NET-1 is a key chemotropic ligand that signals commissural axon migration and change in direction. NET-1 and its receptor UNC-5B switch axon growth cones from attraction to repulsion. The biophysical properties of the NET-1 + UNC-5B complex have been poorly characterized. Using multi-wavelength-AUC by adding a fluorophore to UNC-5B, we were able to separate the UNC-5B sedimentation from NET-1. Using both multi-wavelength- and single-wavelength AUC, we investigated NET-1 and UNC-5B hydrodynamic parameters and complex formation. The sedimentation velocity experiments show that NET-1 exists in a monomer–dimer equilibrium. A close study of the association shows that NET-1 forms a pH-sensitive dimer that interacts in an anti-parallel orientation. UNC-5B can form equimolar NET-1 + UNC-5B heterocomplexes with both monomeric and dimeric NET-1.

The voltage-dependent anion-selective channel (VDAC) is a porin in the mitochondrial outer membrane (MOM). Unlike bacterial porins, several mitochondrial β-barrels comprise an odd number of β-strands, as is the case for the 19-β-stranded VDAC. Previously, a variant of a VDAC from Neurospora crassa , VDAC-ΔC, lacking the predicted 19th β-strand, was found to form gated, anion-selective channels in artificial membranes. In vivo , the two C-terminal β-strands (β18 and β19) in VDAC form a β-hairpin necessary for import from the cytoplasm into mitochondria and the β-signal required for assembly in the mitochondrial outer membrane resides in β19. The current study demonstrated that the putative 18-stranded β-barrel formed by VDAC-ΔC can be imported and assembled in the MOM in vivo and can also partially rescue the phenotype associated with the deletion of VDAC from a strain of N. crassa . Furthermore, when expressed and purified from Escherichia coli , VDAC-ΔC can be folded into a β-strand-rich form in decyl-maltoside. Size exclusion chromatography (SEC) alone or combined with multi-angle light scattering (SEC-MALS) and analytical ultracentrifugation revealed that, unlike full-length VDACs, VDAC-ΔC can self-organize into dimers and higher order oligomers in the absence of sterol.

•Proteinaceous nanotube takes up polycyclic aromatic hydrocarbons.•Industrial development of a novel medium for passive sampling devices.•Device provides quantity information on a subset of significant hydrocarbons.•Proteinaceous medium has an improved equilibration time and reuse capabilities in comparison to the current generation of PAH sampling devices.

UNCoordinated-6 (UNC-6) was the first member of the netrin family to be discovered in Caenorhabditis elegans. With homology to human netrin-1, it is a key signaling molecule involved in directing axon migration in nematodes. Similar to netrin-1, UNC-6 interacts with multiple receptors (UNC-5 and UNC-40, specifically) to guide axon migration in development. As a result of the distinct evolutionary path of UNC-6 compared to vertebrate netrins, we decided to employ an integrated approach to study its solution behavior and compare it to the high-resolution structure we previously published on vertebrate netrins. Dynamic light scattering and analytical ultracentrifugation on UNC-6 (with and without its C-domain) solubilized in a low-ionic strength buffer suggested that UNC-6 forms high-order oligomers. An increase in the buffer ionic strength resulted in a more homogeneous preparation of UNC-6, that was used for subsequent solution x-ray scattering experiments. Our biophysical analysis of UNC-6 ΔC solubilized in a high-ionic strength buffer suggested that it maintains a similar head-to-stalk arrangement as netrins −1 and −4. This phenomenon is thought to play a role in the signaling behavior of UNC-6 and its ability to move throughout the extracellular matrix.

Coiled coils are well described as powerful oligomerization motifs and exhibit a large diversity of functions, including gene regulation, cell division, membrane fusion and drug extrusion. The archaea S-layer originated right-handed coiled coil-RHCC-NT- is characterized by extreme stability and is free of cysteine and histidine moieties. In the current study, we have followed a multidisciplinary approach to investigate the capacity of RHCC-NT to bind a variety of ionic complex metal ions. At the outside of the RHCC-NT, one mercury ion forms an electrostatic interaction with the S-methyl moiety of the single methionine residue present in each coil. We demonstrate that RHCC-NT is reducing and incorporating metallic mercury in the large-sized interior cavities which are lined up along the tetrameric channel.

