The exceptional toxicity of botulinum neurotoxins (BoNTs) is mediated by high avidity binding to complex polysialogangliosides and intraluminal segments of synaptic vesicle proteins embedded in the presynaptic membrane. One peculiarity is an exposed hydrophobic loop in the toxin’s cell binding domain HC, which is located between the ganglioside- and protein receptor-binding sites, and that is particularly pronounced in the serotypes BoNT/B, DC, and G sharing synaptotagmin as protein receptor. Here, we provide evidence that this HC loop is a critical component of their tripartite receptor recognition complex. Binding to nanodisc-embedded receptors and toxicity were virtually abolished in BoNT mutants lacking residues at the tip of the HC loop. Surface plasmon resonance experiments revealed that only insertion of the HC loop into the lipid-bilayer compensates for the entropic penalty inflicted by the dual-receptor binding. Our results represent a new paradigm of how BoNT/B, DC, and G employ ternary interactions with a protein, ganglioside, and lipids to mediate their extraordinary neurotoxicity.

Botulinum neurotoxins (BoNTs) are the most potent toxins known and cause the life threatening disease botulism. Sensitive and broad detection is extremely challenging due to the toxins’ high potency and molecular heterogeneity with several serotypes and more than 40 subtypes. The toxicity of BoNT is mediated by enzymatic cleavage of different synaptic proteins involved in neurotransmitter release at serotype-specific cleavage sites. Hence, active BoNTs can be monitored and distinguished in vitro by detecting their substrate cleavage products. In this work, we developed a comprehensive panel of monoclonal neoepitope antibodies (Neo-mAbs) highly specific for the newly generated N- and/or C-termini of the substrate cleavage products of BoNT serotypes A to F. The Neo-mAbs were implemented in a set of three enzymatic assays for the simultaneous detection of two BoNT serotypes each by monitoring substrate cleavage on colour-coded magnetic Luminex-beads. For the first time, all relevant serotypes could be detected in parallel by a routine in vitro activity assay in spiked serum and food samples yielding excellent detection limits in the range of the mouse bioassay or better (0.3–80 pg/mL). Therefore, this work represents a major step towards the replacement of the mouse bioassay for botulism diagnostics.

Although Rho GTPases are essential molecular switches involved in many cellular processes, an unbiased experimental comparison of their interaction partners was not yet performed. Here, we develop quantitative GTPase affinity purification (qGAP) to systematically identify interaction partners of six Rho GTPases (Cdc42, Rac1, RhoA, RhoB, RhoC, and RhoD), depending on their nucleotide loading state. The method works with cell line or tissuederived protein lysates in combination with SILAC-based or label-free quantification, respectively. We demonstrate that qGAP identifies known and novel binding partners that can be validated in an independent assay. Our interaction network for six Rho GTPases contains many novel binding partners, reveals highly promiscuous interaction of several effectors, and mirrors evolutionary relationships among Rho GTPases. The Ras superfamily of small guanosine triphosphatases (Ras GTPases) consists of more than 150 members in mammals and conserved orthologs in all eukaryotes (1). As molecular switches, they cycle between an active GTP-and an inactive GDP-bound state. GTPase-activating proteins (GAPs) 1 stimulate the slow intrinsic GTPase activity, which inactivates the GTPase. Conversely, guanine nucleotide exchange factors (GEFs) promote release of GDP, which is replaced by GTP, thereby transforming the GTPase into the active state. The GTP-bound form binds to downstream effector proteins to initiate their specific cellular function. In this manner, GTPases control numerous biological processes, including cytoskeletal rearrangements, membrane dynamics, and gene expression. Rho GTPases form a subfamily of the Ras superfamily (2). Of its 22 mammalian members, RhoA, Rac1, and Cdc42 have been studied most intensively and are best known for their role in regulating the actin cytoskeleton (3).Identifying effector proteins is key to understanding Rho GTPase function. More than 70 effector proteins have already been identified for each of the three prototypical family members, RhoA, Rac1, and Cdc42 (4). However, new effector proteins are still being discovered, and little is known about effectors of less well studied family members. Systematic screens for GTPase effectors employed the yeast two-hybrid approach (5) or immobilized GTPases for affinity purification (6, 7). However, these approaches are semiquantitative at best, which makes it difficult to distinguish loading-statespecific binders from constitutive interactors and nonspecific contaminants. Due to these challenges, an unbiased interactor screen for multiple Rho GTPases has not yet been reported.We sought to systematically identify proteins that interact with Rho GTPases in a loading-state-specific manner. Quantitative affinity purification combined with mass spectrometry is a powerful technology that can be used to identify proteinprotein interactions (PPIs) in an unbiased way (8 -11). Here, we develop quantitative GTPase affinity purification (qGAP) as a novel variant of quantitative affinity purification combined with mass spectro...

The detection of catalytically active botulinum neurotoxins (BoNTs) can be achieved by monitoring the enzymatic cleavage of soluble NSF (N-ethylmaleimide-sensitive-factor) attachment protein receptor (SNARE) proteins by the toxins’ light chains (LC) in cleavage-based assays. Thus, for sensitive BoNT detection, optimal cleavage conditions for the clinically relevant A–F serotypes are required. Until now, a systematic evaluation of cleavage conditions for the different BoNT serotypes is still lacking. To address this issue, we optimized cleavage conditions for BoNT/A–F using the Taguchi design-of-experiments (DoE) method. To this aim, we analyzed the influence of buffer composition (pH, Zn2+, DTT (dithiothreitol), NaCl) as well as frequently used additives (BSA (bovine serum albumin), Tween 20, trimethylamine N-oxide (TMAO)) on BoNT substrate cleavage. We identified major critical factors (DTT, Zn2+, TMAO) and were able to increase the catalytic efficiency of BoNT/B, C, E, and F when compared to previously described buffers. Moreover, we designed a single consensus buffer for the optimal cleavage of all tested serotypes. Our optimized buffers are instrumental to increase the sensitivity of cleavage-based assays for BoNT detection. Furthermore, the application of the Taguchi DoE approach shows how the method helps to rationally improve enzymatic assays.

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