Structural basis for specific inhibition of salicylate synthase from Mycobacterium abscessus.

Blocking iron uptake and metabolism has been emerging as a promising therapeutic strategy for the development of novel antimicrobial compounds. Like all mycobacteria, M. abscessus (Mab) has evolved several countermeasures to scavenge iron from host carrier proteins, including the production of siderophores, which play a crucial role in these processes. In this study, we solved, for the first time, the crystal structure of Mab-SaS, the first enzyme involved in the biosynthesis of siderophores. Moreover, we screened a small, focused library and identified a compound exhibiting a potent inhibitory effect against Mab-SaS (IC50 ≈ 2 µM). Its binding mode was investigated by means of Induced Fit Docking simulations, performed on the crystal structure presented herein. Furthermore, cytotoxicity data and pharmacokinetic predictions revealed the safety and drug-likeness of this class of compounds. Finally, the crystallographic data were used to optimize the model for future virtual screening campaigns. Taken together, the findings of our study pave the way for the identification of potent Mab-SaS inhibitors, based on both established and unexplored chemotypes.

Targeting Siderophore-Mediated Iron Uptake in M. abscessus: A New Strategy to Limit the Virulence of Non-Tuberculous Mycobacteria.

Targeting pathogenic mechanisms, rather than essential processes, represents a very attractive approach for the development of new antimycobacterial drugs. In this context, iron acquisition routes have recently emerged as potentially druggable pathways. However, the importance of siderophore biosynthesis in the virulence and pathogenicity of M. abscessus (Mab) is still poorly understood. In this study, we investigated the Salicylate Synthase (SaS) of Mab as an innovative molecular target for the development of inhibitors of siderophore production. Notably, Mab-SaS does not have any counterpart in human cells, making it an interesting candidate for drug discovery. Starting from the analysis of the binding of a series of furan-based derivatives, previously identified by our group as inhibitors of MbtI from M. tuberculosis (Mtb), we successfully selected the lead compound 1, exhibiting a strong activity against Mab-SaS (IC50 ≈ 5 µM). Computational studies characterized the key interactions between 1 and the enzyme, highlighting the important roles of Y387, G421, and K207, the latter being one of the residues involved in the first step of the catalytic reaction. These results support the hypothesis that 5-phenylfuran-2-carboxylic acids are also a promising class of Mab-SaS inhibitors, paving the way for the optimization and rational design of more potent derivatives.

Distinct conformational changes occur within the intrinsically unstructured pro-domain of pro-Nerve Growth Factor in the presence of ATP and Mg2.

Nerve growth factor (NGF), the prototypical neurotrophic factor, is involved in the maintenance and growth of specific neuronal populations, whereas its precursor, proNGF, is involved in neuronal apoptosis. Binding of NGF or proNGF to TrkA, p75NTR , and VP10p receptors triggers complex intracellular signaling pathways that can be modulated by endogenous small-molecule ligands. Here, we show by isothermal titration calorimetry and NMR that ATP binds to the intrinsically disordered pro-peptide of proNGF with a micromolar dissociation constant. We demonstrate that Mg2+ , known to play a physiological role in neurons, modulates the ATP/proNGF interaction. An integrative structural biophysics analysis by small angle X-ray scattering and hydrogen-deuterium exchange mass spectrometry unveils that ATP binding induces a conformational rearrangement of the flexible pro-peptide domain of proNGF. This suggests that ATP may act as an allosteric modulator of the overall proNGF conformation, whose likely distinct biological activity may ultimately affect its physiological homeostasis.

Synthesis, Structure, and Characterization of 4,4'-(Anthracene-9,10-diylbis(ethyne-2,1-diyl))bis(1-methyl-1-pyridinium) Bismuth Iodide (C30H22N2)3Bi4I18, an Air, Water, and Thermally Stable 0D Hybrid Perovskite with High Photoluminescence Efficiency.