Neuropilin-1 (NRP1) is the cellular growth factor that interacts with semaphorin 3A, placenta growth factor-2 and vascular endothelial growth factor (VEGF165). The interactions of NRP1 with these proteins initiate signaling pathways that have implications on fundamental cellular processes. NRP1 have been designated as a potential target for the treatment of various types of cancers. It is composed of two CUB domains (a1 and a2) that are connected with F5/8 type C1 (b1) and C2 (b2) domains, followed by a flexible linker, a MAM domain, membrane-anchored region and a cytoplasmic tail. The high-resolution structures of various domains of human NRP1 (a2, b1 and b2 and MAM domains) are available. However, the information of how the a1 domain is connected to the a2, b1 and b2 domains and how the overall assembly behaves is unavailable. Recently, a crystal structure of mouse a1, a2, b1 and b2 (PDB:4Z9) domains suggested that the a1 domain is linked with the rest of the domains via a flexible linker. We characterized the wild-type NRP1 composed of a1, a2, b1 and b2 domains and a mutant version that lacks glycosylation using the dynamic light scattering, analytical ultracentrifugation, and small angle X-ray scattering techniques. The results from all three techniques suggest that the glycosylation is crucial for the stability and homogeneity of NRP1. Furthermore, all though no major difference was found between these two versions of NRP1 in terms of their low-resolution structures obtained using small angle X-ray scattering, their solution conformation differs significantly compared to the crystal structure of the deglycosylated version of mouse a1, a2, b1 and b2 structure. Based on our preliminary data, we hypothesize that the flexible linker between the a1 and a2 domains allow efficient interaction with ligands to initiate signaling pathways.

Laminin is a heterotrimeric molecule composed of an alpha, a beta and a gamma chain.It interacts with various ligands at the extracellular matrix and plays a pivotal role in many biological processes.Netrin4, one of the four secreted netrins, specifically interacts with the laminin gamma-1 chain (Schneiders et al. 2007).We recently highlighted the biological significance of this complex using ex vivo and in vivo studies (Reuten et al. 2016).Furthermore, we also solved a structure of netrin4 at 3.1 Å that allowed us to reason why netrin4 and our previously reported structure of netrin1 (Grandin et al. 2016) interact with different binding partners.To implicate the structure of netrin4/laminin gamma-1 complex and its biological functions, we employed a multidisciplinary approach where we combined the low-resolution shape information of netrin4/laminin gamma-1 complex with the high-resolution structure information of each component to obtain a highresolution model of the complex.This approach revealed that the N-terminal globular domains of netrin4 and laminin gamma-1 are required for the interaction (Reuten et al. 2016).The interaction sites were validated using structure-guided mutations that provided detailed insights on the biological relevance of this complex (Reuten et al. 2016).The highresolution model of the netrin4/laminin gamma-1 complex was validated by comparing the experimentally derived hydrodynamic properties with those calculated from the structures.

The collagen binding integrin α2β1 plays a crucial role in hemostasis, fibrosis, and cancer progression amongst others. It is specifically inhibited by rhodocetin (RC), a C-type lectin-related protein (CLRP) found in Malayan pit viper (Calloselasma rhodostoma) venom. The structure of RC alone reveals a heterotetramer arranged as an αβ and γδ subunit in a cruciform shape. RC specifically binds to the collagen binding A-domain of the integrin α2 subunit, thereby blocking collagen-induced platelet aggregation. However, until now, the molecular basis for this interaction has remained unclear. Here, we present the molecular structure of the RCγδ-α2A complex solved to 3.0 Å resolution. Our findings show that RC undergoes a dramatic structural reorganization upon binding to α2β1 integrin. Besides the release of the nonbinding RCαβ tandem, the RCγ subunit interacts with loop 2 of the α2A domain as result of a dramatic conformational change. The RCδ subunit contacts the integrin α2A domain in the "closed" conformation through its helix C. Combined with epitope-mapped antibodies, conformationally locked α2A domain mutants, point mutations within the α2A loop 2, and chemical modifications of the purified toxin protein, this molecular structure of RCγδ-α2A complex explains the inhibitory mechanism and specificity of RC for α2β1 integrin.

