Structures of the arginine-vasopressin and oxytocin receptor signaling complexes.

Arginine-vasopressin (AVP) and oxytocin (OT) are neurohypophysial hormones which share a high sequence and structure homology. These are two cyclic C-terminally amidated nonapeptides with different residues at position 3 and 8. In mammals, AVP and OT exert their multiple biological functions through a specific G protein-coupled receptor family: four receptors are identified, the V1a, V1b, V2 receptors (V1aR, V1bR and V2R) and the OT receptor (OTR). The chemical structure of AVP and OT was elucidated in the early 1950s. Thanks to X-ray crystallography and cryo-electron microscopy, it took however 70 additional years to determine the three-dimensional structures of the OTR and the V2R in complex with their natural agonist ligands and with different signaling partners, G proteins and ß-arrestins. Today, the comparison of the different AVP/OT receptor structures gives structural insights into their orthosteric ligand binding pocket, their molecular mechanisms of activation, and their interfaces with canonical Gs, Gq and ß-arrestin proteins. It also paves the way to future rational drug design and therapeutic compound development. Indeed, agonist, antagonist, biased agonist, or pharmacological chaperone analogues of AVP and OT are promising candidates to regulate different physiological functions and treat several pathologies.

Structure, dynamics and transferability of the metal-dependent polyhistidine tetramerization motif TetrHis for single-chain Fv antibodies.

The polyhistidine (6XHis) motif is one of the most ubiquitous protein purification tags. The 6XHis motif enables the binding of tagged proteins to various metals, which can be advantageously used for purification with immobilized metal affinity chromatography. Despite its popularity, protein structures encompassing metal-bound 6XHis are rare. Here, we obtained a 2.5 Å resolution crystal structure of a single chain Fv antibody (scFv) bearing a C-terminal sortase motif, 6XHis and TwinStrep tags (LPETGHHHHHHWSHPQFEK[G3S]3WSHPQFEK). The structure, obtained in the presence of cobalt, reveals a unique tetramerization motif (TetrHis) stabilized by 8 Co2+ ions. The TetrHis motif contains four 6 residues-long ß-strands, and each metal center coordinates 3 to 5 residues, including all 6XHis histidines. By combining dynamic light scattering, small angle x-ray scattering and molecular dynamics simulations, We investigated the influence of Co2+ on the conformational dynamics of scFv 2A2, observing an open/close equilibrium of the monomer and the formation of cobalt-stabilized tetramers. By using a similar scFv design, we demonstrate the transferability of the tetramerization property. This novel metal-dependent tetramerization motif might be used as a fiducial marker for cryoelectron microscopy of scFv complexes, or even provide a starting point for designing metal-loaded biomaterials.

Bringing GPCR Structural Biology to Medical Applications: Insights from Both V2 Vasopressin and Mu-Opioid Receptors.

G-protein coupled receptors (GPCRs) are versatile signaling proteins that regulate key physiological processes in response to a wide variety of extracellular stimuli. The last decade has seen a revolution in the structural biology of clinically important GPCRs. Indeed, the improvement in molecular and biochemical methods to study GPCRs and their transducer complexes, together with advances in cryo-electron microscopy, NMR development, and progress in molecular dynamic simulations, have led to a better understanding of their regulation by ligands of different efficacy and bias. This has also renewed a great interest in GPCR drug discovery, such as finding biased ligands that can either promote or not promote specific regulations. In this review, we focus on two therapeutically relevant GPCR targets, the V2 vasopressin receptor (V2R) and the mu-opioid receptor (µOR), to shed light on the recent structural biology studies and show the impact of this integrative approach on the determination of new potential clinical effective compounds.

Structure of the vasopressin hormone-V2 receptor-ß-arrestin1 ternary complex.