4,4'-(Anthracene-9,10-diylbis(ethyne-2,1-diyl))bis(1-methyl-1-pyridinium) bismuth iodide (C30H22N2)3Bi4I18 (AEPyBiI) was obtained as a black powder by a very simple route by mixing an acetone solution of BiI3 and an aqueous solution of C30H22N2I2. This novel perovskite is air and water stable and displays a remarkable thermal stability up to nearly 300 °C. The highly conjugated cation C30H22N2 2+ is hydrolytically stable, being nitrogen atoms quaternarized, and this accounts for the insensitivity of the perovskite toward water and atmospheric oxygen under ambient conditions. The cation in aqueous solution is highly fluorescent under UV irradiation (emitting yellow-orange light). AEPyBiI as well is intensely luminescent, its photoluminescence emission being more than 1 order of magnitude greater than that of high-quality InP epilayers. The crystal structure of AEPyBiI was determined using synchrotron radiation single-crystal X-ray diffraction. AEPyBiI was extensively characterized using a wide range of techniques, such as X-ray powder diffraction, diffuse reflectance UV-vis spectroscopy, Fourier transform infrared (FTIR) and Raman spectroscopies, thermogravimetry-differential thermal analysis (TG-DTA), elemental analysis, electrospray ionization mass spectroscopy (ESI-MS), and photoluminescence spectroscopy. AEPyBiI displays a zero-dimensional (0D) perovskite structure in which the inorganic part is constituted by binuclear units consisting of two face-sharing BiI6 octahedra (Bi2I9 3- units). The C30H22N2 2+ cations are stacked along the a-axis direction in a complex motif. Considering its noteworthy light-emitting properties coupled with an easy synthesis and environmental stability, and its composition that does not contain toxic lead or easily oxidable Sn(II), AEPyBiI is a promising candidate for environmentally friendly light-emitting devices.

Molecular architecture of the glycogen- committed PP1/PTG holoenzyme.

The delicate alternation between glycogen synthesis and degradation is governed by the interplay between key regulatory enzymes altering the activity of glycogen synthase and phosphorylase. Among these, the PP1 phosphatase promotes glycogenesis while inhibiting glycogenolysis. PP1 is, however, a master regulator of a variety of cellular processes, being conveniently directed to each of them by scaffolding subunits. PTG, Protein Targeting to Glycogen, addresses PP1 action to glycogen granules. In Lafora disease, the most aggressive pediatric epilepsy, genetic alterations leading to PTG accumulation cause the deposition of insoluble polyglucosans in neurons. Here, we report the crystallographic structure of the ternary complex PP1/PTG/carbohydrate. We further refine the mechanism of the PTG-mediated PP1 recruitment to glycogen by identifying i) an unusual combination of recruitment sites, ii) their contributions to the overall binding affinity, and iii) the conformational heterogeneity of this complex by in solution SAXS analyses.

Endogenous modulators of neurotrophin signaling: Landscape of the transient ATP-NGF interactions.

The Nerve Growth Factor (NGF) neurotrophin acts in the maintenance and growth of neuronal populations. Despite the detailed knowledge of NGF's role in neuron physiology, the structural and mechanistic determinants of NGF bioactivity modulated by essential endogenous ligands are still lacking. We present the results of an integrated structural and advanced computational approach to characterize the extracellular ATP-NGF interaction. We mapped by NMR the interacting surface and ATP orientation on NGF and revealed the functional role of this interaction in the binding to TrkA and p75NTR receptors by SPR. The role of divalent ions was explored in conjunction with ATP. Our results pinpoint ATP as a likely transient molecular modulator of NGF signaling, in health and disease states.

Unveiling the binding mode of perfluorooctanoic acid to human serum albumin.

