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.

Selection of Natural Compounds with HMGA-Interfering Activities and Cancer Cell Cytotoxicity.

HMGA proteins are intrinsically disordered (ID) chromatin architectural factors characterized by three DNA binding domains (AT-hooks) that allow them to bind into the DNA minor groove of AT-rich stretches. HMGA are functionally involved in regulating transcription, RNA processing, DNA repair, and chromatin remodeling and dynamics. These proteins are highly expressed and play essential functions during embryonic development. They are almost undetectable in adult tissues but are re-expressed at high levels in all cancers where they are involved in neoplastic transformation and cancer progression. We focused on identifying new small molecules capable of binding into the minor groove of AT-rich DNA sequences that could compete with HMGA for DNA binding and, thus, potentially interfere with their activities. Here, a docking-based virtual screening of a unique high diversity in-house library composed of around 1000 individual natural products identified 16 natural compounds as potential minor groove binders that could inhibit the interaction between HMGA and DNA. To verify the ability of these selected compounds to compete with HMGA proteins, we screened them using electrophoretic mobility shift assays. We identified Sorocein C, a Diels-Alder (D-A)-type adducts, isolated from Sorocea ilicifolia and Sorocea bonplandii with an HMGA/DNA-displacing activity and compared its activity with that of two structurally related compounds, Sorocein A and Sorocein B. All these compounds showed a cytotoxicity effect on cancer cells, suggesting that the Sorocein-structural family may provide new and yet unexplored chemotypes for the development of minor groove binders to be evaluated as anticancer agents.

The NGF R100W Mutation, Associated with Hereditary Sensory Autonomic Neuropathy Type V, Specifically Affects the Binding Energetic Landscapes of NGF and of Its Precursor proNGF and p75NTR.

Nerve Growth Factor (NGF), the prototype of the neurotrophin family, stimulates morphological differentiation and regulates neuronal gene expression by binding to TrkA and p75NTR receptors. It plays a critical role in maintaining the function and phenotype of peripheral sensory and sympathetic neurons and in mediating pain transmission and perception during adulthood. A point mutation in the NGFB gene (leading to the amino acid substitution R100W) is responsible for Hereditary Sensory and Autonomic Neuropathy type V (HSAN V), leading to a congenital pain insensitivity with no clear cognitive impairments, but with alterations in the NGF/proNGF balance. The available crystal structures of the p75NTR/NGF and 2p75NTR/proNGF complexes offer a starting point for Molecular Dynamics (MD) simulations in order to capture the impact of the R100W mutation on their binding energetic landscapes and to unveil the molecular determinants that trigger their different physiological and pathological outcomes. The present in silico studies highlight that the stability and the binding energetic fingerprints in the 2p75NTR/proNGF complex is not affected by R100W mutation, which on the contrary, deeply affects the energetic landscape, and thus the stability in the p75NTR/NGF complex. Overall, these findings present insights into the structural basis of the molecular mechanisms beyond the clinical manifestations of HSAN V patients.

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.

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.

Untangling the Conformational Plasticity of V66M Human proBDNF Polymorphism as a Modifier of Psychiatric Disorder Susceptibility.

The human genetic variant BDNF (V66M) represents the first example of neurotrophin family member that has been linked to psychiatric disorders. In order to elucidate structural differences that account for the effects in cognitive function, this hproBDNF polymorph was expressed, refolded, purified, and compared directly to the WT variant for the first time for differences in their 3D structures by DSF, limited proteolysis, FT-IR, and SAXS measurements in solution. Our complementary studies revealed a deep impact of V66M polymorphism on hproBDNF conformations in solution. Although the mean conformation in solution appears to be more compact in the V66M variant, overall, we demonstrated a large increase in flexibility in solution upon V66M mutation. Thus, considering that plasticity in IDR is crucial for protein function, the observed alterations may be related to the functional alterations in hproBDNF binding to its receptors p75NTR, sortilin, HAP1, and SorCS2. These effects can provoke altered intracellular neuronal trafficking and/or affect proBDNF physiological functions, leading to many brain-associated diseases and conditions such as cognitive impairment and anxiety. The structural alterations highlighted in the present study may pave the way to the development of drug discovery strategies to provide greater therapeutic responses and of novel pharmacologic strategy in human populations with this common polymorphism, ultimately guiding personalized medicine for neuropsychiatric disorders.

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.

LuxR Solos from Environmental Fluorescent Pseudomonads.