Arrestins interact with G protein-coupled receptors (GPCRs) to stop G protein activation and to initiate key signaling pathways. Recent structural studies shed light on the molecular mechanisms involved in GPCR-arrestin coupling, but whether this process is conserved among GPCRs is poorly understood. Here, we report the cryo-electron microscopy active structure of the wild-type arginine-vasopressin V2 receptor (V2R) in complex with ß-arrestin1. It reveals an atypical position of ß-arrestin1 compared to previously described GPCR-arrestin assemblies, associated with an original V2R/ß-arrestin1 interface involving all receptor intracellular loops. Phosphorylated sites of the V2R carboxyl terminus are clearly identified and interact extensively with the ß-arrestin1 N-lobe, in agreement with structural data obtained with chimeric or synthetic systems. Overall, these findings highlight a notable structural variability among GPCR-arrestin signaling complexes.

Effect of human secretory calcium-binding phosphoprotein proline-glutamine rich 1 protein on Porphyromonas gingivalis and identification of its active portions.

The mouth environment comprises the second most significant microbiome in the body, and its equilibrium is critical in oral health. Secretory calcium-binding phosphoprotein proline-glutamine rich 1 (SCPPPQ1), a protein normally produced by the gingival epithelium to mediate its attachment to teeth, was suggested to be bactericidal. Our aim was to further explore the antibacterial potential of human SCPPPQ1 by characterizing its mode of action and identifying its active portions. In silico analysis showed that it has molecular parallels with antimicrobial peptides. Incubation of Porphyromonas gingivalis, a major periodontopathogen, with the full-length protein resulted in decrease in bacterial number, formation of aggregates and membrane disruptions. Analysis of SCPPPQ1-derived peptides indicated that these effects are sustained by specific regions of the molecule. Altogether, these data suggest that human SCPPPQ1 exhibits antibacterial capacity and provide new insight into its mechanism of action.

A hydride transfer complex reprograms NAD metabolism and bypasses senescence.

Metabolic rewiring and redox balance play pivotal roles in cancer. Cellular senescence is a barrier for tumorigenesis circumvented in cancer cells by poorly understood mechanisms. We report a multi-enzymatic complex that reprograms NAD metabolism by transferring reducing equivalents from NADH to NADP+. This hydride transfer complex (HTC) is assembled by malate dehydrogenase 1, malic enzyme 1, and cytosolic pyruvate carboxylase. HTC is found in phase-separated bodies in the cytosol of cancer or hypoxic cells and can be assembled in vitro with recombinant proteins. HTC is repressed in senescent cells but induced by p53 inactivation. HTC enzymes are highly expressed in mouse and human prostate cancer models, and their inactivation triggers senescence. Exogenous expression of HTC is sufficient to bypass senescence, rescue cells from complex I inhibitors, and cooperate with oncogenic RAS to transform primary cells. Altogether, we provide evidence for a new multi-enzymatic complex that reprograms metabolism and overcomes cellular senescence.

A proline rich protein from the gingival seal around teeth exhibits antimicrobial properties against Porphyromonas gingivalis.

The gingival seal around teeth prevents bacteria from destroying the tooth-supporting tissues and disseminating throughout the body. Porphyromonas gingivalis, a major periodontopathogen, degrades components of the specialized extracellular matrix that mediates attachment of the gingiva to the tooth. Of these, secretory calcium-binding phosphoprotein proline-glutamine rich 1 (SCPPPQ1) protein has a distinctive resistance to degradation, suggesting that it may offer resistance to bacterial attack. In silico analysis of its amino acid sequence was used to explore its molecular characteristics and to predict its two- and three-dimensional structure. SCPPPQ1 exhibits similarities with both proline-rich and cationic antimicrobial proteins, suggesting a putative antimicrobial potential. A combination of imaging approaches showed that incubation with 20 µM of purified SCPPPQ1 decrease bacterial number (p < 0.01). Fluorescence intensity decreased by 70% following a 2 h incubation of Porphyromonas gingivalis with the protein. Electron microscopy analyses revealed that SCPPPQ1 induced bacterial membrane disruption and breaches. While SCPPPQ1 has no effect on mammalian cells, our results suggest that it is bactericidal to Porphyromonas gingivalis, and that this protein, normally present in the gingival seal, may be exploited to maintain a healthy seal and prevent systemic dissemination of bacteria.

New Insights Into Acidithiobacillus thiooxidans Sulfur Metabolism Through Coupled Gene Expression, Solution Chemistry, Microscopy, and Spectroscopy Analyses.

