PURA syndrome-causing mutations impair PUR-domain integrity and affect P-body association.

Mutations in the human PURA gene cause the neurodevelopmental PURA syndrome. In contrast to several other monogenetic disorders, almost all reported mutations in this nucleic acid-binding protein result in the full disease penetrance. In this study, we observed that patient mutations across PURA impair its previously reported co-localization with processing bodies. These mutations either destroyed the folding integrity, RNA binding, or dimerization of PURA. We also solved the crystal structures of the N- and C-terminal PUR domains of human PURA and combined them with molecular dynamics simulations and nuclear magnetic resonance measurements. The observed unusually high dynamics and structural promiscuity of PURA indicated that this protein is particularly susceptible to mutations impairing its structural integrity. It offers an explanation why even conservative mutations across PURA result in the full penetrance of symptoms in patients with PURA syndrome.

PURA syndrome is a neurodevelopmental disorder that affects about 650 patients worldwide, resulting in a range of symptoms including neurodevelopmental delays, intellectual disability, muscle weakness, seizures, and eating difficulties. The condition is caused by a mutated gene that codes for a protein called PURA. PURA binds RNA ­ the molecule that carries genetic information so it can be translated into proteins ­ and has roles in regulating the production of new proteins. Contrary to other conditions that result from mutations in a single gene, PURA syndrome patients show 'high penetrance', meaning almost every reported mutation in the gene leads to symptoms. Proske, Janowski et al. wanted to understand the molecular basis for this high penetrance. To find out more, the researchers first examined how patient mutations affected the location of the PURA in the cell, using human cells grown in the laboratory. Normally, PURA travels to P-bodies, which are groupings of RNA and proteins involved in regulating which genes get translated into proteins. The researchers found that in cells carrying PURA syndrome mutations, PURA failed to move adequately to P-bodies. To find out how this 'mislocalization' might happen, Proske, Janowski et al. tested how different mutations affected the three-dimensional folding of PURA. These analyses showed that the mutations impair the protein's folding and thereby disrupt PURA's ability to bind RNA, which may explain why mutant PURA cannot localize correctly. Proske, Janowski et al. describe the molecular abnormalities of PURA underlying this disorder and show how molecular analysis of patient mutations can reveal the mechanisms of a disease at the cell level. The results show that the impact of mutations on the structural integrity of the protein, which affects its ability to bind RNA, are likely key to the symptoms of the syndrome. Additionally, their approach used establishes a way to predict and test mutations that will cause PURA syndrome. This may help to develop diagnostic tools for this condition.

Crystal structure of the RNA-recognition motif of Drosophila melanogaster tRNA (uracil-5-)-methyltransferase homolog A.

Human tRNA (uracil-5-)-methyltransferase 2 homolog A (TRMT2A) is the dedicated enzyme for the methylation of uridine 54 in transfer RNA (tRNA). Human TRMT2A has also been described as a modifier of polyglutamine (polyQ)-derived neuronal toxicity. The corresponding human polyQ pathologies include Huntington's disease and constitute a family of devastating neurodegenerative diseases. A polyQ tract in the corresponding disease-linked protein causes neuronal death and symptoms such as impaired motor function, as well as cognitive impairment. In polyQ disease models, silencing of TRMT2A reduced polyQ-associated cell death and polyQ protein aggregation, suggesting this protein as a valid drug target against this class of disorders. In this paper, the 1.6 Šresolution crystal structure of the RNA-recognition motif (RRM) from Drosophila melanogaster, which is a homolog of human TRMT2A, is described and analysed.

Human TRMT2A methylates tRNA and contributes to translation fidelity.

5-Methyluridine (m5U) is one of the most abundant RNA modifications found in cytosolic tRNA. tRNA methyltransferase 2 homolog A (hTRMT2A) is the dedicated mammalian enzyme for m5U formation at tRNA position 54. However, its RNA binding specificity and functional role in the cell are not well understood. Here we dissected structural and sequence requirements for binding and methylation of its RNA targets. Specificity of tRNA modification by hTRMT2A is achieved by a combination of modest binding preference and presence of a uridine in position 54 of tRNAs. Mutational analysis together with cross-linking experiments identified a large hTRMT2A-tRNA binding surface. Furthermore, complementing hTRMT2A interactome studies revealed that hTRMT2A interacts with proteins involved in RNA biogenesis. Finally, we addressed the question of the importance of hTRMT2A function by showing that its knockdown reduces translation fidelity. These findings extend the role of hTRMT2A beyond tRNA modification towards a role in translation.

A novel monoclonal IgG1 antibody specific for Galactose-alpha-1,3-galactose questions alpha-Gal epitope expression by bacteria.