RNA-protein and protein-protein interactions play crucial roles in a number of biological processes. To understand mechanisms underpinning such process, structural information on biomolecular complexes are essential. However, the experimental determination of biomolecular complexes that provides information on position, orientation and interactions of individual domains is often time-consuming and in many cases not possible. As a result, complete high-resolution structures are not yet available for many complexes that are crucial for numerous fundamental cellular processes. An emerging alternative to high-resolution structural techniques is to employ a hybrid approach that combines low-resolution shape information about macromolecules and their complexes from hydrodynamic and SAXS methods, with high-resolution structures (where available), and with computational modeling to obtain atomic-level models. Our laboratories have established such approach and have used for a variety of complexes involving RNA-protein and protein-protein complexes. For example, such hybrid approach was utilized to study human netrin-4/laminin 𝛾;1 complex, revealing that the N-terminal globular domains of proteins mediate interactions. Structural guided mutations provided detailed insights on the biological relevance of this complex.1 Additionally, a high-resolutions structural model between adenovirus VAI RNA and host enzyme, Protein Kinase R (PKR) was also investigated using a hybrid approach2,3, demonstrating that only the apical stem of VAI interacts with the double-stranded RNA binding motifs of PKR. 1. Reuten R.∗ and Patel, T. R.∗ et al. Nature Communications (accepted). 2. Dzananovic, E.∗ and Patel, T. R.∗ et al. Journal of Structural Biology (2014) 185, 48-57. 3. PyRy3D (http://genesilico.pl/pyry3d/).

Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP) generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST), SlyD, and serum albumin (ser alb) tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin β2 mutant protein (congenital nephrotic syndrome), which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems.

Netrin-1 has been shown to be up-regulated in a fraction of human cancers as a mechanism to allow these tumors to escape the pro-apoptotic activity of some of its main dependence receptors, the UNC5 homologs (UNC5H). Here we identify the V-2 domain of netrin-1 to be important for its interaction with the Ig1/Ig2 domains of UNC5H2. We generate a humanized anti-netrin-1 antibody that disrupts the interaction between netrin-1 and UNC5H2 and triggers death of netrin-1-expressing tumor cells in vitro. We also present evidence that combining the anti-netrin-1 antibody with epidrugs such as decitabine could be effective in treating tumors showing no or modest netrin-1 expression. These results support that this antibody is a promising drug candidate.

RNA helicase associated with AU-rich element (RHAU) is an ATP-dependent RNA helicase that demonstrates high affinity for quadruplex structures in DNA and RNA. To elucidate the significance of these quadruplex-RHAU interactions, we have performed RNA co-immunoprecipitation screens to identify novel RNAs bound to RHAU and characterize their function. In the course of this study, we have identified the non-coding RNA BC200 (BCYRN1) as specifically enriched upon RHAU immunoprecipitation. Although BC200 does not adopt a quadruplex structure and does not bind the quadruplex-interacting motif of RHAU, it has direct affinity for RHAU in vitro. Specifically designed BC200 truncations and RNase footprinting assays demonstrate that RHAU binds to an adenosine-rich region near the 3-end of the RNA. RHAU truncations support binding that is dependent upon a region within the C terminus and is specific to RHAU isoform 1. Tests performed to assess whether BC200 interferes with RHAU helicase activity have demonstrated the ability of BC200 to act as an acceptor of unwound quadruplexes via a cytosine-rich region near the 3-end of the RNA. Furthermore, an interaction between BC200 and the quadruplex-containing telomerase RNA was confirmed by pull-down assays of the endogenous RNAs. This leads to the possibility that RHAU may direct BC200 to bind and exert regulatory functions at quadruplex-containing RNA or DNA sequences.

IMPORTANCE New genomic strategies can now be applied to identify a diagnosis in patients and families with previously undiagnosed rare genetic conditions. The large family evaluated in the present study was described in 1966 and now expands the phenotype of a known neuromuscular gene. OBJECTIVE To determine the genetic cause of a slowly progressive, autosomal dominant, scapuloperoneal neuromuscular disorder by using linkage and exome sequencing. DESIGN, SETTING, AND PARTICIPANTS Fourteen affected individuals in a 6-generation family with a progressive scapuloperoneal disorder were evaluated. Participants were examined at pediatric, neuromuscular, and research clinics from March 1, 2005, to May 31, 2014. Exome and linkage were performed in genetics laboratories of research institutions. MAIN OUTCOMES AND MEASURES Examination and evaluation by magnetic resonance imaging, ultrasonography, electrodiagnostic studies, and muscle biopsies (n = 3). Genetic analysis included linkage analysis (n = 17) with exome sequencing (n = 7). RESULTS Clinical findings included progressive muscle weakness in an initially scapuloperoneal and distal distribution, including wrist extensor weakness, finger and foot drop, scapular winging, mild facial weakness, Achilles tendon contractures, and diminished or absent deep tendon reflexes. Both age at onset and progression of the disease showed clinical variability within the family. Muscle biopsy specimens demonstrated type I fiber atrophy and trabeculated fibers without nemaline rods. Analysis of exome sequences within the linkage region (4.8 megabases) revealed missense mutation c.591C>A p.Glu197Asp in a highly conserved residue in exon 4 of ACTA1. The mutation cosegregated with disease in all tested individuals and was not present in unaffected individuals. CONCLUSIONS AND RELEVANCE This family defines a new scapuloperoneal phenotype associated with an ACTA1 mutation. A highly conserved protein, ACTA1 is implicated in multiple muscle diseases, including nemaline myopathy, actin aggregate myopathy, fiber-type disproportion, and rod-core myopathy. To our knowledge, mutations in Glu197 have not been reported previously. This residue is highly conserved and located in an exposed position in the protein; the mutation affects the intermolecular and intramolecular electrostatic interactions as shown by structural modeling. The mutation in this residue does not appear to lead to rod formation or actin accumulation in vitro or in vivo, suggesting a different molecular mechanism from that of other ACTA1 diseases.

