Binding of Pro-Gly-Pro at the active site of leukotriene A4 hydrolase/aminopeptidase and development of an epoxide hydrolase selective inhibitor.

Leukotriene (LT) A4 hydrolase/aminopeptidase (LTA4H) is a bifunctional zinc metalloenzyme that catalyzes the committed step in the formation of the proinflammatory mediator LTB4. Recently, the chemotactic tripeptide Pro-Gly-Pro was identified as an endogenous aminopeptidase substrate for LTA4 hydrolase. Here, we determined the crystal structure of LTA4 hydrolase in complex with a Pro-Gly-Pro analog at 1.72 Å. From the structure, which includes the catalytic water, and mass spectrometric analysis of enzymatic hydrolysis products of Pro-Gly-Pro, it could be inferred that LTA4 hydrolase cleaves at the N terminus of the palindromic tripeptide. Furthermore, we designed a small molecule, 4-(4-benzylphenyl)thiazol-2-amine, denoted ARM1, that inhibits LTB4 synthesis in human neutrophils (IC50 of ∼0.5 µM) and conversion of LTA4 into LTB4 by purified LTA4H with a Ki of 2.3 µM. In contrast, 50- to 100-fold higher concentrations of ARM1 did not significantly affect hydrolysis of Pro-Gly-Pro. A 1.62-Å crystal structure of LTA4 hydrolase in a dual complex with ARM1 and the Pro-Gly-Pro analog revealed that ARM1 binds in the hydrophobic pocket that accommodates the ω-end of LTA4, distant from the aminopeptidase active site, thus providing a molecular basis for its inhibitory profile. Hence, ARM1 selectively blocks conversion of LTA4 into LTB4, although sparing the enzyme's anti-inflammatory aminopeptidase activity (i.e., degradation and inactivation of Pro-Gly-Pro). ARM1 represents a new class of LTA4 hydrolase inhibitor that holds promise for improved anti-inflammatory properties.

Crystal structures of leukotriene C4 synthase in complex with product analogs: implications for the enzyme mechanism.

Leukotriene (LT) C4 synthase (LTC4S) catalyzes the conjugation of the fatty acid LTA4 with the tripeptide GSH to produce LTC4, the parent compound of the cysteinyl leukotrienes, important mediators of asthma. Here we mutated Trp-116 in human LTC4S, a residue proposed to play a key role in substrate binding, into an Ala or Phe. Biochemical and structural characterization of these mutants along with crystal structures of the wild type and mutated enzymes in complex with three product analogs, viz. S-hexyl-, 4-phenyl-butyl-, and 2-hydroxy-4-phenyl-butyl-glutathione, provide new insights to binding of substrates and product, identify a new conformation of the GSH moiety at the active site, and suggest a route for product release, aided by Trp-116.

Repeatability of radiographic assessments for feline hip dysplasia suggest consensus scores in radiology are more uncertain than commonly assumed.

Variation in the diagnostic interpretation of radiographs is a well-recognised problem in human and veterinary medicine. One common solution is to create a 'consensus' score based on a majority or unanimous decision from multiple observers. While consensus approaches are generally assumed to improve diagnostic repeatability, the extent to which consensus scores are themselves repeatable has rarely been examined. Here we use repeated assessments by three radiologists of 196 hip radiographs from 98 cats within a health-screening programme to examine intra-observer, inter-observer, majority-consensus and unanimous-consensus repeatability scores for feline hip dysplasia. In line with other studies, intra-observer and inter-observer repeatability was moderate (63-71%), and related to the reference assessment and time taken to reach a decision. Consensus scores did show reduced variation between assessments compared to individuals, but consensus repeatability was far from perfect. Only 75% of majority consensus scores were in agreement between assessments, and based on Bayesian multinomial modelling we estimate that unanimous consensus scores can have repeatabilities as low as 83%. These results clearly show that consensus scores in radiology can have large uncertainties, and that future studies in both human and veterinary medicine need to include consensus-uncertainty estimates if we are to properly interpret radiological diagnoses and the extent to which consensus scores improve diagnostic accuracy.

Noncooperative folding of subdomains in adenylate kinase.

Conformational change is regulating the biological activity of a large number of proteins and enzymes. Efforts in structural biology have provided molecular descriptions of the interactions that stabilize the stable ground states on the reaction trajectories during conformational change. Less is known about equilibrium thermodynamic stabilities of the polypeptide segments that participate in structural changes and whether the stabilities are relevant for the reaction pathway. Adenylate kinase (Adk) is composed of three subdomains: CORE, ATPlid, and AMPbd. ATPlid and AMPbd are flexible nucleotide binding subdomains where large-scale conformational changes are directly coupled to catalytic activity. In this report, the equilibrium thermodynamic stabilities of Adk from both mesophilic and hyperthermophilic bacteria were investigated using solution state NMR spectroscopy together with protein engineering experiments. Equilibrium hydrogen to deuterium exchange experiments indicate that the flexible subdomains are of significantly lower thermodynamic stability compared to the CORE subdomain. Using site-directed mutagenesis, parts of ATPlid and AMPbd could be selectively unfolded as a result of perturbation of hydrophobic clusters located in these respective subdomains. Analysis of the perturbed Adk variants using NMR spin relaxation and C(alpha) chemical shifts shows that the CORE subdomain can fold independently of ATPlid and AMPbd; consequently, folding of the two flexible subdomains occurs independently of each other. Based on the experimental results it is apparent that the flexible subdomains fold into their native structure in a noncooperative manner with respect to the CORE subdomain. These results are discussed in light of the catalytically relevant conformational change of ATPlid and AMPbd.