Perfluorooctanoic acid (PFOA) is a toxic compound that is absorbed and distributed throughout the body by noncovalent binding to serum proteins such as human serum albumin (hSA). Though the interaction between PFOA and hSA has been already assessed using various analytical techniques, a high resolution and detailed analysis of the binding mode is still lacking. We report here the crystal structure of hSA in complex with PFOA and a medium-chain saturated fatty acid (FA). A total of eight distinct binding sites, four occupied by PFOAs and four by FAs, have been identified. In solution binding studies confirmed the 4:1 PFOA-hSA stoichiometry and revealed the presence of one high and three low affinity binding sites. Competition experiments with known hSA-binding drugs allowed locating the high affinity binding site in sub-domain IIIA. The elucidation of the molecular basis of the interaction between PFOA and hSA might provide not only a better assessment of the absorption and elimination mechanisms of these compounds in vivo but also have implications for the development of novel molecular receptors for diagnostic and biotechnological applications.

Covalent immobilization of delipidated human serum albumin on poly(pyrrole-2-carboxylic) acid film for the impedimetric detection of perfluorooctanoic acid.

The immobilization of biomolecules at screen printed electrodes for biosensing applications is still an open challenge. To enrich the toolbox of bioelectrochemists, graphite screen printed electrodes (G-SPE) were modified with an electropolymerized film of pyrrole-2-carboxilic acid (Py-2-COOH), a pyrrole derivative rich in carboxylic acid functional groups. These functionalities are suitable for the covalent immobilization of biomolecular recognition layers. The electropolymerization was first optimized to obtain stable and conductive polymeric films, comparing two different electrolytes: sodium dodecyl sulphate (SDS) and sodium perchlorate. The G-SPE modified with Py-2-COOH in 0.1 M SDS solution showed the required properties and were further tested. A proof-of-concept study for the development of an impedimetric sensor for perfluorooctanoic acid (PFOA) was carried out using the delipidated human serum albumin (hSA) as bioreceptor. The data interpretation was supported by size exclusion chromatography and small-angle X-ray scattering (SEC-SAXS) analysis of the bioreceptor-target complex and the preliminary results suggest the possibility to further develop this biosensing strategy for toxicological and analytical studies.

A novel water-resistant and thermally stable black lead halide perovskite, phenyl viologen lead iodide C22H18N2(PbI3)2.

A novel black organoammonium iodoplumbate semiconductor, namely phenyl viologen lead iodide C22H18N2(PbI3)2 (PhVPI), was successfully synthesized and characterized. This material showed physical and chemical properties suitable for photovoltaic applications. Indeed, low direct allowed band gap energy (Eg = 1.32 eV) and high thermal stability (up to at least 300 °C) compared to methylammonium lead iodide CH3NH3PbI3 (MAPI, Eg = 1.5 eV) render PhVPI potentially attractive for solar cell fabrication. The compound was extensively characterized by means of X-ray diffraction (performed on both powder and single crystals), UV-Vis diffuse reflectance spectroscopy (UV-Vis DRS), UV-photoelectron spectroscopy (UPS), FT-IR spectroscopy, TG-DTA, and CHNS analysis. Reactivity towards water was monitored through X-ray powder diffraction carried out after prolonged immersion of the material in water at room temperature. Unlike its methyl ammonium counterpart, PhVPI proved to be unaffected by water exposure. The lack of reactivity towards water is to be attributed to the quaternary nature of the nitrogen atoms of the phenyl viologen units that prevents the formation of acid-base equilibria when in contact with water. On the other hand, PhVPI's thermal stability was evaluated by temperature-controlled powder XRD measurements following an hour-long isothermal treatment at 250 and 300 °C. In both cases no signs of decomposition could be detected. However, the compound melted incongruently at 332 °C producing, upon cooling, a mostly amorphous material. PhVPI was found to be slightly soluble in DMF (∼5 mM) and highly soluble in DMSO. Nevertheless, its solubility in DMF can be dramatically increased by adding an equimolar amount of DMSO. Therefore, phenyl viologen lead iodide can be amenable for the fabrication of solar devices by spin coating as actually done for MAPI-based cells. The crystal structure, determined by means of single crystal X-ray diffraction using synchrotron radiation, turned out to be triclinic and consequently differs from the prototypal perovskite structure. In fact, it comprises infinite double chains of corner-sharing PbI6 octahedra along the a-axis direction with phenyl viologen cations positioned between the columns. Finally, the present determination of PhVPI's electronic band structure achieved through UPS and UV-Vis DRS is instrumental in using the material for solar cells.

