High-Throughput Protein Expression Screening of Cell-Surface Protein Ectodomains.

Cell-surface receptors can be difficult to express and purify for structural and biochemical studies due to low expression levels, misfolding, aggregation, and instability. Cell-surface receptor ectodomains are more amenable to large-scale production, but this requires designing and testing various truncation constructs. However, since each protein is unique, testing these constructs individually for many targets is a time-consuming process.

Synthetic integrin antibodies discovered by yeast display reveal αV subunit pairing preferences with β subunits.

Integrins are cell surface receptors that mediate the interactions of cells with their surroundings and play essential roles in cell adhesion, migration, and homeostasis. Eight of the 24 integrins bind to the tripeptide Arg-Gly-Asp (RGD) motif in their extracellular ligands, comprising the RGD-binding integrin subfamily. Despite similarity in recognizing the RGD motif and some redundancy, these integrins can selectively recognize RGD-containing ligands to fulfill specific functions in cellular processes.

CKLF instigates a "cold" microenvironment to promote MYCN-mediated tumor aggressiveness.

Solid tumors, especially those with aberrant MYCN activation, often harbor an immunosuppressive microenvironment to fuel malignant growth and trigger treatment resistance. Despite this knowledge, there are no effective strategies to tackle this problem. We found that chemokine-like factor () is highly expressed by various solid tumor cells and transcriptionally up-regulated by MYCN.

Structural insights into the formation of repulsive netrin guidance complexes.

Netrins dictate attractive and repulsive responses during axon growth and cell migration, where the presence of the receptor Uncoordinated-5 (UNC-5) on target cells results in repulsion. Here, we showed that UNC-5 is a heparin-binding protein, determined its structure bound to a heparin fragment, and could modulate UNC-5-heparin affinity using a directed evolution platform or structure-based rational design. We demonstrated that UNC-5 and UNC-6/netrin form a large, stable, and rigid complex in the presence of heparin, and heparin and UNC-5 exclude the attractive UNC-40/DCC receptor from binding to UNC-6/netrin to a large extent.

Synthetic integrin antibodies discovered by yeast display reveal αV subunit pairing preferences with β subunits.

Eight of the 24 integrin heterodimers bind to the tripeptide Arg-Gly-Asp (RGD) motif in their extracellular ligands, and play essential roles in cell adhesion, migration, and homeostasis. Despite similarity in recognizing the RGD motif and some redundancy, these integrins can selectively recognize RGD-containing ligands including fibronectin, vitronectin, fibrinogen, nephronectin and the prodomain of the transforming growth factors to fulfill specific functions in cellular processes. Subtype-specific antibodies against RGD-binding integrins are desirable for investigating their specific functions.

Form factor determination of biological molecules with X-ray free electron laser small-angle scattering (XFEL-SAS).

Free-electron lasers (FEL) are revolutionizing X-ray-based structural biology methods. While protein crystallography is already routinely performed at FELs, Small Angle X-ray Scattering (SAXS) studies of biological macromolecules are not as prevalent. SAXS allows the study of the shape and overall structure of proteins and nucleic acids in solution, in a quasi-native environment.

X-ray structure and function of fibronectin domains two and three of the neural cell adhesion molecule L1.

The cell adhesion molecule L1 (L1CAM, L1 in short) plays crucial roles during neural development, regeneration after injury, synapse formation, synaptic plasticity and tumor cell migration. L1 belongs to the immunoglobulin superfamily and comprises in its extracellular part six immunoglobulin (Ig)-like domains and five fibronectin type III homologous repeats (FNs). The second Ig-like domain has been validated for self- (so-called homophilic) binding between cells.

Homogeneously N-glycosylated proteins derived from the GlycoDelete HEK293 cell line enable diffraction-quality crystallogenesis.

Structural studies of glycoproteins and their complexes provide critical insights into their roles in normal physiology and disease. Most glycoproteins contain N-linked glycosylation, a key post-translation modification that critically affects protein folding and stability and the binding kinetics underlying protein interactions. However, N-linked glycosylation is often an impediment to yielding homogeneous protein preparations for structure determination by X-ray crystallography or other methods.

Further insights from structural mass spectrometry into endocytosis adaptor protein assemblies.

As a fundament in many biologically relevant processes, endocytosis in its different guises has been arousing interest for decades and still does so. This is true for the actual transport and its initiation alike. In clathrin-mediated endocytosis, a comparatively well understood endocytic pathway, a set of adaptor proteins bind specific lipids in the plasma membrane, subsequently assemble and thus form a crucial bridge from clathrin to actin for the ongoing process.

Effect of Lactococcus lactis expressing phage endolysin on the late blowing defect of cheese caused by Clostridium tyrobutyricum.

Clostridium tyrobutyricum has been identified as a major species associated with the late blowing defect (LBD) of semi-hard and hard cheeses, due to undesirable butyric acid fermentation. To find new strategies to control this spoilage bacterium, we investigated the delivery of a bacteriophage endolysin by a cheese starter culture. The nisin producer Lactococcus lactis subsp.

