)SO and additive solution produces acceptable in vitro platelet quality.

The cyclin box fold: protein recognition in cell-cycle and transcription control.

Regulation of both the cell cycle and gene transcription is essential for orderly progression of cell growth and division. Recent results on the structures of two cyclins, cyclin A and cyclin H, and two transcription factor mediator proteins, TFIIB and the A pocket region of the retinoblastoma tumour suppressor protein (Rb), show that they share domains with a strikingly similar alpha-helical topology, despite remote sequence identity.

Control by phosphorylation.

The two examples of phospho and dephospho proteins for which structural data were previously available (glycogen phosphorylase and isocitrate dehydrogenase) demonstrated two different mechanisms for control. In glycogen phosphorylase, activation by phosphorylation results in long-range allosteric changes. In isocitrate dehydrogenase, inhibition by phosphorylation is achieved by an electrostatic blocking mechanism with no conformational changes. During the past year, the structures of the phospho and dephospho forms of two more proteins, the cell cycle protein kinase CDK2 and yeast glycogen phosphorylase, have been determined. The new results highlight the importance of the phosphoamino acids both in the organization of local regions of protein structure through phosphate-arginine interactions and in the promotion of long-range conformational responses.

Two structures of the catalytic domain of phosphorylase kinase: an active protein kinase complexed with substrate analogue and product.

BACKGROUND: Control of intracellular events by protein phosphorylation is promoted by specific protein kinases. All the known protein kinase possess a common structure that defines a catalytically competent entity termed the 'kinase catalytic core'. Within this common structural framework each kinase displays its own unique substrate specificity, and a regulatory mechanism that may be modulated by association with other proteins. Structural studies of phosphorylase kinase (Phk), the major substrate of which is glycogen phosphorylase, may be expected to shed light on its regulation. RESULTS: We report two crystal structures of the catalytic core (residues 1-298; Phk gamma trnc) of the gamma-subunit of rabbit muscle phosphorylase kinase: the binary complex with Mn2+/beta-gamma-imidoadenosine 5'-triphosphate (AMPPNP) to a resolution of 2.6 A and the binary complex with Mg2+/ADP to a resolution of 3.0 A. The structures were solved by molecular replacement using the cAMP-dependent protein kinase (cAPK) as a model. CONCLUSIONS: The overall structure of Phk gamma trnc is similar to that of the catalytic core of other protein kinases. It consists of two domians joined on one edge by a 'hinge', with the catalytic site located in the cleft between the domains. Phk gamma trnc is constitutively active, and lacks the need for an activatory phosphorylation event that is essential for many kinases. The structure exhibits an essentially 'closed' conformation of the domains which is similar to that of cAPK complexed with substrates. The phosphorylated residue that is located at the domain interface in many protein kinases and that is believed to stabilize an active conformation is substituted by a glutamate in Phk gamma trnc. The glutamate, in a similar manner to the phosphorylated residue in other protein kinases, interacts with an arginine adjacent to the catalytic aspartate but does not participate in interdomain contacts. The interactions between the enzyme and the nucleotide product of its activity, Mg2+/ADP, explain the inhibitory properties of the nucleotides that are observed in kinetic studies.

A new allosteric site in glycogen phosphorylase b as a target for drug interactions.

BACKGROUND: In muscle and liver, glycogen concentrations are regulated by the coordinated activities of glycogen phosphorylase (GP) and glycogen synthase. GP exists in two forms: the dephosphorylated low-activity form GPb and the phosphorylated high-activity form GPa. In both forms, allosteric effectors can promote equilibrium between a less active T state and a more active R state. GP is a possible target for drugs that aim to prevent unwanted glycogen breakdown and to stimulate glycogen synthesis in non-insulin-dependent diabetes. As a result of a data bank search, 5-chloro-1H-indole-2-carboxylic acid (1-(4-fluorobenzyl)-2-(4-hydroxypiperidin-1-yl)-2-oxoethy l)amide, CP320626, was identified as a potent inhibitor of human liver GP. Structural studies have been carried out in order to establish the mechanism of this unusual inhibitor. RESULTS: The structure of the cocrystallised GPb-CP320626 complex has been determined to 2.3 A resolution. CP320626 binds at a site located at the subunit interface in the region of the central cavity of the dimeric structure. The site has not previously been observed to bind ligands and is some 15 A from the AMP allosteric site and 33 A from the catalytic site. The contacts between GPb and CP320626 comprise six hydrogen bonds and extensive van der Waals interactions that create a tight binding site in the T-state conformation of GPb. In the R-state conformation of GPa these interactions are significantly diminished. CONCLUSIONS: CP320626 inhibits GPb by binding at a new allosteric site. Although over 30 A from the catalytic site, the inhibitor exerts its effects by stabilising the T state at the expense of the R state and thereby shifting the allosteric equilibrium between the two states. The new allosteric binding site offers a further recognition site in the search for improved GP inhibitors.