Synthesis, physico-chemical characterization and structure of the elusive hydroxylammonium lead iodide perovskite NH3OHPbI3.

The synthesis of hydroxylammonium lead iodide NH3OHPbI3 was accomplished by means of the reaction between water solutions of HI and NH2OH with PbI2 in sulfolane in conjunction with either crystallization by CH2Cl2 vapor diffusion or sulfolane extraction with toluene. The appropriate choice of the solvent was found to be crucial in order to attain the desired material. The synthesized compound was extensively characterized by single crystal and powder X-ray diffraction, UV-Vis diffuse reflectance spectroscopy, FT-IR spectroscopy, 1H-NMR spectroscopy, TG-DTA-QMS EGA (Evolved Gas Analysis), ESI-MS, and CHNS analysis. NH3OHPbI3 is an extremely reactive, deliquescent solid that easily oxidizes in air releasing iodine. Furthermore, it is the first reported perovskite to melt (m.p. around 80 °C) before decomposing exothermally at 103 °C. Such a chemical behavior, together with its optical absorption properties (i.e. yellow-colored perovskite), renders this material totally unsuitable for photovoltaic applications. The deliquescence of the material is to be ascribed to the strong hydrophilicity of hydroxylammonium ion. On the other hand, the relatively high Brønsted acidity of hydroxylammonium (pKa = 5.97) compared to other ammonium cations, promotes the reduction of atmospheric oxygen to water and the NH3OHPbI3 oxidation. The crystal structure, determined by single crystal X-ray diffraction with synchrotron radiation, is orthorhombic, but differs from the prototypal perovskite structure. Indeed it comprises infinite chains of face-sharing PbI6 octahedra along the c-axis direction with hydroxylammonium cations positioned between the columns, forming layers on the ac plane. The solvent intercalates easily between the layers. The crystal structure is apparently anomalous considering that the expected Goldschmidt's tolerance factor for the system (0.909) lies in the range of a stable prototypal perovskite structure. Therefore, the strong hydrogen bond forming tendency of hydroxylamine is likely to account for the apparent structural anomaly.

Calibration of rotation axes for multi-axis goniometers in macromolecular crystallography.

The installation of multi-axis goniometers such as the ESRF/EMBL miniKappa goniometer system has allowed the increased use of sample reorientation in macromolecular crystallography. Old and newly appearing data collection methods require precision and accuracy in crystal reorientation. The proper use of such multi-axis systems has necessitated the development of rapid and easy to perform methods for establishing and evaluating device calibration. A new diffraction-based method meeting these criteria has been developed for the calibration of the motors responsible for rotational motion. This method takes advantage of crystal symmetry by comparing the orientations of a sample rotated about a given axis and checking that the magnitude of the real rotation fits the calculated angle between these two orientations. Hence, the accuracy and precision of rotational motion can be assessed. This rotation calibration procedure has been performed on several beamlines at the ESRF and other synchrotrons. Some resulting data are presented here for reference.

A dystroglycan mutation (p.Cys667Phe) associated to muscle-eye-brain disease with multicystic leucodystrophy results in ER-retention of the mutant protein.

