Botulinum neurotoxins (BoNTs) are highly poisonous substances that are also effective medicines. Accidental BoNT poisoning often occurs through ingestion of Clostridium botulinum-contaminated food. Here, we present the crystal structure of a BoNT in complex with a clostridial non-toxic non-hemagglutinin (NTNHA) protein at 2.7 angstrom. Biochemical and functional studies show that NTNHA provides large and multivalent binding interfaces to protect BoNT from gastrointestinal degradation. Moreover, the structure highlights key residues in BoNT that regulate complex assembly in a pH-dependent manner. Collectively, our findings define the molecular mechanisms by which NTNHA shields BoNT in the hostile gastrointestinal environment and releases it upon entry into the circulation. These results will assist in the design of small molecules for inhibiting oral BoNT intoxication, and of delivery vehicles for oral administration of biologics.

Fast excitatory neurotransmission is mediated largely by ionotropic glutamate receptors (iGluRs), tetrameric, ligand-gated ion channel proteins comprised of three subfamilies, AMPA, kainate and NMDA receptors, with each subfamily sharing a common, modular-domain architecture. For all receptor subfamilies, active channels are exclusively formed by assemblages of subunits within the same subfamily, a molecular process principally encoded by the amino-terminal domain (ATD). However, the molecular basis by which the ATD guides subfamily-specific receptor assembly is not known. Here we show that AMPA receptor GluR1-and GluR2-ATDs form tightly associated dimers and, by the analysis of crystal structures of the GluR2-ATD, propose mechanisms by which the ATD guides subfamily-specific receptor assembly.

Nicotine exerts its behavioral and additive actions through a family of brain nicotinic acetylcholine receptors (nAChRs). Enhancing α7-type nAChR signaling improves symptoms in Alzheimer's disease and schizophrenia. The pharmaceutical study of α7 receptors is hampered because these receptors do not form their functional pentameric structure in cell lines, and mechanisms that underlie α7 receptor assembly in neurons are not understood. Here, a genomic screening strategy solves this long-standing puzzle and identifies NACHO, a transmembrane protein of neuronal endoplasmic reticulum that mediates assembly of α7 receptors. NACHO promotes α7 protein folding, maturation through the Golgi complex, and expression at the cell surface. Knockdown of NACHO in cultured hippocampal neurons or knockout of NACHO in mice selectively and completely disrupts α7 receptor assembly and abolishes α7 channel function. This work identifies NACHO as an essential, client-specific chaperone for nAChRs and has implications for physiology and disease associated with these widely distributed neurotransmitter receptors.

Synapses are the fundamental units of neural circuits that enable complex behaviors. The neuromuscular junction (NMJ), a synapse formed between a motoneuron and a muscle fiber, has contributed greatly to understanding of the general principles of synaptogenesis as well as of neuromuscular disorders. NMJ formation requires neural agrin, a motoneuron-derived protein, which interacts with LRP4 (low-density lipoprotein receptor-related protein 4) to activate the receptor tyrosine kinase MuSK (muscle-specific kinase). However, little is known of how signals are transduced from agrin to MuSK. Here, we present the first crystal structure of an agrin-LRP4 complex, consisting of two agrin-LRP4 heterodimers. Formation of the initial binary complex requires the z8 loop that is specifically present in neuronal, but not muscle, agrin and that promotes the synergistic formation of the tetramer through two additional interfaces. We show that the tetrameric complex is essential for neuronal agrin-induced acetylcholine receptor (AChR) clustering. Collectively, these results provide new insight into the agrin-LRP4-MuSK signaling cascade and NMJ formation and represent a novel mechanism for activation of receptor tyrosine kinases.

Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the fatal disease botulism, a flaccid paralysis of the muscle. BoNTs are released together with several auxiliary proteins as progenitor toxin complexes (PTCs) to become highly potent oral poisons. Here, we report the structure of a ∼760 kDa 14-subunit large PTC of serotype A (L-PTC/A) and reveal insight into its absorption mechanism. Using a combination of X-ray crystallography, electron microscopy, and functional studies, we found that L-PTC/A consists of two structurally and functionally independent sub-complexes. A hetero-dimeric 290 kDa complex protects BoNT, while a hetero-dodecameric 470 kDa complex facilitates its absorption in the harsh environment of the gastrointestinal tract. BoNT absorption is mediated by nine glycan-binding sites on the dodecameric sub-complex that forms multivalent interactions with carbohydrate receptors on intestinal epithelial cells. We identified monosaccharides that blocked oral BoNT intoxication in mice, which suggests a new strategy for the development of preventive countermeasures for BoNTs based on carbohydrate receptor mimicry.

How botulinum neurotoxins (BoNTs) cross the host intestinal epithelial barrier in foodborne botulism is poorly understood. Here, we present the crystal structure of a clostridial hemagglutinin (HA) complex of serotype BoNT/A bound to the cell adhesion protein E-cadherin at 2.4 Ångströms. The HA complex recognizes E-cadherin with high specificity involving extensive intermolecular interactions and also binds to carbohydrates on the cell surface. Binding of HA complex sequesters E-cadherin in the monomeric state thereby compromising the E-cadherin-mediated intercellular barrier and facilitating paracellular absorption of BoNT/A. We reconstituted the complete 14-subunit BoNT/A complex using recombinantly-produced components and demonstrated that abolishing either E-cadherin- or carbohydrate-binding of HA complex drastically reduces oral toxicity of BoNT/A complex in vivo. Together, these studies establish the molecular mechanism of how HAs contribute to the oral toxicity of BoNT/A.

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Botulinum neurotoxins (BoNTs) are among the most poisonous substances known to man, but paradoxically, BoNT-containing medicines and cosmetics have been used with great success in the clinic. Accidental BoNT poisoning mainly occurs through oral ingestion of food contaminated with Clostridium botulinum. BoNTs are naturally produced in the form of progenitor toxin complexes, which are high molecular weight (up to ~900 kDa) multi-protein complexes composed of BoNT and several non-toxic neurotoxin-associated proteins (NAPs). NAPs protect the inherently fragile BoNTs against the hostile environment of the gastrointestinal tract and help BoNTs pass through the intestinal epithelial barrier before they are released into the general circulation. These events are essential for ingested BoNTs to gain access to motoneurons, where they inhibit neurotransmitter release and cause muscle paralysis. In this review, we discuss the structural basis for assembly of NAPs and BoNT into the progenitor toxin complexes that protect BoNT and facilitate its delivery into the bloodstream.