Shin-Ichiro Miyashita

Massively parallel de novo protein design for targeted therapeutics

De novo protein design holds promise for creating small stable proteins with shapes customized to bind therapeutic targets. We describe a massively parallel approach for designing, manufacturing and screening mini-protein binders, integrating large-scale computational design, oligonucleotide synthesis, yeast display screening and next-generation sequencing. We designed and tested 22,660 mini-proteins of 37–43 residues that target influenza haemagglutinin and botulinum neurotoxin B, along with 6,286 control sequences to probe contributions to folding and binding, and identified 2,618 high-affinity binders. Comparison of the binding and non-binding design sets, which are two orders of magnitude larger than any previously investigated, enabled the evaluation and improvement of the computational model. Biophysical characterization of a subset of the binder designs showed that they are extremely stable and, unlike antibodies, do not lose activity after exposure to high temperatures. The designs elicit little or no immune response and provide potent prophylactic and therapeutic protection against influenza, even after extensive repeated dosing.

Identification and characterization of a novel botulinum neurotoxin

Botulinum neurotoxins are known to have seven serotypes (BoNT/A–G). Here we report a new BoNT serotype, tentatively named BoNT/X, which has the lowest sequence identity with other BoNTs and is not recognized by antisera against known BoNTs. Similar to BoNT/B/D/F/G, BoNT/X cleaves vesicle-associated membrane proteins (VAMP) 1, 2 and 3, but at a novel site (Arg66-Ala67 in VAMP2). Remarkably, BoNT/X is the only toxin that also cleaves non-canonical substrates VAMP4, VAMP5 and Ykt6. To validate its activity, a small amount of full-length BoNT/X was assembled by linking two non-toxic fragments using a transpeptidase (sortase). Assembled BoNT/X cleaves VAMP2 and VAMP4 in cultured neurons and causes flaccid paralysis in mice. Thus, BoNT/X is a novel BoNT with a unique substrate profile. Its discovery posts a challenge to develop effective countermeasures, provides a novel tool for studying intracellular membrane trafficking, and presents a new potential therapeutic toxin for modulating secretions in cells.

Small-angle X-ray scattering reveals structural dynamics of the botulinum neurotoxin associating protein, nontoxic nonhemagglutinin

In cell culture supernatants, the botulinum neurotoxin (BoNT) exists as part of a toxin complex (TC) in which nontoxic nonhemagglutinin (NTNHA) and/or hemagglutinins (HAs) are assembled onto the BoNT. A series of investigations indicated that formation of the TC is vital for delivery of the toxin to nerve cells through the digestive tract. In the assembly process, BoNT binds to NTNHA yielding M-TC, and it then matures into L-TC by further association with the HAs via NTNHA in the M-TC. Here, we report a crystal structure of the NTNHA from Clostridium botulinum serotype D strain 4947. Additionally, we performed small-angle X-ray scattering (SAXS) analysis of the NTNHA and the M-TC to elucidate the solution structure. The crystal structure of D-4947 NTNHA revealed that BoNT and NTNHA share a closely related structure consisting of three domains. The SAXS image indicated that, even though the N-terminal two-thirds of the NTNHA molecule had an apparently similar conformation in both the crystal and solution structures, the C-terminal third of the molecule showed a more extended structure in the SAXS image than that seen in the crystallographic image. The discrepancy between the crystal and solution structures implies a high flexibility of the C-terminal third domain of NTNHA, which is involved in binding to BoNT. Structural dynamics of the NTNHA molecule revealed by SAXS may explain its binding to BoNT to form the BoNT/NTNHA complex.

Toxic and nontoxic components of botulinum neurotoxin complex are evolved from a common ancestral zinc protein

Zinc atoms play an essential role in a number of enzymes. Botulinum neurotoxin (BoNT), the most potent toxin known in nature, is a zinc-dependent endopeptidase. Here we identify the nontoxic nonhemagglutinin (NTNHA), one of the BoNT-complex constituents, as a zinc-binding protein, along with BoNT. A protein structure classification database search indicated that BoNT and NTNHA share a similar domain architecture, comprising a zinc-dependent metalloproteinase-like, BoNT coiled-coil motif and concanavalin A-like domains. Inductively coupled plasma-mass spectrometry analysis demonstrated that every single NTNHA molecule contains a single zinc atom. This is the first demonstration of a zinc atom in this protein, as far as we know. However, the NTNHA molecule does not possess any known zinc-coordinating motif, whereas all BoNT serotypes possess the classical HEXXH motif. Homology modeling of the NTNHA structure implied that a consensus K-C-L-I-K-X(35)-D sequence common among all NTNHA serotype molecules appears to coordinate a single zinc atom. These findings lead us to propose that NTNHA and BoNT may have evolved distinct functional specializations following their branching out from a common ancestral zinc protein.

Sugar-induced conformational change found in the HA-33/HA-17 trimer of the botulinum toxin complex

Large-sized botulinum toxin complex (L-TC) is formed by conjugation of neurotoxin, nontoxic nonhemagglutinin and hemagglutinin (HA) complex. The HA complex is formed by association of three HA-70 molecules and three HA-33/HA-17 trimers, comprised of a single HA-17 and two HA-33 proteins. The HA-33/HA-17 trimer isolated from serotype D L-TC has the ability to bind to and penetrate through the intestinal epithelial cell monolayer in a sialic acid-dependent manner, and thus it plays an important role in toxin delivery through the intestinal cell wall. In this study, we determined the solution structure of the HA-33/HA-17 trimer by using small-angle X-ray scattering (SAXS). The SAXS image of HA-33/HA-17 exhibited broadly similar appearance to the crystal image of the complex. On the other hand, in the presence of N-acetylneuraminic acid, glucose and galactose, the solution structure of the HA-33/HA-17 trimer was drastically altered compared to the structure in the absence of the sugars. Sugar-induced structural change of the HA-33/HA-17 trimer may contribute to cell binding and subsequent transport across the intestinal cell layer.