Conformational flexibility and dynamics of two (1-->6)-linked disaccharides related to an oligosaccharide epitope expressed on malignant tumour cells.

The conformational flexibility and dynamics of two (1-->6)-linked disaccharides that are related to the action of the glycosyl transferase GnT-V have been investigated. NMR NOE and T-ROE spectroscopy experiments, conformation-dependent coupling constants and molecular dynamics (MD) simulations were used in the analyses. To facilitate these studies, the compounds were synthesised as alpha-d-[6-(13)C]-Manp-OMe derivatives, which reduced the (1)H NMR spectral overlap and facilitated the determination of two- and three-bond (1)H,(1)H, (1)H,(13)C and (13)C,(13)C-coupling constants. The population distribution for the glycosidic omega torsion angle in alpha-d-Manp-(1-->6)-alpha-d-Manp-OMe for gt/gg/tg was equal to 45:50:5, whereas in alpha-d-Manp-OMe it was determined to be 56:36:8. The dynamic model that was generated for beta-d-GlcpNAc-(1-->6)-alpha-d-Manp-OMe by MD simulations employing the PARM22/SU01 CHARMM-based force field was in very good agreement with experimental observations. beta-d-GlcpNAc-(1-->6)-alpha-d-Manp-OMe is described by an equilibrium of populated states in which the phi torsion angle has the exo-anomeric conformation, the psi torsion angle an extended antiperiplanar conformation and the omega torsion angle a distribution of populations predominantly between the gauche-trans and the gauche-gauche conformational states (i.e., gt/gg/tg) is equal to 60:35:5, respectively. The use of site-specific (13)C labelling in these disaccharides leads to increased spectral dispersion, thereby making NMR spectroscopy based conformational analysis possible that otherwise might be difficult to attain.

An integrated approach to NMR spin relaxation in flexible biomolecules: application to beta-D-glucopyranosyl-(1-->6)-alpha-D-mannopyranosyl-OMe.

The description of the reorientational dynamics of flexible molecules is a challenging task, in particular when the rates of internal and global motions are comparable. The commonly used simple mode-decoupling models are based on the assumption of statistical independence between these motions. This assumption is not valid when the time scale separation between their rates is small, a situation that was found to arise in oligosaccharides in the context of certain internal motions. To make possible the interpretation of NMR spin relaxation data from such molecules, we developed a comprehensive approach generally applicable to flexible rotators with one internal degree of freedom. This approach integrates a stochastic description of coupled global tumbling and internal torsional motion, quantum chemical calculations of the local potential and the local geometry at the site of the restricted torsion, and hydrodynamics-based calculations of the diffusive properties. The method is applied to the disaccharide beta-D-Glcp-(1-->6)-alpha-D-[6-(13)C]-Manp-OMe dissolved in a DMSO-d(6)/D(2)O cryosolvent. The experimental NMR relaxation parameters, associated with the (13)CH(2) probe residing at the glycosidic linkage, include (13)C T(1) and T(2) and (13)C-{(1)H} nuclear Overhauser enhancement (NOE) as well as longitudinal and transverse dipole-dipole cross-correlated relaxation rates, acquired in the temperature range of 253-293 K. These data are predicted successfully by the new theory with only the H-C-H angle allowed to vary. Previous attempts to fit these data using mode-decoupling models failed.

Demography, heritability and genetic correlation of feline hip dysplasia and response to selection in a health screening programme.

Feline hip dysplasia (FHD) is a debilitating condition affecting the hip joints of millions of domestic cats worldwide. Despite this, little is known about FHD except that it is relatively common in the large breed Maine Coon. We used 20 years of data from 5038 pedigree-registered Maine Coon cats in a radiographic health screening programme for FHD to determine, for the first time, its heritability, genetic correlation to body mass and response to selection. FHD prevalence was 37.4%, with no sex predilection; however, FHD severity increased with age and body mass. Heritability of the radiographic categories used to classify FHD severity was 0.36 (95%CI: 0.30-0.43). The severity of FHD symptoms was also genetically correlated with body mass (0.285), suggesting that selection for a large body type in this breed concurrently selects for FHD. Support for this was found by following generational responses to selective breeding against FHD. Not only did selective breeding successfully reduce the severity of FHD symptoms in descendants, but these cats were also smaller than their ancestors (-33g per generation). This study highlights the value of breeding programmes against FHD and cautions against breed standards that actively encourage large bodied cats.

Conformational analysis of beta-glycosidic linkages in 13C-labeled glucobiosides using inter-residue scalar coupling constants.