The alpha-Gal epitope (α-Gal) with the determining element galactose-α1,3-galactose can lead to clinically relevant allergic reactions and rejections in xenotransplantation. These immune reactions can develop because humans are devoid of this carbohydrate due to evolutionary loss of the enzyme α1,3-galactosyltransferase (GGTA1). In addition, up to 1% of human IgG antibodies are directed against α-Gal, but the stimulus for the induction of anti-α-Gal antibodies is still unclear. Commensal bacteria have been suggested as a causal factor for this induction as α-Gal binding tools such as lectins were found to stain cultivated bacteria isolated from the intestinal tract. Currently available tools for the detection of the definite α-Gal epitope, however, are cross-reactive, or have limited affinity and, hence, offer restricted possibilities for application. In this study, we describe a novel monoclonal IgG1 antibody (27H8) specific for the α-Gal epitope. The 27H8 antibody was generated by immunization of Ggta1 knockout mice and displays a high affinity towards synthetic and naturally occurring α-Gal in various applications. Using this novel tool, we found that intestinal bacteria reported to be α-Gal positive cannot be stained with 27H8 questioning whether commensal bacteria express the native α-Gal epitope at all.

Presence and function of Hbl B', the fourth protein component encoded by the hbl operon in Bacillus cereus.

The genes hblC, hblD and hblA encode the components Hbl L2, L1 and B of the pore forming enterotoxin haemolysin BL of Bacillus cereus. Two variants of the operon existand the more common one additionally contains hblB downstream of hblCDA. Up to now, it was completely unclear whether the corresponding protein, Hbl B', is widely expressed among B. cereus strains and if it has a distinct function. In the present study, it was shown that the hblB gene is indeed expressed and the Hbl B' protein is secreted by nearly all analysed B. cereus strains. For the latter, a detection system was developed based on monoclonal antibody 11A5. Further, a distinct reduction of cytotoxic and haemolytic activity was observed when recombinant (r)Hbl B' was applied simultaneously with L2, L1 and B. This effect was due to direct interaction of rHbl B' with L1. D-6B. cereusAltogether, we present the first simple tool for the detection of Hbl B' in B. cereus culture supernatants. Moreover, an important regulatory function of Hbl B' in the mechanism of Hbl was determined, which is best described as an additional control of complex formation, balancing the amounts of Hbl B-L1 complexes and the corresponding free subunits.

Small-molecule modulators of TRMT2A decrease PolyQ aggregation and PolyQ-induced cell death.

Polyglutamine (polyQ) diseases are characterized by an expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats encoding for an uninterrupted prolonged polyQ tract. We previously identified TRMT2A as a strong modifier of polyQ-induced toxicity in an unbiased large-scale screen in Drosophila melanogaster. This work aimed at identifying and validating pharmacological TRMT2A inhibitors as treatment opportunities for polyQ diseases in humans. Computer-aided drug discovery was implemented to identify human TRMT2A inhibitors. Additionally, the crystal structure of one protein domain, the RNA recognition motif (RRM), was determined, and Biacore experiments with the RRM were performed. The identified molecules were validated for their potency to reduce polyQ aggregation and polyQ-induced cell death in human HEK293T cells and patient derived fibroblasts. Our work provides a first step towards pharmacological inhibition of this enzyme and indicates TRMT2A as a viable drug target for polyQ diseases.

Genetically encoded photo-switchable molecular sensors for optoacoustic and super-resolution imaging.

Reversibly photo-switchable proteins are essential for many super-resolution fluorescence microscopic and optoacoustic imaging methods. However, they have yet to be used as sensors that measure the distribution of specific analytes at the nanoscale or in the tissues of live animals. Here we constructed the prototype of a photo-switchable Ca2+ sensor based on GCaMP5G that can be switched with 405/488-nm light and describe its molecular mechanisms at the structural level, including the importance of the interaction of the core barrel structure of the fluorescent protein with the Ca2+ receptor moiety. We demonstrate super-resolution imaging of Ca2+ concentration in cultured cells and optoacoustic Ca2+ imaging in implanted tumor cells in mice under controlled Ca2+ conditions. Finally, we show the generalizability of the concept by constructing examples of photo-switching maltose and dopamine sensors based on periplasmatic binding protein and G-protein-coupled receptor-based sensors.

UGT76B1, a promiscuous hub of small molecule-based immune signaling, glucosylates N-hydroxypipecolic acid, and balances plant immunity.