Transport of the botulinum neurotoxin–associating protein, nontoxic nonhemagglutinin, across the rat small intestinal epithelial cell monolayer

Botulinum neurotoxin (BoNT) associates with nontoxic nonhemagglutinin (NTNHA) yielding a complex in culture. BoNT and NTNHA have similar domain organizations, implying that they share common functions, although this remains unclear. Here, we examined cell monolayer transport of serotype D NTNHA in the rat intestinal epithelial cell line IEC-6. NTNHA and BoNT both bound to the cell and were transported across the cell layer. NTNHA contains a QXW motif and a β-trefoil fold, both common in sugar chain-recognizing proteins, whereas the QXW motif is absent in all BoNT serotypes. This could explain the distinct sugar chain-recognizing properties of NTNHA and BoNT.

Hemagglutinin gene shuffling among Clostridium botulinum serotypes C and D yields distinct sugar recognition of the botulinum toxin complex

Clostridium botulinum strains produce a large-sized toxin complex (TC) that is composed of botulinum neurotoxin (BoNT), non-toxic non-hemagglutinin and three different hemagglutinins (HA-70, HA-33 and HA-17). HA components enhance toxin delivery across the intestinal cell wall in a sugar chain-dependent manner. Here we characterized the sugar recognition of serotype D strain 1873 (D-1873) botulinum L-TC. Most L-TCs produced by serotype C and D strains bind to cells via interactions between HA-33 and cell surface sialo-oligosaccharides. However, like the previously reported L-TC produced by serotype C strain Yoichi (C-Yoichi), D-1873 L-TC binds only to cells that have been treated with neuraminidase, indicating that they recognize asialo-oligosaccharides. The D-1873 HA-33 amino acid sequence is similar to that of C-Yoichi, but had lower similarity to the majority of serotype C and D HA-33s. A comparison of TC component primary structures for 12 serotype C and D strains suggested that at least three types of HA-33 genes exist, and these are shuffled among the serotype C and D strains independently of BoNT serotype. This shuffling produces the distinct sugar recognition of serotype C and D botulinum TCs.

Host-cell specificity and transcytosis of nontoxic nonhemagglutinin protein of botulinum neurotoxin serotype D.

Serotype D botulinum toxin (BoNT) complex (TC), a causative agent of foodborne botulism in animals, traverses the gastrointestinal tract and circulation, eventually becoming localized in neuromuscular junctions, where the serotype D BoNT cleaves SNARE substrate synaptobrevin II involved in neurotransmitter release. During this process, BoNT must pass through cells, thus from the intestinal lumen to the cells of the intestinal tract and blood vessels. The botulinum TC is formed by association of the BoNT with at least one nontoxic protein, which may be a nontoxic nonhemagglutinin (NTNHA). In this work, we examined the binding and transcytosis of serotype D NTNHA protein in epithelial and endothelial cells to clarify the role played by the protein in toxin delivery. Our studies showed that NTNHA bound to and transcytosed across rat intestinal epithelial (IEC-6) and bovine aortic endothelial (BAEC) cells. While NTNHA also bound to canine renal (MDCK) or human colon carcinoma (Caco-2) cells, but it did not traverse across MDCK or Caco-2 cells. Such specificity of NTNHA protein transcytosis may explain why only some animals are sensitive to botulinum toxin. The sensitivity depends on the toxin serotype in play, and the route of toxin delivery.

Crystallization and preliminary X-ray analysis of the Clostridium botulinum type D nontoxic nonhaemagglutinin.

Clostridium botulinum produces botulinum neurotoxin (BoNT) as a large toxin complex assembled with nontoxic nonhaemagglutinin (NTNHA) and/or haemagglutinin components. Complex formation with NTNHA is considered to be critical in eliciting food poisoning because the complex shields the BoNT from the harsh conditions in the digestive tract. In the present study, NTNHA was expressed in Escherichia coli and crystallized. Diffraction data were collected to 3.9 Å resolution. The crystal belonged to the trigonal space group P321 or P3(1)21/P3(2)21, with unit-cell parameters a = b = 147.85, c = 229.74 Å. The structure of NTNHA will provide insight into the assembly mechanism that produces the unique BoNT-NTNHA complex.

“Non-Toxic” Proteins of the Botulinum Toxin Complex Exert In-vivo Toxicity

The botulinum neurotoxin (BoNT) causes muscle paralysis and is the most potent toxin in nature. BoNT is associated with a complex of auxiliary “Non-Toxic” proteins, which constitute a large-sized toxin complex (L-TC). However, here we report that the “Non-Toxic” complex of serotype D botulinum L-TC, when administered to rats, exerts in-vivo toxicity on small-intestinal villi. Moreover, Serotype C and D of the “Non-Toxic” complex, but not BoNT, induced vacuole-formation in a rat intestinal epithelial cell line (IEC-6), resulting in cell death. Our results suggest that the vacuole was formed in a manner distinct from the mechanism by which Helicobacter pylori vacuolating toxin (VacA) and Vibrio cholerae haemolysin induce vacuolation. We therefore hypothesise that the serotype C and D botulinum toxin complex is a functional hybrid of the neurotoxin and vacuolating toxin (VT) which arose from horizontal gene transfer from an ancestral BoNT-producing bacterium to a hypothetical VT-producing bacterium.