The crystal structure of cyclin A.

BACKGROUND: Eukaryotic cell cycle progression is regulated by cyclin dependent protein kinases (CDKs) whose activity is regulated by association with cyclins and by reversible phosphorylation. Cyclins also determine the subcellular location and substrate specificity of CDKs. Cyclins exhibit diverse sequences but all share homology over a region of approximately 100 amino acids, termed the cyclin box. From the determination of the structure of cyclin A, together with results from biochemical and genetic analyses, we can identify which parts of the cyclin molecular may contribute to cyclin A structure and function. RESULTS: We have solved the crystal structure, at 2.0 A resolution, of an active recombinant fragment of bovine cyclin A, cyclin A-3, corresponding to residues 171-432 of human cyclin A. The cyclin box has an alpha-helical fold comprising five alpha helices. This fold is repeated in the C-terminal region, although this region shares negligible sequence similarity with the cyclin box. CONCLUSIONS: Analysis of residues that are conserved throughout the A, B, and E cyclins identifies two exposed clusters of residues, one of which has recently been shown to be involved in the association with human CDK2. The second cluster may identify another site of cyclin A-protein interaction. Comparison of the structure of the unbound cyclin with the structure of cyclin A complexed with CDK2 reveals that cyclin A does not undergo any significant conformational changes on complex formation. Threading analysis shows that the cyclin-box fold is consistent with the sequences of the transcription factor TFIIB and other functionally related proteins. The structural results indicate a role for the cyclin-box fold both as a template for the cyclin family and as a generalised adaptor molecule in the regulation of transcription.

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.

Evaluation of owner experiences and adherence to home-cooked diet recipes for dogs.

OBJECTIVES: To evaluate owner experiences and adherence to home-cooked diet recipes for dogs. METHODS: Clients of a veterinary teaching hospital clinical nutrition service who had a home-cooked diet recipe formulated for their dogs between March 2011 and December 2013 were given a survey by email, postal mail and telephone. Survey questions addressed motivations, positive and negative aspects of feeding home-cooked diets and current feeding practices. Responses were compared to animals' medical records to determine adherence. RESULTS: Of the 93 owners who were contacted, 53 (57%) completed the survey. Of the 53 respondents, 43 owners (81%) reported that they were still feeding an home-cooked diet or had fed an home-cooked diet until the time of their dogs' deaths. The most common motivation for feeding a home-cooked diet was suitability for specific medical needs. Of the 30 surveys that included a complete diet history, only four (13%) demonstrated exact adherence to home-cooked diet recipes. CLINICAL SIGNIFICANCE: Most respondents liked and continued to feed a home-cooked diet, but few owners adhered to prescribed recipes and many dogs required recipe modifications. It is important to counsel dog owners about benefits and drawbacks of feeding home-cooked diets, importance of recipe adherence and necessity for follow-up after diet formulation with a board-certified veterinary nutritionist.

Crystallization of pig skeletal phosphorylase b. Purification, physical and catalytic characterization.