Dystroglycan (DG) is a cell adhesion complex composed by two subunits, the highly glycosylated α-DG and the transmembrane ß-DG. In skeletal muscle, DG is involved in dystroglycanopathies, a group of heterogeneous muscular dystrophies characterized by a reduced glycosylation of α-DG. The genes mutated in secondary dystroglycanopathies are involved in the synthesis of O-mannosyl glycans and in the O-mannosylation pathway of α-DG. Mutations in the DG gene (DAG1), causing primary dystroglycanopathies, destabilize the α-DG core protein influencing its binding to modifying enzymes. Recently, a homozygous mutation (p.Cys699Phe) hitting the ß-DG ectodomain has been identified in a patient affected by muscle-eye-brain disease with multicystic leucodystrophy, suggesting that other mechanisms than hypoglycosylation of α-DG could be implicated in dystroglycanopathies. Herein, we have characterized the DG murine mutant counterpart by transfection in cellular systems and high-resolution microscopy. We observed that the mutation alters the DG processing leading to retention of its uncleaved precursor in the endoplasmic reticulum. Accordingly, small-angle X-ray scattering data, corroborated by biochemical and biophysical experiments, revealed that the mutation provokes an alteration in the ß-DG ectodomain overall folding, resulting in disulfide-associated oligomerization. Our data provide the first evidence of a novel intracellular mechanism, featuring an anomalous endoplasmic reticulum-retention, underlying dystroglycanopathy.

The effect of the pathological V72I, D109N and T190M missense mutations on the molecular structure of α-dystroglycan.

Dystroglycan (DG) is a highly glycosylated protein complex that links the cytoskeleton with the extracellular matrix, mediating fundamental physiological functions such as mechanical stability of tissues, matrix organization and cell polarity. A crucial role in the glycosylation of the DG α subunit is played by its own N-terminal region that is required by the glycosyltransferase LARGE. Alteration in this O-glycosylation deeply impairs the high affinity binding to other extracellular matrix proteins such as laminins. Recently, three missense mutations in the gene encoding DG, mapped in the α-DG N-terminal region, were found to be responsible for hypoglycosylated states, causing congenital diseases of different severity referred as primary dystroglycanopaties.To gain insight on the molecular basis of these disorders, we investigated the crystallographic and solution structures of these pathological point mutants, namely V72I, D109N and T190M. Small Angle X-ray Scattering analysis reveals that these mutations affect the structures in solution, altering the distribution between compact and more elongated conformations. These results, supported by biochemical and biophysical assays, point to an altered structural flexibility of the mutant α-DG N-terminal region that may have repercussions on its interaction with LARGE and/or other DG-modifying enzymes, eventually reducing their catalytic efficiency.

Structural flexibility of human α-dystroglycan.

Dystroglycan (DG), composed of α and ß subunits, belongs to the dystrophin-associated glycoprotein complex. α-DG is an extracellular matrix protein that undergoes a complex post-translational glycosylation process. The bifunctional glycosyltransferase like-acetylglucosaminyltransferase (LARGE) plays a crucial role in the maturation of α-DG, enabling its binding to laminin. We have already structurally analyzed the N-terminal region of murine α-DG (α-DG-Nt) and of a pathological single point mutant that may affect recognition of LARGE, although the structural features of the potential interaction between LARGE and DG remain elusive. We now report on the crystal structure of the wild-type human α-DG-Nt that has allowed us to assess the reliability of our murine crystallographic structure as a α-DG-Nt general model. Moreover, we address for the first time both structures in solution. Interestingly, small-angle X-ray scattering (SAXS) reveals the existence of two main protein conformations ensembles. The predominant species is reminiscent of the crystal structure, while the less populated one assumes a more extended fold. A comparative analysis of the human and murine α-DG-Nt solution structures reveals that the two proteins share a common interdomain flexibility and population distribution of the two conformers. This is confirmed by the very similar stability displayed by the two orthologs as assessed by biochemical and biophysical experiments. These results highlight the need to take into account the molecular plasticity of α-DG-Nt in solution, as it can play an important role in the functional interactions with other binding partners.

Structural basis for inhibition of 17ß-hydroxysteroid dehydrogenases by phytoestrogens: The case of fungal 17ß-HSDcl.