Glucosylation modulates the biological activity of small molecules and frequently leads to their inactivation. The Arabidopsis thaliana glucosyltransferase UGT76B1 is involved in conjugating the stress hormone salicylic acid (SA) as well as isoleucic acid (ILA). Here, we show that UGT76B1 also glucosylates N-hydroxypipecolic acid (NHP), which is synthesized by FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1) and activates systemic acquired resistance (SAR). Upon pathogen attack, Arabidopsis leaves generate two distinct NHP hexose conjugates, NHP-O-ß-glucoside and NHP glucose ester, whereupon only NHP-O-ß-glucoside formation requires a functional SA pathway. The ugt76b1 mutants specifically fail to generate the NHP-O-ß-glucoside, and recombinant UGT76B1 synthesizes NHP-O-ß-glucoside in vitro in competition with SA and ILA. The loss of UGT76B1 elevates the endogenous levels of NHP, SA, and ILA and establishes a constitutive SAR-like immune status. Introgression of the fmo1 mutant lacking NHP biosynthesis into the ugt76b1 background abolishes this SAR-like resistance. Moreover, overexpression of UGT76B1 in Arabidopsis shifts the NHP and SA pools toward O-ß-glucoside formation and abrogates pathogen-induced SAR. Our results further indicate that NHP-triggered immunity is SA-dependent and relies on UGT76B1 as a common metabolic hub. Thereby, UGT76B1-mediated glucosylation controls the levels of active NHP, SA, and ILA in concert to balance the plant immune status.

Challenging a Preconception: Optoacoustic Spectrum Differs from the Optical Absorption Spectrum of Proteins and Dyes for Molecular Imaging.

Optoacoustic (photoacoustic) imaging has seen marked advances in detection and data analysis, but there is less progress in understanding the photophysics of common optoacoustic contrast agents. This gap blocks the development of novel agents and the accurate analysis and interpretation of multispectral optoacoustic images. To close it, we developed a multimodal laser spectrometer (MLS) to enable the simultaneous measurement of optoacoustic, absorbance, and fluorescence spectra. Herein, we employ MLS to analyze contrast agents (methylene blue, rhodamine 800, Alexa Fluor 750, IRDye 800CW, and indocyanine green) and proteins (sfGFP, mCherry, mKate, HcRed, iRFP720, and smURFP). We found that the optical absorption spectrum does not correlate with the optoacoustic spectrum for the majority of the analytes. We determined that for dyes, the transition underlying an aggregation state has more optoacoustic signal generation efficiency than the monomer transition. For proteins we found a favored optoacoustic relaxation that stems from the neutral or zwitterionic chromophores and unreported photoswitching behavior of tdTomato and HcRed. We then crystalized HcRed in its photoswitch optoacoustic state, confirming structurally the change in isomerization with respect to HcReds' fluorescence state. Finally, on the example of the widely used label tdTomato and the dye indocyanine green, we show the importance of correct photophysical (e.g., spectral and kinetic) information as a prerequisite for spectral-unmixing for in vivo imaging.

Structural basis for the recognition of transiently structured AU-rich elements by Roquin.

Adenylate/uridylate-rich elements (AREs) are the most common cis-regulatory elements in the 3'-untranslated region (UTR) of mRNAs, where they fine-tune turnover by mediating mRNA decay. They increase plasticity and efficacy of mRNA regulation and are recognized by several ARE-specific RNA-binding proteins (RBPs). Typically, AREs are short linear motifs with a high content of complementary A and U nucleotides and often occur in multiple copies. Although thermodynamically rather unstable, the high AU-content might enable transient secondary structure formation and modify mRNA regulation by RBPs. We have recently suggested that the immunoregulatory RBP Roquin recognizes folded AREs as constitutive decay elements (CDEs), resulting in shape-specific ARE-mediated mRNA degradation. However, the structural evidence for a CDE-like recognition of AREs by Roquin is still lacking. We here present structures of CDE-like folded AREs, both in their free and protein-bound form. Moreover, the AREs in the UCP3 3'-UTR are additionally bound by the canonical ARE-binding protein AUF1 in their linear form, adopting an alternative binding-interface compared to the recognition of their CDE structure by Roquin. Strikingly, our findings thus suggest that AREs can be recognized in multiple ways, allowing control over mRNA regulation by adapting distinct conformational states, thus providing differential accessibility to regulatory RBPs.

The large family of PC4-like domains - similar folds and functions throughout all kingdoms of life.

RNA- and DNA-binding domains are essential building blocks for specific regulation of gene expression. While a number of canonical nucleic acid binding domains share sequence and structural conservation, others are less obviously linked by evolutionary traits. In this review, we describe a protein fold of about 150 aa in length, bearing a conserved ß-ß-ß-ß-α-linker-ß-ß-ß-ß-α topology and similar nucleic acid binding properties but no apparent sequence conservation. The same overall fold can also be achieved by dimerization of two proteins, each bearing a ß-ß-ß-ß-α topology. These proteins include but are not limited to the transcription factors PC4 and P24 from humans and plants, respectively, the human RNA-transport factor Pur-α (also termed PURA), as well as the ssDNA-binding SP_0782 protein from Streptococcus pneumonia and the bacteriophage coat proteins PP7 and MS2. Besides their common overall topology, these proteins share common nucleic acids binding surfaces and thus functional similarity. We conclude that these PC4-like domains include proteins from all kingdoms of life and are much more abundant than previously known.

