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Graphical abstract, Highlights • High-resolution solution NMR structure of rhNGF has been determined. • Quinary interactions characterize ATP binding to rhNGF. • SPR, ITC and STD-NMR reveal ATP binding to rhNGF with mM affinity. • NMR and MD analysis pinpoint to the presence of two binding sites of ATP on rhNGF. • Stoichiometry of ATP-Mg2+ or Zn2+-rhNGF mixtures affects KD affinity to TrkA/p75NTR., 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.

Graphical abstract, Highlights • Bioinformatics analysis unveiled the structural conservation domains of proNTs. • The dynamic behavior in solution of human proNTs has been exploited by SAXS and DSF. • The functional plasticity and evolutionary adaptation of proNTs have been assessed., The neurotrophins, i.e., Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin 3 (NT3) and Neurotrophin 4 (NT4), are known to play a range of crucial functions in the developing and adult peripheral and central nervous systems. Initially synthesized as precursors, i.e., proneurotrophins (proNTs), that are cleaved to release C-terminal mature forms, they act through two types of receptors, the specific Trk receptors (Tropomyosin-related kinases) and the pan-neurotrophin receptor p75NTR, to initiate survival and differentiative responses. Recently, all the proNTs but proNT4 have been demonstrated to be not just inactive precursors, but signaling ligands that mediate opposing actions in fundamental aspects of the nervous system with respect to the mature counterparts through dual-receptor complexes formation with a member of the VPS10 family and p75NTR. Despite the functional relevance, the molecular determinants underpinning the interactions between the pro-domains and their receptors are still elusive probably due to their intrinsically disordered nature. Here we present an evolutionary approach coupled to an experimental study aiming to uncover the structural and dynamical basis of the biological function displayed by proNGF, proBDNF and proNT3 but missing in proNT4. A bioinformatic analysis allowed to elucidate the functional adaptability of the proNTs family in vertebrates, identifying conserved key structural features. The combined biochemical and SAXS experiments shed lights on the structure and dynamic behavior of the human proNTs in solution, giving insights on the evolutionary conserved structural motifs, essential for the multifaceted roles of proNTs in physiological as well as in pathological contexts.

Dystroglycan (DG) is a cell adhesion complex composed by two subunits, the highly glycosylated alpha-DG and the transmembrane beta-DG. In skeletal muscle, DG is involved in dystroglycanopathies, a group of heterogeneous muscular dystrophies characterized by a reduced glycosylation of alpha-DG. The genes mutated in secondary dystroglycanopathies are involved in the synthesis of O-mannosyl glycans and in the O-mannosylation pathway of alpha-DG. Mutations in the DG gene (DAG1), causing primary dystroglycanopathies, destabilize the alpha-DG core protein influencing its binding to modifying enzymes. Recently, a homozygous mutation (p.Cys699Phe) hitting the beta-DG ectodomain has been identified in a patient affected by muscle-eye-brain disease with multicystic leucodystrophy, suggesting that other mechanisms than hypoglycosylation of alpha-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 beta-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.

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.

Nerve Growth Factor (NGF), the prototype of the neurotrophin family, is essential for maintenance and growth of different neuronal populations. The X-ray crystal structure of NGF has been known since the early ‘90s and shows a β-sandwich fold with extensive loops that are involved in the interaction with its binding partners. Understanding the dynamical properties of these loops is thus important for molecular recognition. We present here a combined solution NMR/molecular dynamics study which addresses the question of whether and how much the long loops of NGF are flexible and describes the N-terminal intrinsic conformational tendency of the unbound NGF molecule. NMR titration experiments allowed identification of a previously undetected epitope of the anti-NGF antagonist antibody aD11 which will be of crucial importance for future drug lead discovery. The present study thus recapitulates all the available structural information and unveils the conformational versatility of the relatively rigid NGF loops upon functional ligand binding.

MNAC13, a mouse monoclonal antibody, recognizes with high affinity and specificity the neurotrophin receptor TrkA and displays a neutralizing activity toward the NGF/TrkA interaction. Detailed knowledge of the molecular basis determining the specificity of this antibody is of importance because of its potential use as a modulator of the TrkA-mediated NGF activity. Here, we report a full biochemical and structural characterization of the MNAC13 antibody. Epitope mapping studies, by serial deletion mutants and by phage display, reveal a conformational epitope that is localized on the carboxy-terminal region of the first immunoglobulin-like domain (d4) of TrkA. The X-ray crystal structure of the MNAC13 Fab fragment has been determined and refined to 1.8 A resolution. The antigen-binding site is characterized by a crevice, surrounded by hydrophilic-charged residues on either side, dipping deep toward three mainly hydrophobic subsites. Remarkably an isopropanol molecule has been found to bind in one of the hydrophobic crevices. Overall, the surface topology (shape and electrostatic potential) of the combining site is consistent with the binding data on TrkA ECD serial deletions mutants. The structure of the MNAC13 Fab fragment may assist in the rational structure-based design of high affinity humanized forms of MNAC13, appropriate for therapeutic approaches in neuropathy and inflammatory pain states.

