Sara Košenina

Insecticidal bacterial proteins play key roles in insect-bacteria interactions and have been used as biopesticides. Here, we identify two insecticidal proteins in Paeniclostridium ghonii , designated PG-toxin 1 (PG1) and PG-toxin 2 (PG2), which are homologs of botulinum neurotoxins (BoNTs). Unlike BoNTs, PG1 and PG2 contain two separate proteins: One is the protease light chain (LC), and the other is the heavy chain containing the translocation domain and the receptor binding domain. Crystal and cryo–electron microscopy structures show a conserved BoNT-like architecture but without an interchain disulfide bond. Functional characterizations establish that the LCs of PG1 and PG2 cleave insect synaptosomal–associated protein 25 (SNAP25), but not human or rat SNAP25, and microinjection of PG1 and PG2 caused paralysis and death in Drosophila and Aedes mosquitoes. These findings identified unique two-component BoNT-like insecticidal proteins, revealing insights into the evolution of the BoNT family of toxins, and broadening our understanding of bacteria that can be used for biopest controls.

Botulinum neurotoxin serotype B1 (BoNT/B) is a highly potent neurotoxin and therapeutic agent. Here, we present the structure of the complete 14-subunit (780 kDa) progenitor toxin complex (L-PTC) and of five subcomplexes. The structures show how the toxin interacts with its associated components in their role to protect and deliver BoNT/B across epithelial barriers. Each subcomplex, including the M-PTC, M-PTC–HA70, NTNH-HA70, and HA70 trimer, provides detailed understanding of the assembly mechanism, in which the NTNH-nLoop adopts a unique fold that locks the M-PTC into a central pore formed by HA70. The HA subcomplex presents a tripod architecture with flexible legs that may adapt to the rugged cell surface. Mass photometry reveals the pH dependence of BoNT/B release from the complex which is unexpectedly influenced by the presence of HA70. This study provides the complete L-PTC structure, offering insights into its assemblage and supporting the development of countermeasures and therapeutic applications.

Fragment-based screening can catalyze drug discovery by identifying novel scaffolds, but this approach is limited by the small chemical libraries studied by biophysical experiments and the challenging optimization process. To expand the explored chemical space, we employ structure-based docking to evaluate orders-of-magnitude larger libraries than those used in traditional fragment screening. We computationally dock a set of 14 million fragments to 8-oxoguanine DNA glycosylase (OGG1), a difficult drug target involved in cancer and inflammation, and evaluate 29 highly ranked compounds experimentally. Four of these bind to OGG1 and X-ray crystallography confirms the binding modes predicted by docking. Furthermore, we show how fragment elaboration using searches among billions of readily synthesizable compounds identifies submicromolar inhibitors with anti-inflammatory and anti-cancer effects in cells. Comparisons of virtual screening strategies to explore a chemical space of 10²² compounds illustrate that fragment-based design enables enumeration of all molecules relevant for inhibitor discovery. Virtual fragment screening is hence a highly efficient strategy for navigating the rapidly growing combinatorial libraries and can serve as a powerful tool to accelerate drug discovery efforts for challenging therapeutic targets.

The botulinum neurotoxin‐like toxin from Weissella oryzae (BoNT/Wo) is one of the BoNT‐like toxins recently identified outside of the Clostridium genus. We show that, like the canonical BoNTs, BoNT/Wo forms a complex with its non‐toxic non‐hemagglutinin (NTNH) partner, which in traditional BoNT serotypes protects the toxin from proteases and the acidic environment of the hosts' guts. We here report the cryo‐EM structure of the 300 kDa BoNT/Wo‐NTNH/Wo complex together with pH stability studies of the complex. The structure reveals molecular details of the toxin's interactions with its protective partner. The overall structural arrangement is similar to other reported BoNT‐NTNH complexes, but NTNH/Wo uniquely contains two extra bacterial immunoglobulin‐like (Big) domains on the C‐terminus. Although the function of these Big domains is unknown, they are structurally most similar to bacterial proteins involved in adhesion to host cells. In addition, the BoNT/Wo protease domain contains an internal disulfide bond not seen in other BoNTs. Mass photometry analysis revealed that the BoNT/Wo‐NTNH/Wo complex is stable under acidic conditions and may dissociate at neutral to basic pH. These findings established that BoNT/Wo‐NTNH/Wo shares the general fold of canonical BoNT–NTNH complexes. The presence of unique structural features suggests that it may have an alternative mode of activation, translocation and recognition of host cells, raising interesting questions about the activity and the mechanism of action of BoNT/Wo as well as about its target environment, receptors and substrates.

Paraclostridial mosquitocidal protein 1 (PMP1) is a member of the clostridial neurotoxin (CNT) family, which includes botulinum and tetanus neurotoxins. PMP1 has unique selectivity for anopheline mosquitos and is the only known member of the family that targets insects. PMP1 is encoded in an orfX gene cluster, which in addition to the toxin, consists of OrfX1, OrfX2, OrfX3, P47 and NTNH, which have been shown to aid in PMP1 toxicity. We here show that OrfX1 and OrfX3 form a complex and present its structure at 2.7 angstrom. The OrfX1-OrfX3 complex mimics the structure of full-length OrfX2 and belongs to the lipid-binding TULIP protein superfamily. With this report, the structures of all proteins encoded in the orfX gene cluster of CNTs are now determined.