Here, we experimentally expand understanding of the reactions and enzymes involved in Acidithiobacillus thiooxidans ATCC 19377 S0 and S 2 ⁢ O 3 2 - metabolism by developing models that integrate gene expression analyzed by RNA-Seq, solution sulfur speciation, electron microscopy and spectroscopy. The A. thiooxidans S 2 ⁢ O 3 2 - metabolism model involves the conversion of S 2 ⁢ O 3 2 - to SO 4 2 - , S0 and S 4 ⁢ O 6 2 - , mediated by the sulfur oxidase complex (Sox), tetrathionate hydrolase (TetH), sulfide quinone reductase (Sqr), and heterodisulfate reductase (Hdr) proteins. These same proteins, with the addition of rhodanese (Rhd), were identified to convert S0 to SO 3 2 - , S 2 ⁢ O 3 2 - and polythionates in the A. thiooxidans S0 metabolism model. Our combined results shed light onto the important role specifically of TetH in S 2 ⁢ O 3 2 - metabolism. Also, we show that activity of Hdr proteins rather than Sdo are likely associated with S0 oxidation. Finally, our data suggest that formation of intracellular S 2 ⁢ O 3 2 - is a critical step in S0 metabolism, and that recycling of internally generated SO 3 2 - occurs, through comproportionating reactions that result in S 2 ⁢ O 3 2 - . Electron microscopy and spectroscopy confirmed intracellular production and storage of S0 during growth on both S0 and S 2 ⁢ O 3 2 - substrates.

Nanoporosity Stimulates Cell Spreading and Focal Adhesion Formation in Cells with Mutated Paxillin.

We have evaluated the response to nanotopography of CHO-K1 cells that express wild-type paxillin or paxillin with mutations at serine 273 that inhibit phosphorylation. Cells were grown on nanoporous and polished titanium surfaces. With all cell types, immunofluorescence showed that adhesion and spreading were minimally affected on the treated surface and that the actin filaments were more abundant and well-aligned. Scanning electron microscopy revealed changes in cell shape and abundant filopodia with lateral nanoprotrusions in response to nanoporosity. Gene expression of proteins associated with cellular adhesion and protrusions was significantly increased on the nanoporous surface regardless of the cell type. In particular, α-actinin, Rac1, Cdc42, and ITGα1 were upregulated in S273 cells with alanine substitutions, whereas FAK, Pxn, and Src were downregulated, leading to improved focal adhesion formation. These findings suggest that the surface nanoporosity can "compensate for" the genetic mutations that affect the biomechanical relationship of cells to surfaces.

Selective bacterial degradation of the extracellular matrix attaching the gingiva to the tooth.

The junctional epithelium (JE) is a specialized portion of the gingiva that seals off the tooth-supporting tissues from the oral environment. This relationship is achieved via a unique adhesive extracellular matrix that is, in fact, a specialized basal lamina (sBL). Three unique proteins - amelotin (AMTN), odontogenic ameloblast-associated (ODAM), and secretory calcium-binding phosphoprotein proline-glutamine rich 1 (SCPPPQ1) - together with laminin-332 structure the supramolecular organization of this sBL and determine its adhesive capacity. Despite the constant challenge of the JE by the oral microbiome, little is known of the susceptibility of the sBL to bacterial degradation. Assays with trypsin-like proteases, as well as incubation with Porphyromonas gingivalis, Prevotella intermedia, and Treponema denticola, revealed that all constituents, except SCPPPQ1, were rapidly degraded. Porphyromonas gingivalis was also shown to alter the supramolecular network of reconstituted and native sBLs. These results provide evidence that proteolytic enzymes and selected gram-negative periodontopathogenic bacteria can attack this adhesive extracellular matrix, intimating that its degradation could contribute to progression of periodontal diseases.

High-yield production of human Dicer by transfection of human HEK293-EBNA1 cells grown in suspension.

