GRID1/GluD1 homozygous variants linked to intellectual disability and spastic paraplegia impair mGlu1/5 receptor signaling and excitatory synapses.

The ionotropic glutamate delta receptor GluD1, encoded by the GRID1 gene, is involved in synapse formation, function, and plasticity. GluD1 does not bind glutamate, but instead cerebellin and D-serine, which allow the formation of trans-synaptic bridges, and trigger transmembrane signaling. Despite wide expression in the nervous system, pathogenic GRID1 variants have not been characterized in humans so far.

High-Level Production of Recombinant Eukaryotic Proteins from Mammalian Cells Using Lentivirus.

Mammalian protein expression systems are ideally suited for the high-level production of recombinant eukaryotic secreted and membrane proteins for structural biology applications. Here, we present genetic transduction of HEK293-derived cells using lentivirus as a robust and cost-efficient method for the rapid generation of stable expression cell lines. We describe the features of the lentiviral transfer plasmid pHR-CMV-TetO, as well as detailed protocols for production of lentiviral particles, determination of functional lentiviral titer, infection of expression cells, culture and expansion of the resulting stable cell lines, their adaptation to adherent and suspension growth, and constitutive or inducible milligram-scale protein production.

Simultaneous binding of Guidance Cues NET1 and RGM blocks extracellular NEO1 signaling.

During cell migration or differentiation, cell surface receptors are simultaneously exposed to different ligands. However, it is often unclear how these extracellular signals are integrated. Neogenin (NEO1) acts as an attractive guidance receptor when the Netrin-1 (NET1) ligand binds, but it mediates repulsion via repulsive guidance molecule (RGM) ligands.

A synthetic synaptic organizer protein restores glutamatergic neuronal circuits.

Neuronal synapses undergo structural and functional changes throughout life, which are essential for nervous system physiology. However, these changes may also perturb the excitatory-inhibitory neurotransmission balance and trigger neuropsychiatric and neurological disorders. Molecular tools to restore this balance are highly desirable.

Pumping the brakes: suppression of synapse development by MDGA-neuroligin interactions.

Synapse development depends on a dynamic balance between synapse promoters and suppressors. MDGAs, immunoglobulin superfamily proteins, negatively regulate synapse development through blocking neuroligin-neurexin interactions. Recent analyses of MDGA-neuroligin complexes revealed the structural basis of this activity and indicate that MDGAs interact with all neuroligins with differential affinities.

Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins.

Structural, biochemical and biophysical studies of eukaryotic soluble and membrane proteins require their production in milligram quantities. Although large-scale protein expression strategies based on transient or stable transfection of mammalian cells are well established, they are associated with high consumable costs, limited transfection efficiency or long and tedious selection of clonal cell lines. Lentiviral transduction is an efficient method for the delivery of transgenes to mammalian cells and unifies the ease of use and speed of transient transfection with the robust expression of stable cell lines.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software.

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187.

nandb-number and brightness in R with a novel automatic detrending algorithm.

Summary: An R package for performing number and brightness image analysis, with the implementation of a novel automatic detrending algorithm.

Availability And Implementation: Available at https://github.com/rorynolan/nandb for all platforms.

Structural dissection of Shewanella oneidensis old yellow enzyme 4 bound to a Meisenheimer complex and (nitro)phenolic ligands.

Shewanella oneidensis, a Gram-negative γ-proteobacterium with an extensive redox capacity, possesses four old yellow enzyme (OYE) homologs. Of these, Shewanella yellow enzyme 4 (SYE4) is implicated in resistance to oxidative stress. Here, we present a series of high-resolution crystal structures for SYE4 in the oxidized and reduced states, and in complex with phenolic ligands and the nitro-aromatic explosive picric acid.

Structural basis for integration of GluD receptors within synaptic organizer complexes.

Ionotropic glutamate receptor (iGluR) family members are integrated into supramolecular complexes that modulate their location and function at excitatory synapses. However, a lack of structural information beyond isolated receptors or fragments thereof currently limits the mechanistic understanding of physiological iGluR signaling. Here, we report structural and functional analyses of the prototypical molecular bridge linking postsynaptic iGluR δ2 (GluD2) and presynaptic β-neurexin 1 (β-NRX1) via Cbln1, a C1q-like synaptic organizer.

Structure and Assembly Mechanism of the Signaling Complex Mediated by Human CSF-1.

Human colony-stimulating factor 1 receptor (hCSF-1R) is unique among the hematopoietic receptors because it is activated by two distinct cytokines, CSF-1 and interleukin-34 (IL-34). Despite ever-growing insights into the central role of hCSF-1R signaling in innate and adaptive immunity, inflammatory diseases, and cancer, the structural basis of the functional dichotomy of hCSF-1R has remained elusive. Here, we report crystal structures of ternary complexes between hCSF-1 and hCSF-1R, including their complete extracellular assembly, and propose a mechanism for the cooperative human CSF-1:CSF-1R complex that relies on the adoption by dimeric hCSF-1 of an active conformational state and homotypic receptor interactions.

Structure and functional properties of Norrin mimic Wnt for signalling with Frizzled4, Lrp5/6, and proteoglycan.

Wnt signalling regulates multiple processes including angiogenesis, inflammation, and tumorigenesis. Norrin (Norrie Disease Protein) is a cystine-knot like growth factor. Although unrelated to Wnt, Norrin activates the Wnt/β-catenin pathway.

Repulsive guidance molecule is a structural bridge between neogenin and bone morphogenetic protein.

Repulsive guidance molecules (RGMs) control crucial processes including cell motility, adhesion, immune-cell regulation and systemic iron metabolism. RGMs signal via the neogenin (NEO1) and the bone morphogenetic protein (BMP) pathways. Here, we report crystal structures of the N-terminal domains of all human RGM family members in complex with the BMP ligand BMP2, revealing a new protein fold and a conserved BMP-binding mode.