Four beta-linked glucobioses selectively (13)C labeled at C1' or C2' have been prepared. The inter-residue coupling constants, J(CH), and J(CC), have been determined and related to the solution conformations of the disaccharides using Karplus-type relationships. Relying only on the experimental coupling constants, glycosidic linkage conformation in methyl alpha-sophoroside (methyl 2-O-beta-D-glucopyranosyl-alpha-D-glucopyranoside), methyl alpha-laminarabioside (methyl 3-O-beta-D-glucopyranosyl-alpha-D-glucopyranoside), and methyl alpha-cellobioside (methyl 4-O-beta-D-glucopyranosyl-alpha-D-glucopyranoside) were found to be close to those observed in the solid state (39 degrees < phi(H) < 41 degrees , -24 degrees < psi(H) < -36 degrees ). The laminarabioside and cellobioside were found to have conformations that accommodate an intramolecular hydrogen bond to O5' that is observed in the solid state. In all compounds, the exocyclic hydroxymethyl groups retain a conformation close to that observed in unsubstituted glucose (gt/gg 1:1). Methyl alpha-gentiobioside (methyl 6-O-beta-D-glucopyranosyl-alpha-D-glucopyranoside) shows greater flexibility at the psi-torsion than the other disaccharides, but the population distribution around the C5-C6 bond is essentially unaffected by substitution. None of the O2' hydroxyl groups of the beta-D-glucopyranosyl residues in any of the disaccharides appear to be involved in inter-residue hydrogen bonding since (1)JCH, (1)JCC, and (2)JCH values sensitive to C2'-O2' rotamer distribution remain close to those observed in methyl beta-D-glucopyranoside.

Structural determination of the O-antigenic polysaccharide from the verocytotoxin-producing Escherichia coli O176.

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O176 has been determined. Component analysis together with 1H and 13C NMR spectroscopy was employed to elucidate the structure. Inter-residue correlations were determined by 1H, 1H NOESY and 1H, 13C heteronuclear multiple-bond correlation experiments. The PS is composed of tetrasaccharide repeating units with the following structure: [Formula: see text] Cross-peaks of low intensity from alpha-linked mannopyranosyl residues were present in the 1H, 1H TOCSY NMR spectra and further analysis of these showed that they originate from the terminal part of the polysaccharide. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-galactosamine residue at its reducing end. The repeating unit of the E. coli O176 O-antigen is similar to those from E. coli O17 and O77, thereby explaining the reported cross-reactivities between the strains, and identical to that of Salmonella cerro (O:6, 14, 18).

Structural analysis of the O-antigen polysaccharide from Escherichia coli O152.

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O152 has been determined. Component analysis together with 1H, 13C and 31P NMR spectroscopy were used to elucidate the structure. Inter-residue correlations were determined by 1H,31P COSY, 1H,1H NOESY and 1H,13C heteronuclear multiple-bond correlation experiments. The PS is composed of pentasaccharide repeating units with the following structure: [structure: see text]. The structure is similar to that of the O-antigen polysaccharide from E. coli O173. The cross-reactivity between E. coli O152 and E. coli O3 may be explained by structural similarities in the branching region of their O-antigen polysaccharides.

Efficient and specific conversion of 9-lipoxygenase hydroperoxides in the beetroot. Formation of pinellic acid.

The linoleate 9-lipoxygenase product 9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid was stirred with a crude enzyme preparation from the beetroot (Beta vulgaris ssp. vulgaris var. vulgaris) to afford a product consisting of 95% of 9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoic acid (pinellic acid). The linolenic acid-derived hydroperoxide 9(S)-hydroperoxy-10(E),12(Z),15(Z)-octadecatrienoic acid was converted in an analogous way into 9(S),12(S),13(S)-trihydroxy-10(E),15(Z)-octadecadienoic acid (fulgidic acid). On the other hand, the 13-lipoxygenase-generated hydroperoxides of linoleic or linolenic acids failed to produce significant amounts of trihydroxy acids. Short-time incubation of 9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid afforded the epoxy alcohol 12(R),13(S)-epoxy-9(S)-hydroxy-10(E)-octadecenoic acid as the main product indicating the sequence 9-hydroperoxide → epoxy alcohol → trihydroxy acid catalyzed by epoxy alcohol synthase and epoxide hydrolase activities, respectively. The high capacity of the enzyme system detected in beetroot combined with a simple isolation protocol made possible by the low amounts of endogenous lipids in the enzyme preparation offered an easy access to pinellic and fulgidic acids for use in biological and medical studies.

Overlap between folding and functional energy landscapes for adenylate kinase conformational change.

Enzyme function is often dependent on fluctuations between inactive and active structural ensembles. Adenylate kinase isolated from Escherichia coli (AK(e)) is a small phosphotransfer enzyme in which interconversion between inactive (open) and active (closed) conformations is rate limiting for catalysis. AK(e) has a modular three-dimensional architecture with two flexible substrate-binding domains that interact with the substrates AMP, ADP and ATP. Here, we show by using a combination of biophysical and mutagenic approaches that the interconversion between open and closed states of the ATP-binding subdomain involves partial subdomain unfolding/refolding in an otherwise folded enzyme. These results provide a novel and, possibly general, molecular mechanism for the switch between open and closed conformations in AK(e).