BACKGROUND: Dicer is a 219-kDa protein that plays key roles in gene regulation, particularly as the ribonuclease III enzyme responsible for cleaving precursor miRNA substrates. Its enzymatic activity is highly regulated by protein factors, and this regulation can impact on the levels of miRNAs and modulate the behavior of a cell. To better understand the underlying mechanisms of regulation, detailed enzymatic and structural characterization of Dicer are needed. However, these types of studies generally require several milligrams of recombinant protein, and efficient preparation of such quantities of pure human Dicer remains a challenge. To prepare large quantities of human Dicer, we have optimized transfection in HEK293-6E cells grown in suspension and streamlined a purification procedure. RESULTS: Transfection conditions were first optimized to achieve expression levels between 10 and 18 mg of recombinant Dicer per liter of culture. A three-step purification protocol was then developed that yields 4-9 mg of purified Dicer per liter of culture in a single day. From SEC-MALS/RI analysis and negative stain TEM, we confirmed that the purified protein is monomerically pure ( ≥ 98%) and folds with the characteristic L-shape geometry. Using an electrophoretic mobility shift assay, a dissociation constant (Kd) of 5 nM was measured for Dicer binding to pre-let-7a-1, in agreement with previous reports. However, when probing the cleavage activity of Dicer for pre-let-7a-1, we measured kcat (7.2 ± 0.5 min- 1) and KM (1.2 ± 0.3 µM) values that are much higher than previously reported due to experimental conditions that better respect the steady-state assumption. CONCLUSIONS: The expression and purification protocols described here provide high yields of monomerically pure and active human Dicer. Cleavage studies of a pre-let-7 substrate with this purified Dicer reveal higher kcat and KM values than previously reported and support the current view that conformational changes are associated with substrate binding. Large quantities of highly pure Dicer will be valuable for future biochemical, biophysical and structural investigations of this key protein of the miRNA pathway.

Interaction via the N terminus of the type IV secretion system (T4SS) protein VirB6 with VirB10 is required for VirB2 and VirB5 incorporation into T-pili and for T4SS function.

Many bacterial pathogens employ multicomponent protein complexes such as type IV secretion systems (T4SSs) to transfer virulence factors into host cells. Here we studied the interaction between two essential T4SS components: the very hydrophobic inner membrane protein VirB6, which may be a component of the translocation channel, and VirB10, which links the inner and outer bacterial membranes. To map the interaction site between these two T4SS components, we conducted alanine scanning and deleted six-amino acid stretches from the N-terminal periplasmic domain of VirB6 from Brucella suis Using the bacterial two-hybrid system to analyze the effects of these alterations on the VirB6-VirB10 interaction, we identified the amino acid regions 16-21 and 28-33 and Leu-18 in VirB6 as being required for this interaction. SDS-PAGE coupled with Western blotting of cell lysates and native PAGE of detergent-extracted membrane proteins revealed that the corresponding VirB6 residues in Agrobacterium tumefaciens (Phe-20 and amino acids 18-23 and 30-35) modulate the stability of both VirB6 and VirB5. However, the results from immuno-EM and super-resolution microscopy suggested that these regions and residues are not required for membrane association or for polar localization of VirB6. The six-amino acid deletions in the N terminus of VirB6 abolished pilus formation and virulence of A. tumefaciens, and the corresponding deletions in the VirB6 homolog TraD from the plasmid pKM101-T4SS abrogated plasmid transfer. Our results indicate that specific residues of the VirB6 N-terminal domain are required for VirB6 stabilization, its interaction with VirB10, and the incorporation of VirB2 and VirB5 into T-pili.

VirB8 homolog TraE from plasmid pKM101 forms a hexameric ring structure and interacts with the VirB6 homolog TraD.

Type IV secretion systems (T4SSs) are multiprotein assemblies that translocate macromolecules across the cell envelope of bacteria. X-ray crystallographic and electron microscopy (EM) analyses have increasingly provided structural information on individual T4SS components and on the entire complex. As of now, relatively little information has been available on the exact localization of the inner membrane-bound T4SS components, notably the mostly periplasmic VirB8 protein and the very hydrophobic VirB6 protein. We show here that the membrane-bound, full-length version of the VirB8 homolog TraE from the plasmid pKM101 secretion system forms a high-molecular-mass complex that is distinct from the previously characterized periplasmic portion of the protein that forms dimers. Full-length TraE was extracted from the membranes with detergents, and analysis by size-exclusion chromatography, cross-linking, and size exclusion chromatography (SEC) multiangle light scattering (MALS) shows that it forms a high-molecular-mass complex. EM and small-angle X-ray scattering (SAXS) analysis demonstrate that full-length TraE forms a hexameric complex with a central pore. We also overproduced and purified the VirB6 homolog TraD and show by cross-linking, SEC, and EM that it binds to TraE. Our results suggest that TraE and TraD interact at the substrate translocation pore of the secretion system.