A new method for purification and crystallization of pig skeletal muscle phosphorylase b is presented. The ease of crystallization in the presence of 1 mM AMP and 1 mM spermine has permitted the study of some physical, chemical and enzymatic properties of the enzyme. The crystalline pig phosphorylase b gave a single band on SDS polyacrylamide gels of the same mobility as rabbit muscle phosphorylase subunit. Ultracentrifugation experiments showed that pig phosphorylase b exists in a dimeric form (S20,w = 8.4 S). No association occurred at 20 degrees C under conditions where rabbit phosphorylase b can be tetramerized; pig phosphorylase b was only 30% associated from dimer to tetramer at 13 degrees C. Pig phosphorylase b is highly stable to freezing and its specific activity did not change appreciably upon prolonged storage in the cold. Pig and rabbit phosphorylases b have comparable Vmax and Km values towards the substrate and the activator. However, there is an essential difference between the two enzymes in that pig phosphorylase b is not significantly inhibited by glucose 6-phosphate, which is a powerful inhibitor of the rabbit enzyme. Two different crystal forms of pig phosphorylase b were obtained which are small for X-ray diffraction studies. Diffusion of spermine into tetragonal crystals of rabbit phosphorylase b resulted in a difference Fourier synthesis at 3 A resolution that showed no strong indication of specific binding.

Structural mechanism for glycogen phosphorylase control by phosphorylation and AMP.

The crystal structures of activated R state glycogen phosphorylase a (GPa) and R and T state glycogen phosphorylase b (GPb) complexed with AMP have been solved at 2.9 A, 2.9 A and 2.2 A resolution, respectively. The structure of R state GPa is nearly identical to the structure of sulphate-activated R state GPb, except in the region of Ser14, where there is a covalently attached phosphate group in GPa and a non-covalently attached sulphate group in GPb. The contacts made by the N-terminal tail residues in R state GPa at the subunit interface of the functionally active dimer are similar to those observed previously for T state GPa. 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 GPa. The transition from the T state GPb structure to the R state GPa 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 GPa and T state GPb is 50 A. One of the important subunit-subunit interactions in the dimer molecule involves contacts between the helix alpha 2 and the cap' (residues 35' to 45' that form a loop between the 1st and 2nd alpha helices, alpha 1' and alpha 2' 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'/alpha 2 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 A 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 A. AMP binds to R state GPb 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 A2 greater solvent accessible surface area buried compared with AMP bound to T state GPb.(ABSTRACT TRUNCATED AT 400 WORDS)

Enzymatic catalysis in crystals of Escherichia coli maltodextrin phosphorylase.

The bacterial enzyme maltodextrin phosphorylase (MalP) catalyses the phosphorolysis of an alpha-1,4-glycosidic bond in maltodextrins, removing the non-reducing glucosyl residues of linear oligosaccharides as glucose-1-phosphate (Glc1P). In contrast to the well-studied muscle glycogen phosphorylase (GP), MalP exhibits no allosteric properties and has a higher affinity for linear oligosaccharides than GP. We have used MalP as a model system to study catalysis in the crystal in the direction of maltodextrin synthesis. The 2.0A crystal structure of the MalP/Glc1P binary complex shows that the Glc1P substrate adopts a conformation seen previously with both inactive and active forms of mammalian GP, with the phosphate group not in close contact with the 5'-phosphate group of the essential pyridoxal phosphate (PLP) cofactor. In the active MalP enzyme, the residue Arg569 stabilizes the negative-charged Glc1P, whereas in the inactive form of GP this key residue is held away from the catalytic site by loop 280s and an allosteric transition of the mammalian enzyme is required for activation. The comparison between MalP structures shows that His377, through a hydrogen bond with the 6-hydroxyl group of Glc1P substrate, triggers a conformational change of the 380s loop. This mobile region folds over the catalytic site and contributes to the specific recognition of the oligosaccharide and to the synergism between substrates in promoting the formation of the MalP ternary complex. The structures solved after the diffusion of oligosaccharides (either maltotetraose, G4 or maltopentaose, G5) into MalP/Glc1P crystals show the formation of phosphate and elongation of the oligosaccharide chain. These structures, refined at 1.8A and at 2.2A, confirm that only when an oligosaccharide is bound to the catalytic site will Glc1P bend its phosphate group down so it can contact the PLP 5' phosphate group and promote catalysis. The relatively large oligosaccharide substrates can diffuse quickly into the MalP/Glc1P crystals and the enzymatic reaction can occur without significant crystal damage. These structures obtained before and after catalysis have been used as frames of a molecular movie. This movie reveals the relative positions of substrates in the catalytic channel and shows a minimal movement of the protein, involving mainly Arg569, which tracks the substrate phosphate group.