NMDA receptors (NMDARs) are multimeric proteins, the biological and functional characteristics of which depend on differential subunit assembly during postnatal development. In the present paper, we investigated whether the expression of NMDAR subunits NR1, NR2A, NR2B is influenced by neurotrophins in rat visual cortex. We used a soluble form of the TrkB receptor engineered as an immunoadhesin (TrkB-IgG) in order to block TrkB ligands. TrkB-IgG was released through a cannula implanted in the occipital pole and connected to a mini-osmotic pump. TrkB-IgG was continuously released from postnatal day 20-21 (P20-21) to P36-37. In a different group of animals used as controls, osmotic pumps were filled with saline. Different antibodies were used to stain neurons expressing NR1, NR2A and NR2B. We counted the number of neurons stained for NR2A and NR2B subunits and expressed this as percentage with respect to the total number of cresyl-violet stained neurons in each cortical layer. In the visual cortex of TrkB-IgG-treated rats, the percentage of neurons expressing NR2A was significantly increased in all cortical layers. Concerning the NR2B subunit, the percentage of stained neurons was not significantly different between TrkB-IgG-treated and control rats. The staining level for both NR2A and NR2B, but not NR1, was reduced in all cortical layers in TrkB-IgG-treated animals. In agreement with this result, the endogenous levels of NR2A and NR2B subunits were reduced in TrkB-IgG-treated animals as shown by Western blotting. Thus, TrkB signalling controls the cellular expression of NMDAR subunits in visual cortical neurons during postnatal development.

Objective: ADAMTS5 (aggrecanase-2) has been demonstrated to be crucial in the development of osteoarthritis (OA), by use of several mouse mutants carrying either truncated, catalytically inactive enzymes or aggrecanase-resistant mutant aggrecan. We have selected recombinant monoclonal antibodies directed against ADAMTS5, by using Intracellular Antibody Capture Technology (IACT). CRB0017 revealed very high affinity for the enzyme in Biacore analyses and very good specificity in a panel of binding assays. Therefore, we tested CRB0017 in a relevant spontaneous OA model, the STR/ort mouse. Design: STR/ort male mice were recruited at 5 months of age, and treated intra-articularly in each knee with CRB0017 1.2μg, CRB0017 12μg, or vehicle. After 6 weeks, the intra-articular administration of CRB0017 was repeated with the same doses. After 3 months from recruitment, the animals were sacrificed and the femorotibial joints processed for histology and scored in a blind fashion according to both Mankin's and the OARSI methods. Results and conclusions: All histological scores were significantly decreased in the CRB0017 12μg/knee group compared to vehicle, while administration of CRB0017 1.2μg was associated with a trend to a decrease in the same parameters. Therefore, CRB0017 administered twice in 3 months could modify the course of OA in the STR/ort mouse, by delaying cartilage breakdown as assessed histologically. The procedure of blind scoring of the histological samples clearly showed that knee intra-articular administration of CRB0017, an anti-ADAMTS5 antibody, dose-dependently improved disease progression in a relevant animal model of OA. © 2013 Osteoarthritis Research Society International.

SbmA protein has been proposed as a dimeric secondary transporter. The protein is involved in the transport of microcins B17 and J25, bleomycin, proline-rich antimicrobial peptides, antisense peptide phosphorodiamidate morpholino oligomers, and peptide nucleic acids into the Escherichia coli cytoplasm. The sbmA homologue is found in a variety of bacteria, though the physiological role of the protein is hitherto unknown. In this work, we carried out a functional and structural analysis to determine which amino acids are critical for the transport properties of SbmA. We created a set of 15 site-directed sbmA mutants in which single conserved amino acids were replaced by glycine residues. Our work demonstrated that strains carrying the site-directed mutants V102G, F219G, and E276G had a null phenotype for SbmA transport functions. In contrast, strains carrying the single point mutants W19G, W53G, F60G, S69G, N155G, R190, L233G, A344G, T255G, N308G, and R385G showed transport capacities indistinguishable from those of strains harboring a wild-type sbmA. The strain carrying the Y116G mutant exhibited mixed phenotypic characteristics. We also demonstrated that those sbmA mutants with severely impaired transport capacity showed a dominant negative phenotype. Electron microscopy data and in silico three-dimensional (3D) homology modeling support the idea that SbmA forms a homodimeric complex, closely resembling the membrane-spanning region of the ATP-binding cassette transporter family. Direct mapping of the sbmA single point mutants on the protein surface allowed us to explain the observed phenotypic differences in transport ability. Fil: Corbalan, Natalia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Runti, Giulia. Università degli Studi di Trieste; Italia Fil: Adler, Conrado. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Covaceuszach, Sonia. Consiglio Nazionale delle Ricerche; Italia Fil: Ford, Robert C.. University of Manchester; Reino Unido Fil: Lamba, Doriano. Consiglio Nazionale delle Ricerche; Italia Fil: Beis, Konstantinos. Imperial College London; Reino Unido Fil: Scocchi, Marco. Università degli Studi di Trieste; Italia Fil: Vincent, Paula Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina

During adulthood, the neurotrophin Nerve Growth Factor (NGF) sensitizes nociceptors, thereby increasing the response to noxious stimuli. The relationship between NGF and pain is supported by genetic evidence: mutations in the NGF TrkA receptor in patients affected by an hereditary rare disease (Hereditary Sensory and Autonomic Neuropathy type IV, HSAN IV) determine a congenital form of severe pain insensitivity, with mental retardation, while a mutation in NGFB gene, leading to the aminoacid substitution R100W in mature NGF, determines a similar loss of pain perception, without overt cognitive neurological defects (HSAN V). The R100W mutation provokes a reduced processing of proNGF to mature NGF in cultured cells and a higher percentage of neurotrophin secreted is in the proNGF form. Moreover, using Surface Plasmon Resonance we showed that the R100W mutation does not affect NGF binding to TrkA, while it abolishes NGF binding to p75NTR receptors. However, it remains to be clarified whether the major impact of the mutation is on the biological function of proNGF or of mature NGF and to what extent the effects of the R100W mutation on the HSAN V clinical phenotype are developmental, or whether they reflect an impaired effectiveness of NGF to regulate and mediate nociceptive transmission in adult sensory neurons. Here we show that the R100 mutation selectively alters some of the signaling pathways activated downstream of TrkA NGF receptors. NGFR100 mutants maintain identical neurotrophic and neuroprotective properties in a variety of cell assays, while displaying a significantly reduced pain-inducing activity in vivo (n = 8–10 mice/group). We also show that proNGF has a significantly reduced nociceptive activity, with respect to NGF. Both sets of results jointly contribute to elucidating the mechanisms underlying the clinical HSAN V manifestations, and to clarifying which receptors and intracellular signaling cascades participate in the pain sensitizing action of NGF.

Nerve Growth Factor (NGF) signalling is mediated by the TrkA and p75NTR receptors. Besides its neurotrophic and survival activities, NGF displays a potent pro-nociceptive activity. Recently, a missense point mutation was found in the NGFB gene (C661T, leading to the aminoacid substitution R100W) of individuals affected by a form of hereditary loss of pain perception (hereditary sensory and autonomic neuropathy type V, HSAN V). In order to gain insights into the functional consequences of the HSAN V NGF mutation, two sets of hNGFR100 mutants were expressed in Escherichia coli and purified, as mature NGF or proNGF, for in vitro receptor binding studies. Here, we show by Surface Plasmon Resonance analysis that the R100 mutation selectively disrupts binding of hNGF to p75NTR receptor, to an extent which depends on the substituting residue at position 100, while the affinity of hNGFR100 mutants for TrkA receptor is not affected. As for unprocessed hproNGF, the binding of the R100 variants to p75NTR receptor shows only a limited impairment, showing that the impact of the R100 mutation on p75NTR receptor binding is greater in the context of mature, processed hNGF. These results provide a basis for elucidating the mechanisms underlying the clinical manifestations of HSAN V patients, and provide a basis for the development of "painless" hNGF molecules with therapeutic potential.