Structural basis for extracellular cis and trans RPTPσ signal competition in synaptogenesis.

Receptor protein tyrosine phosphatase sigma (RPTPσ) regulates neuronal extension and acts as a presynaptic nexus for multiple protein and proteoglycan interactions during synaptogenesis. Unknown mechanisms govern the shift in RPTPσ function, from outgrowth promotion to synaptic organization. Here, we report crystallographic, electron microscopic and small-angle X-ray scattering analyses, which reveal sufficient inter-domain flexibility in the RPTPσ extracellular region for interaction with both cis (same cell) and trans (opposite cell) ligands.

The bacterial antitoxin HipB establishes a ternary complex with operator DNA and phosphorylated toxin HipA to regulate bacterial persistence.

Nearly all bacteria exhibit a type of phenotypic growth described as persistence that is thought to underlie antibiotic tolerance and recalcitrant chronic infections. The chromosomally encoded high-persistence (Hip) toxin-antitoxin proteins HipASO and HipBSO from Shewanella oneidensis, a proteobacterium with unusual respiratory capacities, constitute a type II toxin-antitoxin protein module. Here we show that phosphorylated HipASO can engage in an unexpected ternary complex with HipBSO and double-stranded operator DNA that is distinct from the prototypical counterpart complex from Escherichia coli.

Human IL-34 and CSF-1 establish structurally similar extracellular assemblies with their common hematopoietic receptor.

The discovery that hematopoietic human colony stimulating factor-1 receptor (CSF-1R) can be activated by two distinct cognate cytokines, colony stimulating factor-1 (CSF-1) and interleukin-34 (IL-34), created puzzling scenarios for the two possible signaling complexes. We here employ a hybrid structural approach based on small-angle X-ray scattering (SAXS) and negative-stain EM to reveal that bivalent binding of human IL-34 to CSF-1R leads to an extracellular assembly hallmarked by striking similarities to the CSF-1:CSF-1R complex, including homotypic receptor-receptor interactions. Thus, IL-34 and CSF-1 have evolved to exploit the geometric requirements of CSF-1R activation.

Allosteric competitive inactivation of hematopoietic CSF-1 signaling by the viral decoy receptor BARF1.

Hematopoietic human colony-stimulating factor 1 (hCSF-1) is essential for innate and adaptive immunity against viral and microbial infections and cancer. The human pathogen Epstein-Barr virus secretes the lytic-cycle protein BARF1 that neutralizes hCSF-1 to achieve immunomodulation. Here we show that BARF1 binds the dimer interface of hCSF-1 with picomolar affinity, away from the cognate receptor-binding site, to establish a long-lived complex featuring three hCSF-1 at the periphery of the BARF1 toroid.

Extracellular complexes of the hematopoietic human and mouse CSF-1 receptor are driven by common assembly principles.

The hematopoietic colony stimulating factor-1 receptor (CSF-1R or FMS) is essential for the cellular repertoire of the mammalian immune system. Here, we report a structural and mechanistic consensus for the assembly of human and mouse CSF-1:CSF-1R complexes. The EM structure of the complete extracellular assembly of the human CSF-1:CSF-1R complex reveals how receptor dimerization by CSF-1 invokes a ternary complex featuring extensive homotypic receptor contacts and striking structural plasticity at the extremities of the complex.

Inducible production of recombinant human Flt3 ectodomain variants in mammalian cells and preliminary crystallographic analysis of Flt3 ligand-receptor complexes.

The extracellular complex between the haematopoietic receptor Flt3 and its cytokine ligand (FL) is the cornerstone of signalling cascades that are central to early haematopoiesis and the immune system. Here, efficient protocols for the production of two ectodomain variants of human Flt3 receptor, Flt3D1-D5 and Flt3D1-D4, for structural studies are reported based on tetracycline-inducible stable cell lines in HEK293S cells deficient in N-acetylglycosaminyltransferase I (GnTI-/-) that can secrete the target proteins with limited and homogeneous N-linked glycosylation to milligram amounts. The ensuing preparative purification of Flt3 receptor-ligand complexes yielded monodisperse complex preparations that were amenable to crystallization.

Structural insights into the extracellular assembly of the hematopoietic Flt3 signaling complex.

The class III receptor tyrosine kinase (RTKIII) Fms-like tyrosine kinase receptor 3 (Flt3) and its cytokine ligand (FL) play central roles in hematopoiesis and the immune system, by establishing signaling cascades crucial for the development and homeostasis of hematopoietic progenitors and antigen-presenting dendritic cells. However, Flt3 is also one of the most frequently mutated receptors in hematologic malignancies and is currently a major prognostic factor and clinical target for acute myeloid leukemia. Here, we report the structural basis for the Flt3 ligand-receptor complex and unveil an unanticipated extracellular assembly unlike any other RTKIII/V complex characterized to date.

Glutathione import in Haemophilus influenzae Rd is primed by the periplasmic heme-binding protein HbpA.

Glutathione (GSH) is a vital intracellular cysteine-containing tripeptide across all kingdoms of life and assumes a plethora of cellular roles. Such pleiotropic behavior relies on a finely tuned spatiotemporal distribution of glutathione and its conjugates, which is not only controlled by synthesis and breakdown, but also by transport. Here, we show that import of glutathione in the obligate human pathogen Haemophilus influenzae, a glutathione auxotrophe, is mediated by the ATP-binding cassette (ABC)-like dipeptide transporter DppBCDF, which is primed for glutathione transport by a dedicated periplasmic-binding protein (PBP).