No abstract

In drug discovery, efficient screening of protein-drug interactions (PDIs) is hampered by the limitations of current biophysical approaches. Here, we develop a biological nanopore sensor for single-molecule detection of proteins and PDIs using the pore-forming toxin YaxAB. Using this YaxAB nanopore, we demonstrate label-free, single-molecule detection of interactions between the anticancer Bcl-xL protein and small-molecule drugs as well as the Bak-BH3 peptide. The long funnel-shaped structure and nanofluidic characteristics of the YaxAB nanopore enable the electro-osmotic trapping of diverse folded proteins and high-resolution monitoring of PDIs. Distinctive nanopore event distributions observed in the two-dimensional (ΔI/Io-versus-IN) plot illustrate the ability of the YaxAB nanopore to discriminate individual small-molecule drugs bound to Bcl-xL from non-binders. Taken together, our results present the YaxAB nanopore as a robust platform for label-free, ultrasensitive, single-molecule detection of PDIs, opening up a possibility for low-cost, highly efficient drug discovery against diverse drug targets.

Angiopoietin (Angpt)-Tie receptor 2 (Tie2) plays key roles in vascular development and homeostasis as well as pathological vascular remodeling. Therefore, Tie2-agonistic antibody and engineered Angpt1 variants have been developed as potential therapeutics for ischemic and inflammatory vascular diseases. However, their underlying mechanisms for Tie2 clustering and activation remain elusive and the poor manufacturability and stability of Angpt1 variants limit their clinical application. Here, we develop a human Tie2-agonistic antibody (hTAAB), which targets the membrane proximal fibronectin type III domain of Tie2 distinct from the Angpt-binding site. Our Tie2/hTAAB complex structures reveal that hTAAB tethers the preformed Tie2 homodimers into polygonal assemblies through specific binding to Tie2 Fn3 domain. Notably, the polygonal Tie2 clustering induced by hTAAB is critical for Tie2 activation and are resistant to antagonism by Angpt2. Our results provide insight into the molecular mechanism of Tie2 clustering and activation mediated by hTAAB, and the structure-based humanization of hTAAB creates a potential clinical application.

No abstract

Cross-reactive material 197 (CRM 197 ) is a non-toxic mutant of diphtheria toxin containing a single amino acid substitution of glycine 52 with glutamic acid. CRM 197 has been used as a carrier protein for poorly immunogenic polysaccharide antigens to improve immune responses. In this study, to develop a sandwich ELISA that can detect CRM 197 and CRM 197 conjugate vaccines, we generated a human anti-CRM 197 monoclonal antibody (mAb) 3F9 using a phage-displayed human synthetic Fab library and produced mouse anti-CRM 197 polyclonal antibody. The affinity (K D ) of 3F9 for CRM 197 was 3.55 nM, based on Bio-Layer interferometry, and it bound specifically to the B fragment of CRM 197 . The sandwich ELISA was carried out using 3F9 as a capture antibody and the mouse polyclonal antibody as a detection antibody. The detection limit of the sandwich ELISA was <1 ng/ml CRM 197 . In addition, the 3F9 antibody bound to the CRM 197 -polysaccharide conjugates tested in a dose-dependent manner. This ELISA system will be useful for the quantification and characterization of CRM 197 and CRM 197 conjugate vaccines. To our knowledge, this study is the first to generate a human monoclonal antibody against CRM 197 and to develop a sandwich ELISA for CRM 197 conjugate vaccines.

Current approaches to design monodisperse protein assemblies require rigid, tight, and symmetric interactions between oligomeric protein units.H erein, we introduce an ew multivalent-interaction-driven assembly strategy that allows flexible,s paced, and asymmetric assembly between protein oligomers.Wediscoveredthat two polygonal protein oligomers (ranging from triangle to hexagon) dominantly form adiscrete and stable two-layered protein prism nanostructure via multivalent interactions between fused binding pairs.W ed emonstrated that protein nano-prisms with long flexible peptide linkers (over 80 amino acids) between protein oligomer layers could be discretely formed. Oligomers with different structures could also be monodispersely assembled into two-layered but asymmetric protein nano-prisms.F urthermore,p roducing higher-order architectures with multiple oligomer layers,f or example,3-layeredn ano-prisms or nanotubes,w as also feasible.

Vibrio cholerae , cause of the life-threatening diarrheal disease cholera, can be divided into different serogroups based on the structure of its lipopolysaccharide (LPS), which consists of lipid-A, corepolysaccharide and O-antigen polysaccharide (O-PS). The O1 serogroup, the predominant cause of cholera, includes two major serotypes, Inaba and Ogawa. These serotypes are differentiated by the presence of a single 2- O -methyl group in the upstream terminal perosamine of the Ogawa O-PS, which is absent in the Inaba O-PS. To ensure the consistent quality and efficacy of the current cholera vaccines, accurate measurement and characterization of each of these two serotypes is highly important. In this study, we efficiently screened a phage-displayed human synthetic Fab library by bio-panning against Ogawa LPS and finally selected three unique mAbs (D9, E11, and F7) that specifically react with Ogawa LPS. The mAbs bound to Vibrio cholerae vaccine in a dose-dependent fashion. Sequence and structure analyses of antibody paratopes suggest that IgG D9 might have the same fine specificity as that of the murine mAbs, which were shown to bind to the upstream terminal perosamine of Ogawa O-PS, whereas IgGs F7 and E11 showed some different characteristics in the paratopes. To our knowledge, this study is the first to demonstrate the generation of Ogawa-specific mAbs using phage display technology. The mAbs will be useful for identification and quantification of Ogawa LPS in multivalent V. cholerae vaccines.

Current approaches to design monodisperse protein assemblies require rigid, tight, and symmetric interactions between oligomeric protein units.H erein, we introduce an ew multivalent-interaction-driven assembly strategy that allows flexible,s paced, and asymmetric assembly between protein oligomers.Wediscoveredthat two polygonal protein oligomers (ranging from triangle to hexagon) dominantly form adiscrete and stable two-layered protein prism nanostructure via multivalent interactions between fused binding pairs.W ed emonstrated that protein nano-prisms with long flexible peptide linkers (over 80 amino acids) between protein oligomer layers could be discretely formed. Oligomers with different structures could also be monodispersely assembled into two-layered but asymmetric protein nano-prisms.F urthermore,p roducing higher-order architectures with multiple oligomer layers,f or example,3-layeredn ano-prisms or nanotubes,w as also feasible.