Expression, purification and crystallisation of phosphorylase kinase catalytic domain.

The catalytic subunit of phosphorylase kinase is composed of a kinase catalytic core domain (residues 1 to 298), which has a 33% identity with the kinase core of the cyclic AMP-dependent protein kinase, and a C-terminal calmodulin binding domain. The kinase domain of the catalytic subunit has been expressed in Escherichia coli, purified and crystallised in the presence of ATP and magnesium from 5% (w/v) polyethylene glycol 8000, 10% (v/v) glycerol, 50 mM Hepes/NaOH (pH 6.9). A three-fold excess of magnesium to ATP was used for crystal growth. The inclusion of glycerol in the crystallization medium produced a marked reduction in mosaic spread of the diffraction spots from greater than 1 degree to 0.3 degree. The crystals are orthorhombic, space group P2(1)2(1)2(1) with unit cell dimensions a = 47.1 A, b = 69.1 A, c = 112.9 A and one molecule per asymmetric unit. Data to 3 A resolution have been collected and structure determination is in progress.

Crystallographic binding studies on the allosteric inhibitor glucose-6-phosphate to T state glycogen phosphorylase b.

Glucose-6-phosphate is an important allosteric inhibitor of glycogen phosphorylase b that restrains the enzyme in the inactive state in resting muscle. A crystallographic binding study by diffusion of glucose-6-phosphate into performed crystals of T state phosphorylase b has been carried out at 2.3 A resolution and the structure refined by restrained crystallographic least-squares and simulated annealing to give a crystallographic R-value of 0.203. The inhibitor binds at the AMP allosteric effector site at the subunit-subunit interface of the dimer. The phosphate groups of the glucose-6-phosphate and AMP occupy partially overlapping sites and make similar contacts to two arginine residues (Arg309 and Arg310) but in glucose-6-phosphate there is a contact to a third arginine (Arg242). The glucopyranose of glucose-6-phosphate and the adenine ribose of AMP occupy different positions. Including the contacts to the three arginine residues by the phosphate group, the glucose-6-phosphate makes a total of 11 hydrogen-bonds to the enzyme and all but one of these are to charged groups. The O-2 hydroxyl hydrogen-bonds to the main-chain carbonyl oxygen of Val40' from the other subunit and this interaction appears important for the allosteric response. There are substantial conformational changes both in the vicinity of the glucose-6-phosphate (involving for example Phe196 and Arg309) and at the subunit interface (involving residues 42' to 51' and 192 to 196). These shifts tighten the binding of the inhibitor and the interface. Comparison of the glucose-6-phosphate complex with the T state native phosphorylase b and the R state phosphorylase a structures shows that there is a graded response from T state glucose-6-phosphate complex through T state phosphorylase b to R state phosphorylase a that suggests that glucose-6-phosphate promotes a tight structure that is more "tensed" than native T state phosphorylase b. The results show how the same allosteric effector site can exhibit a tight binding site for the activator AMP in the R state structure and a tight binding site for glucose-6-phosphate in the modified T state structure.

The binding of 2-deoxy-D-glucose 6-phosphate to glycogen phosphorylase b: kinetic and crystallographic studies.