The unprocessed precursor of the Nerve Growth Factor (NGF), proNGF, has additional functions, besides its initially described role as a chaperone for NGF folding. The precursor protein endows apoptotic and/or neurotrophic properties, in contrast to the mature part. The structural and molecular basis for such distinct activities are presently unknown. Aiming to gain insights into the specific molecular interactions that govern rm-proNGF biological activities versus those of its mature counterpart, a structural study by synchrotron Small Angle X-ray Scattering (SAXS) in solution was carried out. The different binding properties of the two proteins were investigated by Surface Plasmon Resonance (SPR) using, as structural probes, a panel of anti-NGF antibodies and the soluble forms of TrkA and p75NTR receptors. SAXS measurements revealed the rm-proNGF to be dimeric and anisometric, with the pro-peptide domain being intrinsically unstructured. Ab initio reconstructions assuming two-fold symmetry generated two types of structural models, a globular crab-like and an elongated shape, that resulted in equally good fits of the scattering data. A novel method accounting for possible co-existence of different conformations contributing to the experimental scattering pattern, with no symmetry constraints, suggests the crab-like to be a more likely proNGF conformation. In order to exploit the potential of chemical stabilizers affecting the existing conformational protein populations, SAXS data were also collected in the presence of ammonium sulphate. An increase of the proNGF compactness was observed. SPR data pinpoints that the pro-peptide of proNGF may act as an intrinsically unstructured protein domain, characterized by a molecular promiscuity in the interaction/binding to multiple partners (TrkA and p75NTR receptors and a panel of neutralizing anti-NGF antibodies) depending on the physiological conditions of the cell. These data provide a first insight into the structural basis for the selectivity of mouse short proNGF, versus NGF, towards its binding partners.

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.

The rat monoclonal neuroantibody alphaD11 is a potent antagonist that prevents the binding of nerve growth factor (NGF) to its tyrosine kinase A receptor (TrkA) in a variety of systems, most notably in two in vivo systems linked to crucial pathological states, such as Alzheimer's disease and HIV infection. To provide further insights into the mechanism of action of this potentially therapeutic monoclonal antibody, structural studies of the antigen-binding fragment (Fab) of alphaD11 were performed. alphaD11 IgG2a immunoglobulin was obtained from hybridomas by in vitro tissue culture. The alphaD11 Fab crystallizes in two crystal forms. Form I belongs to space group P1, with unit-cell parameters a = 42.7, b = 50.6, c = 102.7 A, alpha = 82.0, beta = 89.1, gamma = 86.0 degrees. With two molecules in the asymmetric unit, V(M) is 2.3 A(3) Da(-1) and the solvent content is 46%. A complete data set has been collected at 2.7 A resolution on beamline XRD-1 (ELETTRA, Trieste, Italy). Form II belongs to space group C2, with unit-cell parameters a = 114.8, b = 69.4, c = 64.10 A, beta = 117.0 degrees. With one molecule in the asymmetric unit, V(M) is 2.4 A(3) Da(-1) and the solvent content is 48%. A complete data set has been collected at 1.7 A resolution on beamline ID14-1 (ESRF, Grenoble, France). Phasing was successfully performed by Patterson search techniques and refinement of the structures is currently under way. Crystal forms I and II display a close-packing pattern.

Most forms of chronic pain are inadequately treated by present therapeutic options. Compelling evidence has accumulated, demonstrating that Nerve Growth Factor (NGF) is a key modulator of inflammatory and nociceptive responses, and is a promising target for the treatment of human pathologies linked to chronic and inflammatory pain. There is therefore a growing interest in the development of therapeutic molecules antagonising the NGF pathway and its nociceptor sensitization actions, among which function-blocking anti-NGF antibodies are particularly relevant candidates.In this respect, the rat anti-NGF alphaD11 monoclonal antibody (mAb) is a potent antagonist, able to effectively antagonize rodent and human NGF in a variety of in vitro and in vivo systems. Here we show that mAb alphaD11 displays a significant analgesic effect in two different models of persistent pain in mice, with a remarkable long-lasting activity. In order to advance alphaD11 mAb towards its clinical application in man, anti-NGF alphaD11 mAb was humanized by applying a novel single cycle strategy based on the a priori experimental determination of the crystal and molecular structure of the parental Fragment antigen-binding (Fab). The humanized antibody (hum-alphaD11) was tested in vitro and in vivo, showing that the binding mode and the NGF neutralizing biological activities of the parental antibody are fully preserved, with even a significant affinity improvement. The results firmly establish hum-alphaD11 as a lead candidate for clinical applications in a therapeutic area with a severe unmet medical need. More generally, the single-cycle structure-based humanization method represents a considerable improvement over the standard humanization methods, which are intrinsically empirical and require several refinement cycles.

The monoclonal neuroantibody ?D11 is a potent antagonist that prevents the binding of the neurotrophin NGF (nerve growth factor) to its receptors, TrkA and p75, in a variety of systems, most notably in two in vivo systems linked to crucial pathological states (i.e. Alzheimer disease and AIDS). To elucidate the mechanism of neutralization, structural and functional studies were performed. The potential therapeutical interest of the antibody was confirmed, demonstrating that it binds both mouse and human NGF with similar affinity. Its epitope was mapped by Sandwich Elisa assay with a pool of mutants. Its Fab fragment was crystallized [1] and the structure was solved at 1.80Å resolution. This structure, that may assist in the humanization without loss in affinity, was docked to NGF on the basis of epitope mapping results.