TDP-43 regulates the alternative splicing of hnRNP A1 to yield an aggregation-prone variant in amyotrophic lateral sclerosis.

See Fratta and Isaacs (doi:10.1093/brain/awy091) for a scientific commentary on this article.The RNA binding proteins TDP-43 (encoded by TARDBP) and hnRNP A1 (HNRNPA1) are each mutated in certain amyotrophic lateral sclerosis cases and are often mislocalized in cytoplasmic aggregates within motor neurons of affected patients. Cytoplasmic inclusions of TDP-43, which are accompanied by a depletion of nuclear TDP-43, are observed in most amyotrophic lateral sclerosis cases and nearly half of frontotemporal dementia cases. Here, we report that TDP-43 binds HNRNPA1 pre-mRNA and modulates its splicing, and that depletion of nuclear TDP-43 results in increased inclusion of a cassette exon in the HNRNPA1 transcript, and consequently elevated protein levels of an isoform containing an elongated prion-like domain, referred to as hnRNP A1B. Combined in vivo and in vitro approaches demonstrated greater fibrillization propensity for hnRNP A1B, which drives protein aggregation and is toxic to cells. Moreover, amyotrophic lateral sclerosis patients with documented TDP-43 pathology showed neuronal hnRNP A1B cytoplasmic accumulation, indicating that TDP-43 mislocalization may contribute to neuronal vulnerability and loss via altered HNRNPA1 pre-mRNA splicing and function. Given that TDP-43 and hnRNP A1 each bind, and thus modulate, a third of the transcriptome, our data suggest a much broader disruption in RNA metabolism than previously considered.

A nanoporous titanium surface promotes the maturation of focal adhesions and formation of filopodia with distinctive nanoscale protrusions by osteogenic cells.

While topography is a key determinant of the cellular response to biomaterials, the mechanisms implicated in the cell-surface interactions are complex and still not fully elucidated. In this context, we have examined the effect of nanoscale topography on the formation of filopodia, focal adhesions, and gene expression of proteins associated with cell adhesion and sensing. Commercially pure titanium discs were treated by oxidative nanopatterning with a solution of H2SO4/H2O2 50:50 (v/v). Scanning electron microscopy and atomic force microscopy characterizations showed that this facile chemical treatment efficiently creates a unique nanoporous surface with a root-mean-square roughness of 11.5nm and pore diameter of 20±5nm. Osteogenic cells were cultured on polished (control) and nanotextured discs for periods of 6, 24, and 72h. Immunofluorescence analysis revealed increases in the adhesion formation per cell area, focal adhesion length, and maturity on the nanoporous surface. Gene expression for various focal adhesion markers, including paxillin and talin, and different integrins (e.g. α1, ß1, and α5) was also significantly increased. Scanning electron microscopy revealed the presence of more filopodia on cells grown on the nanoporous surface. These cell extensions displayed abundant and distinctive nanoscale lateral protrusions of 10-15nm diameter that molded the nanopore walls. Together the increase in the focal adhesions and abundance of filopodia and associated protrusions could contribute to strengthening the adhesive interaction of cells with the surface, and thereby, alter the nanoscale biomechanical relationships that trigger cellular cascades that regulate cell behavior. STATEMENT OF SIGNIFICANCE: Oxidative patterning was exploited to create a unique three-dimensional network of nanopores on titanium surfaces. Our study illustrates how a facile chemical treatment can be advantageously used to modulate cellular behavior. The nanoscale lateral protrusions on filopodia elicited by this surface are novel adhesive structures. Altogether, the increases in focal adhesion, length, maturity, and filopodia with distinctive lateral protrusions could substantially increase the contact area and adhesion strength of cells, thereby promoting the activation of cellular signaling cascades that may explain the positive osteogenic outcomes previously achieved with this surface. Such physicochemical cueing offers a simple attractive alternative to the use of bioactive agents for guiding tissue repair/regeneration around implantable metals.