Kinetic and crystallographic studies have characterized the effect of 2-deoxy-glucose 6-phosphate on the catalytic and structural properties of glycogen phosphorylase b. Previous work on the binding of glucose 6-phosphate, a potent physiological inhibitor of the enzyme, to T state phosphorylase b in the crystal showed that the inhibitor binds at the allosteric site and induces substantial conformational changes that affect the subunit-subunit interface. The hydrogen-bond from the O-2 hydroxyl of glucose 6-phosphate to the main-chain oxygen of Val40' represents the only hydrogen bond from the sugar to the other subunit, and this interaction appears important for promoting a more "tensed" structure than native T state phosphorylase b. 2-Deoxy-glucose 6-phosphate acts competitively with both the activator AMP and the substrate glucose 1-phosphate, with Ki values of 0.53 mM and 1.23 mM, respectively. The binding of 2-deoxy-glucose 6-phosphate to T state glycogen phosphorylase b in the crystal, has been investigated and the complex phosphorylase b: 2-deoxy-glucose 6-phosphate has been refined to give a crystallographic R factor of 17.3%, for data between 8 A and 2.3 A. 2-Deoxy-glucose 6-phosphate binds at the allosteric site as the a anomer and adopts a different conformation compared to glucose 6-phosphate. The two conformations differ by 160 degrees in the torsion angle about the C-5-C-6 bond. The contacts from the phosphate group are essentially identical to those made by the phosphate of glucose 6-phosphate but the 2-deoxy glucosyl moiety binds in a quite different orientation compared to the glucosyl of glucose 6-phosphate. 2-Deoxy-glucose 6-phosphate can be accommodated in the allosteric site with very little change in the protein, while structural comparisons show that the phosphorylase b: 2-deoxy-glucose 6-phosphate complex structure is overall more similar to a glucose-like complex than to the Glc-6-P complex structure.

Refined crystal structure of the phosphorylase-heptulose 2-phosphate-oligosaccharide-AMP complex.

The crystal structure of phosphorylase b-heptulose 2-phosphate complex with oligosaccharide and AMP bound has been refined by molecular dynamics and crystallographic least-squares with the program XPLOR. Shifts in atomic positions of up to 4 A from the native enzyme structure were correctly determined by the program without manual intervention. The final crystallographic R value for data between 8 and 2.86 A resolution is 0.201, and the overall root-mean-square difference between the native and complexed structure is 0.58 A for all protein atoms. The results confirm the previous observation that there is a direct hydrogen bond between the phosphate of heptulose 2-phosphate and the pyridoxal phosphate 5'-phosphate group. The close proximity of the two phosphates is stabilized by an arginine residue, Arg569, which shifts from a site buried in the protein to a position where it can make contact with the product phosphate. There is a mutual interchange in position between the arginine and an acidic group, Asp283. These movements represent the first stage of the allosteric response which converts the catalytic site from a low to a high-affinity binding site. Communication of these changes to other sites is prevented in the crystal by the lattice forces, which also form the subunit interface. The constellation of groups in the phosphorylase transition state analogue complex provides a structural basis for understanding the catalytic mechanism in which the cofactor pyridoxal phosphate 5'-phosphate group functions as a general acid to promote attack by the substrate phosphate on the glycosidic bond when the reaction proceeds in the direction of glycogen degradation. In the direction of glycogen synthesis, stereoelectronic effects contribute to the cleavage of the C-1-O-1 bond. In both reactions the substrate phosphate plays a key role in transition state stabilization. The details of the oligosaccharide, maltoheptaose, interactions with the enzyme at the glycogen storage site are also described.

Comparison of AMP and NADH binding to glycogen phosphorylase b.

The binding sites for the allosteric activator, AMP, to glycogen phosphorylase b are described in detail utilizing the more precise knowledge of the native structure obtained from crystallographic restrained least-squares refinement than has hitherto been available. Localized conformational changes are seen at the allosteric effector site that include shifts of between 1 and 2 A for residues Tyr75 and Arg309 and very small shifts for the region of residues 42 to 44 from the symmetry-related subunit. Kinetic studies demonstrate that NADH inhibits the AMP activation of glycogen phosphorylase b. Crystallographic binding studies at 3.5 A resolution show that NADH binds to the same sites on the enzyme as AMP, i.e. the allosteric effector site N, which is close to the subunit-subunit interface, and the nucleoside inhibitor site I, which is some 12 A from the catalytic site. The conformations of NADH at the two sites are different but both conformations are "folded" so that the nicotinamide ring is close (approx. 6 A) to the adenine ring. These conformations are compared with those suggested from solution studies and with the extended conformations observed in the single crystal structure of NAD+ and for NAD bound to dehydrogenases. Possible mechanisms for NADH inhibition of phosphorylase activation are discussed.