Phytoestrogens are plant-derived compounds that functionally and structurally mimic mammalian estrogens. Phytoestrogens have broad inhibitory activities toward several steroidogenic enzymes, such as the 17ß-hydroxysteroid dehydrogenases (17ß-HSDs), which modulate the biological potency of androgens and estrogens in mammals. However, to date, no crystallographic data are available to explain phytoestrogens binding to mammalian 17ß-HSDs. NADP(H)-dependent 17ß-HSD from the filamentous fungus Cochliobolus lunatus (17ß-HSDcl) has been the subject of extensive biochemical, kinetic and quantitative structure-activity relationship studies that have shown that the flavonols are the most potent inhibitors. In the present study, we investigated the structure-activity relationships of the ternary complexes between the holo form of 17ß-HSDcl and the flavonols kaempferol and 3,7-dihydroxyflavone, in comparison with the isoflavones genistein and biochanin A. Crystallographic data are accompanied by kinetic analysis of the inhibition mechanisms for six flavonols (3-hydroxyflavone, 3,7-dihydroxyflavone, kaempferol, quercetin, fisetin, myricetin), one flavanone (naringenin), one flavone (luteolin), and two isoflavones (genistein, biochanin A). The kinetics analysis shows that the degree of hydroxylation of ring B significantly influences the overall inhibitory efficacy of the flavonols. A distinct binding mode defines the interactions between 17ß-HSDcl and the flavones and isoflavones. Moreover, the complex with biochanin A reveals an unusual binding mode that appears to account for its greater inhibition of 17ß-HSDcl with respect to genistein. Overall, these data provide a blueprint for identification of the distinct molecular determinants that underpin 17ß-HSD inhibition by phytoestrogens.

The Structure of the T190M Mutant of Murine α-Dystroglycan at High Resolution: Insight into the Molecular Basis of a Primary Dystroglycanopathy.

The severe dystroglycanopathy known as a form of limb-girdle muscular dystrophy (LGMD2P) is an autosomal recessive disease caused by the point mutation T192M in α-dystroglycan. Functional expression analysis in vitro and in vivo indicated that the mutation was responsible for a decrease in posttranslational glycosylation of dystroglycan, eventually interfering with its extracellular-matrix receptor function and laminin binding in skeletal muscle and brain. The X-ray crystal structure of the missense variant T190M of the murine N-terminal domain of α-dystroglycan (50-313) has been determined, and showed an overall topology (Ig-like domain followed by a basket-shaped domain reminiscent of the small subunit ribosomal protein S6) very similar to that of the wild-type structure. The crystallographic analysis revealed a change of the conformation assumed by the highly flexible loop encompassing residues 159-180. Moreover, a solvent shell reorganization around Met190 affects the interaction between the B1-B5 anti-parallel strands forming part of the floor of the basket-shaped domain, with likely repercussions on the folding stability of the protein domain(s) and on the overall molecular flexibility. Chemical denaturation and limited proteolysis experiments point to a decreased stability of the T190M variant with respect to its wild-type counterpart. This mutation may render the entire L-shaped protein architecture less flexible. The overall reduced flexibility and stability may affect the functional properties of α-dystroglycan via negatively influencing its binding behavior to factors needed for dystroglycan maturation, and may lay the molecular basis of the T190M-driven primary dystroglycanopathy.

The conundrum of the high-affinity NGF binding site formation unveiled?

The homodimer NGF (nerve growth factor) exerts its neuronal activity upon binding to either or both distinct transmembrane receptors TrkA and p75(NTR). Functionally relevant interactions between NGF and these receptors have been proposed, on the basis of binding and signaling experiments. Namely, a ternary TrkA/NGF/p75(NTR) complex is assumed to be crucial for the formation of the so-called high-affinity NGF binding sites. However, the existence, on the cell surface, of direct extracellular interactions is still a matter of controversy. Here, supported by a small-angle x-ray scattering solution study of human NGF, we propose that it is the oligomerization state of the secreted NGF that may drive the formation of the ternary heterocomplex. Our data demonstrate the occurrence in solution of a concentration-dependent distribution of dimers and dimer of dimers. A head-to-head molecular assembly configuration of the NGF dimer of dimers has been validated. Overall, these findings prompted us to suggest a new, to our knowledge, model for the transient ternary heterocomplex, i.e., a TrkA/NGF/p75(NTR) ligand/receptors molecular assembly with a (2:4:2) stoichiometry. This model would neatly solve the problem posed by the unconventional orientation of p75(NTR) with respect to TrkA, as being found in the crystal structures of the TrkA/NGF and p75(NTR)/NGF complexes.