Malignant glioma are often fatal and pose a significant therapeutic challenge. Here we have employed alpha-helical right handed coiled coils (RHCC) which self-assemble into tetrameric nanotubes that stably associate with platinum (Pt) (IV) compound. This Pt(IV)-RHCC complex showed superior in vitro and in vivo toxicity in human malignant glioma cells at up to 5 fold lower platinum concentrations when compared to free Pt(IV). Pt(IV)-RHCC nanotubes activated multiple cell death pathways in GB cells without affecting astrocytes in vitro or causing damage to normal mouse brain. This Pt(IV)-RHCC nanotubes may serve as a promising new therapeutic tool for low dose Pt(IV) prodrug application for highly efficient and selective treatment of human brain tumors. (C) 2015 Elsevier Inc. All rights reserved.

Collagen XXII, a FACIT (fibril-associated collagen with interrupted triple helices), is expressed at the myotendinous junction and the articular surface of joint cartilage. Cellular receptors like collagen-binding integrins are known to bind collagens with distinct binding motifs following the sequence GXOGER. In the present study, we demonstrate the sequences GLQGER and GFKGER as novel binding motifs between collagen XXII and collagen-binding integrins, especially α2β1 integrin. Solid-phase assays and surface plasmon resonance spectroscopy revealed a direct interaction between α2β1 integrin and the motif GFKGER. In addition, immunohistochemical analysis demonstrated partial co-localization of collagen XXII, α2β1 integrin and α11β1 integrin at the myotendinous junction. Furthermore, computational modelling of the motifs GLQGER and GFKGER showed perfect fitting of the sequences into the binding pocket of collagen-binding integrins. Taken together, we demonstrated that collagen XXII interacts with collagen-binding integrins via the new motifs GLQGER and GFKGER.

The human gyrovirus derived protein Apoptin (HGV-Apoptin) a homologue of the chicken anemia virus Apoptin (CAV-Apoptin), a protein with high cancer cells selective toxicity, triggers apoptosis selectively in cancer cells. In this paper, we show that HGV-Apoptin acts independently from the death receptor pathway as it induces apoptosis in similar rates in Jurkat cells deficient in either FADD (fas-associated death domain) function or caspase-8 (key players of the extrinsic pathway) and their parental clones. HGV-Apoptin induces apoptosis via the activation of the mitochondrial intrinsic pathway. It induces both mitochondrial inner and outer membrane permebilization, characterized by the loss of the mitochondrial potential and the release into cytoplasm of the pro-apoptotic molecules including apoptosis inducing factor and cytochrome c. HGV-Apoptin acts via the apoptosome, as lack of expression of apoptotic protease-activating factor 1 in murine embryonic fibroblast strongly protected the cells from HGV-Apoptin–induced apoptosis. Moreover, QVD-oph a broad-spectrum caspase inhibitor delayed HGV-Apoptin–induced death. On the other hand, overexpression of the anti-apoptotic BCL-XL confers resistance to HGV-Apoptin–induced cell death. In contrast, cells that lack the expression of the pro-apoptotic BAX and BAK are protected from HGV-Apoptin induced apoptosis. Furthermore, HGV-Apoptin acts independently from p53 signal but triggers the cytoplasmic translocation of Nur77. Taking together these data indicate that HGV-Apoptin acts through the mitochondrial pathway, in a caspase-dependent manner but independently from the death receptor pathway.