Structures of peptide-free and partially loaded MHC class I molecules reveal mechanisms of peptide selection.

Major Histocompatibility Complex (MHC) class I molecules selectively bind peptides for presentation to cytotoxic T cells. The peptide-free state of these molecules is not well understood. Here, we characterize a disulfide-stabilized version of the human class I molecule HLA-A*02:01 that is stable in the absence of peptide and can readily exchange cognate peptides.

Netrin Synergizes Signaling and Adhesion through DCC.

Netrin is a prototypical axon guidance cue. Structural studies have revealed how netrin interacts with the deleted in colorectal cancer (DCC) receptor, other receptors, and co-factors for signaling. Recently, genetic studies suggested that netrin is involved in neuronal haptotaxis, which requires a reversible adhesion process.

High-throughput peptide-MHC complex generation and kinetic screenings of TCRs with peptide-receptive HLA-A*02:01 molecules.

Major histocompatibility complex (MHC) class I molecules present short peptide ligands on the cell surface for interrogation by cytotoxic CD8 T cells. MHC class I complexes presenting tumor-associated peptides such as neoantigens represent key targets of cancer immunotherapy approaches currently in development, making them important for efficacy and safety screenings. Without peptide ligand, MHC class I complexes are unstable and decay quickly, making the production of soluble monomers for analytical purposes labor intensive.

Empty peptide-receptive MHC class I molecules for efficient detection of antigen-specific T cells.

The peptide-dependent stability of MHC class I molecules poses a substantial challenge for their use in peptide-MHC multimer-based approaches to comprehensively analyze T cell immunity. To overcome this challenge, we demonstrate the use of functionally empty MHC class I molecules stabilized by a disulfide bond to link the α and α helices close to the F pocket. Peptide-loaded disulfide-stabilized HLA-A*02:01 shows complete structural overlap with wild-type HLA-A*02:01.

Roles of glycosaminoglycans as regulators of ligand/receptor complexes.

Glycosaminoglycans (GAGs) play a widespread role in embryonic development, as deletion of enzymes that contribute to GAG synthesis lead to deficiencies in cell migration and tissue modelling. Despite the biochemical and structural characterization of individual protein/GAG interactions, there is no concept available that links the molecular mechanisms of GAG/protein engagements to tissue development. Here, we focus on the role of GAG polymers in mediating interactions between cell surface receptors and their ligands.

Structural Basis for Draxin-Modulated Axon Guidance and Fasciculation by Netrin-1 through DCC.

Axon guidance involves the spatiotemporal interplay between guidance cues and membrane-bound cell-surface receptors, present on the growth cone of the axon. Netrin-1 is a prototypical guidance cue that binds to deleted in colorectal cancer (DCC), and it has been proposed that the guidance cue Draxin modulates this interaction. Here, we present structural snapshots of Draxin/DCC and Draxin/Netrin-1 complexes, revealing a triangular relationship that affects Netrin-mediated haptotaxis and fasciculation.

Multi-channel in situ dynamic light scattering instrumentation enhancing biological small-angle X-ray scattering experiments at the PETRA III beamline P12.

Small-angle X-ray scattering (SAXS) analysis of biomolecules is increasingly common with a constantly high demand for comprehensive and efficient sample quality control prior to SAXS experiments. As monodisperse sample suspensions are desirable for SAXS experiments, latest dynamic light scattering (DLS) techniques are most suited to obtain non-invasive and rapid information about the particle size distribution of molecules in solution. A multi-receiver four-channel DLS system was designed and adapted at the BioSAXS endstation of the EMBL beamline P12 at PETRA III (DESY, Hamburg, Germany).

The binding of DCC-P3 motif and FAK-FAT domain mediates the initial step of netrin-1/DCC signaling for axon attraction.

Netrin-1 plays a key role in axon guidance through binding to its receptor, Deleted in Colorectal Cancer (DCC). The initial step of signaling inside the cell after netrin-1/DCC ligation is the binding of DCC cytoplasmic P3 motif to focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK). Here we report the crystal structure of P3/FAT complex.

Epsin and Sla2 form assemblies through phospholipid interfaces.

In clathrin-mediated endocytosis, adapter proteins assemble together with clathrin through interactions with specific lipids on the plasma membrane. However, the precise mechanism of adapter protein assembly at the cell membrane is still unknown. Here, we show that the membrane-proximal domains ENTH of epsin and ANTH of Sla2 form complexes through phosphatidylinositol 4,5-bisphosphate (PIP2) lipid interfaces.

Development of a specific fluorescent phage endolysin for in situ detection of Clostridium species associated with cheese spoilage.

Late blowing defect (LBD) is a major cause of spoilage in cheeses, caused by the growth of Clostridium spp. in the cheese matrix. We investigated the application of CTP1L, a bacteriophage endolysin active against Clostridium tyrobutyricum, and its enzymatically active and cell wall-binding domains (EAD and CBD) attached to green fluorescent protein (GFP) to detect dairy-related Clostridium species by fluorescence microscopy.