Interactions of AMTN, ODAM and SCPPPQ1 proteins of a specialized basal lamina that attaches epithelial cells to tooth mineral.

A specialized basal lamina (sBL) mediates adhesion of certain epithelial cells to the tooth. It is distinct because it does not contain collagens type IV and VII, is enriched in laminin-332, and includes three novel constituents called amelotin (AMTN), odontogenic ameloblast-associated (ODAM), and secretory calcium-binding phosphoprotein proline-glutamine rich 1 (SCPPPQ1). The objective of this study was to clarify the structural organization of the sBL. Fluorescence and immunogold labeling showed that the three proteins co-localize. Quantitative analysis of the relative position of gold particles on the sBL demonstrates that the distribution of ODAM is skewed towards the cell while that of AMTN and SCPPPQ1 tends towards the tooth surface. Bacterial two-hybrid analysis and co-immunoprecipitation, gel filtration of purified proteins and transmission electron and atomic force microscopies highlight the propensity of AMTN, ODAM, and SCPPPQ1 to interact with and among themselves and form supramolecular aggregates. These data suggest that AMTN, ODAM and SCPPPQ1 participate in structuring an extracellular matrix with the distinctive capacity of attaching epithelial cells to mineralized surfaces. This unique feature is particularly relevant for the adhesion of gingival epithelial cells to the tooth surface, which forms a protective seal that is the first line of defense against bacterial invasion.

Cag-delta (Cag3) protein from the Helicobacter pylori 26695 cag type IV secretion system forms ring-like supramolecular assemblies.

Helicobacter pylori is an important cause of gastric pathologies and persistent infection can lead to stomach cancer. Virulent H. pylori strains encode a type IV secretion system responsible for translocation of the oncogenic CagA protein into cells of the gastric mucosa. Gene HP0522 encodes the essential component Cagδ (Cag3), and we show by gel filtration and cross-linking that purified Cagδ forms high molecular mass complexes. In contrast, its interaction partner CagT is mostly monomeric, but co-fractionates after gel filtration. Analysis by transmission electron microscopy revealed that purified Cagδ complexes can self-assemble ring-like structures. Cagδ-overexpressing Escherichia coli exhibits membrane-associated circular profiles in regions of the cell envelope with intense immunogold labelling with a Cagδ-specific antiserum. Our results suggest that Cagδ has the capacity to form macromolecular structures contributing to the assembly of the type IV secretion system.

Laminin γ2 knockout mice rescued with the human protein exhibit enamel maturation defects.

The epithelial ameloblasts are separated from the maturing enamel by an atypical basement membrane (BM) that is enriched in laminin 332 (LM-332). This heterotrimeric protein (α3, ß3 and γ2 chains) provides structural integrity to BMs and influences various epithelial cell processes including cell adhesion and differentiation. Mouse models that lack expression of individual LM-332 chains die shortly after birth. The lethal phenotype of laminin γ2 knockout mice can be rescued by human laminin γ2 (LAMC2) expressed using a doxycycline-inducible (Tet-on) cytokeratin 14 promoter-rtTA. These otherwise normal-looking rescued mice exhibit white spot lesions on incisors. We therefore investigated the effect of rescue with human LAMC2 on enamel maturation and structuring of the atypical BM. The maturation stage enamel organ in transgenic mice was severely altered as compared to wild type controls, a structured BM was no longer discernible, dystrophic matrix appeared in the maturing enamel layer, and there was residual enamel matrix late into the maturation stage. Microtomographic scans revealed excessive wear of occlusal surfaces on molars, chipping of enamel on incisor tips, and hypomineralization of the enamel layer. No structural alterations were observed at other epithelial sites, such as skin, palate and tongue. These results indicate that while this humanized mouse model is capable of rescue in various epithelial tissues, it is unable to sustain structuring of a proper BM at the interface between ameloblasts and maturing enamel. This failure may be related to the atypical composition of the BM in the maturation stage and reaffirms that the atypical BM is essential for enamel maturation.