Insights into subtle conformational differences in the substrate-binding loop of fungal 17ß-hydroxysteroid dehydrogenase: a combined structural and kinetic approach.

The 17ß-HSD (17ß-hydroxysteroid dehydrogenase) from the filamentous fungus Cochliobolus lunatus (17ß-HSDcl) is a NADP(H)-dependent enzyme that preferentially catalyses the interconversion of inactive 17-oxo-steroids and their active 17ß-hydroxy counterparts. 17ß-HSDcl belongs to the SDR (short-chain dehydrogenase/reductase) superfamily. It is currently the only fungal 17ß-HSD member that has been described and represents one of the model enzymes of the cP1 classical subfamily of NADPH-dependent SDR enzymes. A thorough crystallographic analysis has been performed to better understand the structural aspects of this subfamily and provide insights into the evolution of the HSD enzymes. The crystal structures of the 17ß-HSDcl apo, holo and coumestrol-inhibited ternary complex, and the active-site Y167F mutant reveal subtle conformational differences in the substrate-binding loop that probably modulate the catalytic activity of 17ß-HSDcl. Coumestrol, a plant-derived non-steroidal compound with oestrogenic activity, inhibits 17ß-HSDcl [IC50 2.8 µM; at 100 µM substrate (4-oestrene-3,17-dione)] by occupying the putative steroid-binding site. In addition to an extensive hydrogen-bonding network, coumestrol binding is stabilized further by π-π stacking interactions with Tyr212. A stopped-flow kinetic experiment clearly showed the coenzyme dissociation as the slowest step of the reaction and, in addition to the low steroid solubility, it prevents the accumulation of enzyme-coenzyme-steroid ternary complexes.

Dissecting NGF interactions with TrkA and p75 receptors by structural and functional studies of an anti-NGF neutralizing antibody.

The anti-nerve growth factor (NGF) monoclonal antibody alphaD11 is a potent antagonist that neutralizes the biological functions of its antigen in vivo. NGF antagonism is expected to be a highly effective and safe therapeutic approach in many pain states. A comprehensive functional and structural analysis of alphaD11 monoclonal antibody was carried out, showing its ability to neutralize NGF binding to either tropomyosine receptor kinase A (TrkA) or p75 receptors. The 3-D structure of the alphaD11 Fab fragment was solved at 1.7 A resolution. A computational docking model of the alphaD11 Fab-NGF complex, based on epitope mapping using a pool of 44 NGF mutants and experimentally validated by small-angle X-ray scattering, provided the structural basis for identifying the residues involved in alphaD11 Fab binding. The present study pinpoints loop II of NGF to be an important structural determinant for NGF biological activity mediated by TrkA receptor.

(3R,5S)-5(3)-Carb-oxy-3,4,5,6-tetra-hydro-2H-1,4-thia-zin-4-ium-3(5)-carboxyl-ate.

The molecule of the zwitterionic title compound, C(6)H(9)NO(4)S, which lies on a mirror plane, shows a puckered chair conformation of the six-membered ring with the S and N atoms out of the mean plane of the other four C atoms by 0.929 (2) and 0.647 (2) Å, respectively. The ionized carboxyl group is equatorially oriented. The hydrogen-bonding network includes very short O-H⋯O [2.470 (2) Å] and N-H⋯S [3.471 (2) and 3.416 (2) Å] inter-molecular contacts.