Crystal structure of the mutant D52S hen egg white lysozyme with an oligosaccharide product.

The crystal structure of a mutant hen egg white lysozyme, in which the key catalytic residue aspartic acid 52 has been changed to a serine residue (D52S HEWL), has been determined and refined to a crystallographic R value of 0.173 for all data F > 0 between 8 and 1.9 A resolution. The D52S HEWL structure is very similar to the native HEWL structure (r.m.s. deviation of main-chain atoms 0.20 A). Small shifts that result from the change in hydrogen bonding pattern on substitution of Asp by Ser were observed in the loop between beta-strands in the region of residues 46 to 49. D52S HEWL exhibits less than 1% activity against the bacterial cell wall substrate. Cocrystallisation experiments with the hexasaccharide substrate beta(1-4) polymer of N-acetyl-D-glucosamine (GlcNAc6) resulted in crystals between 5 days and 14 days after the initial mixing of enzyme and substrate. Analysis by laser absorption mass spectrometry of the oligosaccharides present after incubation with native and D52S HEWL under conditions similar to those used for crystal growth showed that after 14 days with native HEWL complete catalysis to GlcNAc3. GlcNAc2 and GlcNac had occurred but with D52S HEWL only partial catalysis to the major products GlcNAc4 and GlcNAc2 had occurred and at least 50% of the GlcNAc6 remained intact. X-ray analysis of the D52S-oligosaccharide complex crystals showed that they contained the product GlcNAc4. The structure of the D52S HEWL-GlcNAc4 complex has been determined and refined to an R value of 0.160 for data between 8 and 2 A resolution. GlcNAc4 occupies sites A to D in the active site cleft. Careful refinement and examination of 2Fo-Fc electron density maps showed that the sugar in site D has the sofa conformation, a conformation previously observed with the HEWL complex with tetra-N-acetylglucosamine lactone transition state analogue, the HEWL complex with the cell wall trisaccharide and the phage T4 lysozyme complex with a cell wall product. The semi-axial C(5)-C(6) geometry of the sofa is stabilised by hydrogen bonds from the O-6 hydroxyl group to the main-chain N of Val109 and main-chain O of Ala107. The sugar in site D adopts the alpha configuration, seemingly in conflict with the observation that the hydrolysis of beta (1-4) glycosidie linkage by HEWL proceeds with 99.9% retention of beta-configuration.(ABSTRACT TRUNCATED AT 400 WORDS)

Murine synaptosomal lipid raft protein and lipid composition are altered by expression of human apoE 3 and 4 and by increasing age.

Apolipoprotein E (apoE) 4 and aging are risk factors for Alzheimer's disease (AD). Mice expressing human apoE4 and aged wild-type mice show a similarity in the transbilayer distribution of cholesterol in synaptic plasma membranes (SPMs) but differ markedly compared with apoE3 mice and young mice. The largest changes in cholesterol distribution were observed in the SPM exofacial leaflet where there was a doubling of cholesterol. Lipid rafts are thought to be associated with the exofacial leaflet, and we proposed that lipid raft protein and lipid composition would be associated with apoE genotype and age. Lipid rafts were isolated from synaptosomes of different age groups (2, 12, 24 months) of mice expressing human apoE3 and apoE4. Lipid raft markers, alkaline phosphatase (ALP), flotillin-1, cholesterol and sphingomyelin (SM) were examined. Lipid rafts of young apoE4 mice were more similar to older mice as compared with young apoE3 mice in reductions in alkaline phosphatase activity and flotillin-1 abundance. Lipid raft cholesterol and sphingomyelin levels were not significantly different between the young apoE3 and apoE4 mice but cholesterol levels of lipid rafts did increase with age in both genotypes. Results of the present study demonstrate that the two risk factors for Alzheimer's disease, apoE4 genotype and increasing age have similar effects on brain lipid raft protein markers and these findings support the notion that the transbilayer distribution of cholesterol is associated with lipid raft function.