Background: Peptidoglycan metabolites regulate AmpR-mediated control of ampC -lactamase expression. Results: AmpR binds DNA as a tetramer and interacts with up to four copies of its repressor UDP-MurNAc-pentapeptide via its d-Ala-d-Ala motif. Conclusion: d-Ala-d-Ala recognition supports 1,6-anhydroMurNAc-pentapeptide as an AmpR activator, probably through competitive binding with UDP-MurNAc-pentapeptide. Significance: Understanding how peptidoglycan metabolites modulate AmpR provides insight into how -lactam classes differentially induce ampC expression.Inducible expression of chromosomal AmpC -lactamase is a major cause of -lactam antibiotic resistance in the Gram-negative bacteria Pseudomonas aeruginosa and Enterobacteriaceae. AmpC expression is induced by the LysR-type transcriptional regulator (LTTR) AmpR, which activates ampC expression in response to changes in peptidoglycan (PG) metabolite levels that occur during exposure to -lactams. Under normal conditions, AmpR represses ampC transcription by binding the PG precursor UDP-N-acetylmuramic acid (MurNAc)-pentapeptide. When exposed to -lactams, however, PG catabolites (1,6-anhydroMurNAc-peptides) accumulate in the cytosol, which have been proposed to competitively displace UDP-MurNAc-pentapeptide from AmpR and convert it into an activator of ampC transcription. Here we describe the molecular interactions between AmpR (from Citrobacter freundii), its DNA operator, and repressor UDP-MurNAc-pentapeptide. Non-denaturing mass spectrometry revealed AmpR to be a homotetramer that is stabilized by DNA containing the T-N-11-A LTTR binding motif and revealed that it can bind four repressor molecules in an apparently stepwise manner. A crystal structure of the AmpR effector-binding domain bound to UDP-MurNAc-pentapeptide revealed that the terminal d-Ala-d-Ala motif of the repressor forms the primary contacts with the protein. This observation suggests that 1,6-anhydroMurNAc-pentapeptide may convert AmpR into an activator of ampC transcription more effectively than 1,6-anhydroMurNAc-tripeptide (which lacks the d-Ala-d-Ala motif). Finally, small angle x-ray scattering demonstrates that the AmpRDNA complex adopts a flat conformation similar to the LTTR protein AphB and undergoes only a slight conformational change when binding UDP-MurNAc-pentapeptide. Modeling the AmpRDNA tetramer bound to UDP-MurNAc-pentapeptide predicts that the UDP-MurNAc moiety of the repressor participates in modulating AmpR function.

Netrin-1 is a bifunctional guidance cue for migrating axons in vertebrates. Depending on the target receptor family netrin-1 interacts with, it either causes attraction or repulsion of axonal growth cones. Chemoattraction is mediated by adhering to DCC or its homologue neogenin, whereas chemorepulsion results upon binding to the closely related UNC5A, UNC5B, UNC5C or UNC5D receptors. In addition to the nervous system, netrin-1 and its receptors have been detected in developing heart, lung, mammary gland, intestine and pancreas and are believed to be expressed ubiquitously. Both families of receptors have been implicated as dependence receptors, that is receptors that induce apoptosis if they are not bound to their ligand such as netrin-1. Thus they play a pivotal role in the development of the organism and in tumour suppression. Here, we present the 2.5 Å high resolution structure determined by X-ray crystallography that comprises four domains of netrin-1. We mapped the netrin-1/neogenin and the netrin-1/UNC5 binding sites by a series of domain swap experiments where we exchanged individual domains of netrin-1 with a structurally closely related -but non-functional- domain of the laminin γ1 short arm. We probed the binding behaviour of the chimeras by surface plasmon resonance and solid phase binding assays. Once the binding domain had been unambiguously identified, we determined the binding epitopes on netrin-1 by mutational analysis, using structural information and phylogenetic conservation as guides. We found that the DCC family and the UNC5 family of receptors bind to spatially distinct sites within the same domain. Reciprocally, we confirmed that the 5th fibronectin domain of DCC harbours the binding site for netrin-1 and determined the binding epitopes. Finally, we show the conformation of the netrin-1/neogenin complex in solution using SAXS data and present a map of the binding sites.

Nidogen-1 is a key basement membrane protein that is required for many biological activities. It is one of the central elements in organizing basal laminae including those in the skin, muscle, and the nervous system. The self-assembling extracellular matrix that also incorporates fibulins, fibronectin and integrins is clamped together by networks formed between nidogen, perlecan, laminin and collagen IV. To date, the full-length version of nidogen-1 has not been studied in detail in terms of its solution conformation and shape because of its susceptibility to proteolysis. In the current study, we have expressed and purified full-length nidogen-1 and have investigated its solution behavior using size-exclusion chromatography (SEC), dynamic light scattering (DLS) and small angle X-ray scattering (SAXS). The ab initio shape reconstruction of the complex between nidogen-1 and the laminin γ-1 short arm confirms that the interaction is mediated solely by the C-terminal domains: the rest of the domains of both proteins do not participate in complex formation.