LuxR solos are related to quorum sensing (QS) LuxR family regulators; however, they lack a cognate LuxI family protein. LuxR solos are widespread and almost exclusively found in proteobacteria. In this study, we investigated the distribution and conservation of LuxR solos in the fluorescent pseudomonads group. Our analysis of more than 600 genomes revealed that the majority of fluorescent Pseudomonas spp. carry one or more LuxR solos, occurring considerably more frequently than complete LuxI/LuxR archetypical QS systems. Based on the adjacent gene context and conservation of the primary structure, nine subgroups of LuxR solos have been identified that are likely to be involved in the establishment of communication networks. Modeling analysis revealed that the majority of subgroups shows some substitutions at the invariant amino acids of the ligand-binding pocket of QS LuxRs, raising the possibility of binding to non-acyl-homoserine lactone (AHL) ligands. Several mutants and gene expression studies on some LuxR solos belonging to different subgroups were performed in order to shed light on their response. The commonality of LuxR solos among fluorescent pseudomonads is an indication of their important role in cell-cell signaling.IMPORTANCE Cell-cell communication in bacteria is being extensively studied in simple settings and uses chemical signals and cognate regulators/receptors. Many Gram-negative proteobacteria use acyl-homoserine lactones (AHLs) synthesized by LuxI family proteins and cognate LuxR-type receptors to regulate their quorum sensing (QS) target loci. AHL-QS circuits are the best studied QS systems; however, many proteobacterial genomes also contain one or more LuxR solos, which are QS-related LuxR proteins which are unpaired to a cognate LuxI. A few LuxR solos have been implicated in intraspecies, interspecies, and interkingdom signaling. Here, we report that LuxR solo homologs occur considerably more frequently than complete LuxI/LuxR QS systems within the Pseudomonas fluorescens group of species and that they are characterized by different genomic organizations and primary structures and can be subdivided into several subgroups. The P. fluorescens group consists of more than 50 species, many of which are found in plant-associated environments. The role of LuxR solos in cell-cell signaling in fluorescent pseudomonads is discussed.

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.

LuxR Solos in the Plant Endophyte Kosakonia sp. Strain KO348.

Endophytes are microorganisms that live inside plants and are often beneficial for the host. Kosakonia is a novel bacterial genus that includes several species that are diazotrophic and plant associated. This study revealed two quorum sensing-related LuxR solos, designated LoxR and PsrR, in the plant endophyte Kosakonia sp. strain KO348. LoxR modeling and biochemical studies demonstrated that LoxR binds N-acyl homoserine lactones (AHLs) in a promiscuous way. PsrR, on the other hand, belongs to the subfamily of plant-associated-bacterium (PAB) LuxR solos that respond to plant compounds. Target promoter studies as well as modeling and phylogenetic comparisons suggest that PAB LuxR solos are likely to respond to different plant compounds. Finally, LoxR is involved in the regulation of T6SS and PsrR plays a role in root endosphere colonization.IMPORTANCE Cell-cell signaling in bacteria allows a synchronized and coordinated behavior of a microbial community. LuxR solos represent a subfamily of proteins in proteobacteria which most commonly detect and respond to signals produced exogenously by other microbes or eukaryotic hosts. Here, we report that a plant-beneficial bacterial endophyte belonging to the novel genus of Kosakonia possesses two LuxR solos; one is involved in the detection of exogenous N-acyl homoserine lactone quorum sensing signals and the other in detecting a compound(s) produced by the host plant. These two Kosakonia LuxR solos are therefore most likely involved in interspecies and interkingdom signaling.

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.

Negative Regulation of Violacein Biosynthesis in Chromobacterium violaceum.

In Chromobacteium violaceum, the purple pigment violacein is under positive regulation by the N-acylhomoserine lactone CviI/R quorum sensing system and negative regulation by an uncharacterized putative repressor. In this study we report that the biosynthesis of violacein is negatively controlled by a novel repressor protein, VioS. The violacein operon is regulated negatively by VioS and positively by the CviI/R system in both C. violaceum and in a heterologous Escherichia coli genetic background. VioS does not regulate the CviI/R system and apart from violacein, VioS, and quorum sensing regulate other phenotypes antagonistically. Quorum sensing regulated phenotypes in C. violaceum are therefore further regulated providing an additional level of control.

Conformational Rigidity within Plasticity Promotes Differential Target Recognition of Nerve Growth Factor.

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 ß-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 αD11 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.

Studies on synthetic LuxR solo hybrids.

A sub-group of LuxR family of proteins that plays important roles in quorum sensing, a process of cell-cell communication, is widespread in proteobacteria. These proteins have a typical modular structure consisting of N-ter autoinducer binding and C-ter helix-turn-helix (HTH) DNA binding domains. The autoinducer binding domain recognizes signaling molecules which are most often N-acyl homoserine lactones (AHLs) but could also be other novel and yet unidentified molecules. In this study we carried out a series of specific domain swapping and promoter activation experiments as a first step to engineer synthetic signaling modules, taking advantage of the modularity and the versatile/diverse signal specificities of LuxR proteins. In our experiments the N-ter domains from different LuxR homologs were either interchanged or placed in tandem followed by a C-ter domain. The rational design of the hybrid proteins was supported by a structure-based homology modeling studies of three members of the LuxR family (i.e., LasR, RhlR, and OryR being chosen for their unique ligand binding specificities) and of selected chimeras. Our results reveal that these LuxR homologs were able to activate promoter elements that were not their usual targets; we also show that hybrid LuxR proteins retained the ability to recognize the signal specific for their N- ter autoinducer binding domain. However, the activity of hybrid LuxR proteins containing two AHL binding domains in tandem appears to depend on the organization and nature of the introduced domains. This study represents advances in the understanding of the modularity of LuxR proteins and provides additional possibilities to use hybrid proteins in both basic and applied synthetic biology based research.

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.