Crystal Contact Engineering Enables Efficient Capture and Purification of an Oxidoreductase by Technical Crystallization.

Technical crystallization is an attractive method to purify recombinant proteins. However, it is rarely applied due to the limited crystallizability of many proteins. To overcome this limitation, single amino acid exchanges are rationally introduced to enhance intermolecular interactions at the crystal contacts of the industrially relevant biocatalyst Lactobacillus brevis alcohol dehydrogenase (LbADH). The wildtype (WT) and the best crystallizing and enzymatically active LbADH mutants K32A, D54F, Q126H, and T102E are produced with Escherichia coli and subsequently crystallized from cell lysate in stirred mL-crystallizers. Notwithstanding the high host cell protein (HCP) concentrations in the lysate, all mutants crystallize significantly faster than the WT. Combinations of mutations result in double mutants with faster crystallization kinetics than the respective single mutants, demonstrating a synergetic effect. The almost entire depletion of the soluble LbADH fraction at crystallization equilibrium is observed, proving high yields. The HCP concentration is reduced to below 0.5% after crystal dissolution and recrystallization, and thus a 100-fold HCP reduction is achieved after two successive crystallization steps. The combination of fast kinetics, high yields, and high target protein purity highlights the potential of crystal contact engineering to transform technical crystallization into an efficient protein capture and purification step in biotechnological downstream processes.

Ferritin-Displayed GLP-1 with Improved Pharmacological Activities and Pharmacokinetics.

Glucagon-like peptide-1 (GLP-1) is an incretin (a type of metabolic hormone that stimulates a decrease in blood glucose levels), holding great potential for the treatment of type 2 diabetes mellitus (T2DM). However, its extremely short half-life of 1-2 min hampers any direct clinical application. Here, we describe the application of the heavy chain of human ferritin (HFt) nanocage as a carrier to improve the pharmacological properties of GLP-1. The GLP-HFt was designed by genetic fusion of GLP-1 to the N-terminus of HFt and was expressed in inclusion bodies in E. coli. The refolding process was developed to obtain a soluble GLP-HFt protein. The biophysical properties determined by size-exclusion chromatography (SEC), dynamic light scattering (DLS), circular dichroism (CD), transmission electron microscopy (TEM), and X-ray crystallography verified that the GLP-HFt successfully formed a 24-mer nanocage with GLP-1 displayed on the external surface of HFt. The in vivo pharmacodynamic results demonstrated that the GLP-HFt nanocage retained the bioactivity of natural GLP-1, significantly reduced the blood glucose levels for at least 24 h in a dose-dependent manner, and inhibited food intake for at least 8-10 h. The half-life of the GLP-HFt nanocage was approximately 52 h in mice after subcutaneous injection. The prolonged half-life and sustained control of blood glucose levels indicate that the GLP-HFt nanocage can be further developed for the treatment of T2DM. Meanwhile, the HFt nanocage proves its great potential as a universal carrier that improves the pharmacodynamic and pharmacokinetic properties of a wide range of therapeutic peptides and proteins.

Combinatorial recognition of clustered RNA elements by the multidomain RNA-binding protein IMP3.

How multidomain RNA-binding proteins recognize their specific target sequences, based on a combinatorial code, represents a fundamental unsolved question and has not been studied systematically so far. Here we focus on a prototypical multidomain RNA-binding protein, IMP3 (also called IGF2BP3), which contains six RNA-binding domains (RBDs): four KH and two RRM domains. We establish an integrative systematic strategy, combining single-domain-resolved SELEX-seq, motif-spacing analyses, in vivo iCLIP, functional validation assays, and structural biology. This approach identifies the RNA-binding specificity and RNP topology of IMP3, involving all six RBDs and a cluster of up to five distinct and appropriately spaced CA-rich and GGC-core RNA elements, covering a >100 nucleotide-long target RNA region. Our generally applicable approach explains both specificity and flexibility of IMP3-RNA recognition, allows the prediction of IMP3 targets, and provides a paradigm for the function of multivalent interactions with multidomain RNA-binding proteins in gene regulation.

Staufen2-mediated RNA recognition and localization requires combinatorial action of multiple domains.

Throughout metazoans, Staufen (Stau) proteins are core factors of mRNA localization particles. They consist of three to four double-stranded RNA binding domains (dsRBDs) and a C-terminal dsRBD-like domain. Mouse Staufen2 (mStau2)-like Drosophila Stau (dmStau) contains four dsRBDs. Existing data suggest that only dsRBDs 3-4 are necessary and sufficient for mRNA binding. Here, we show that dsRBDs 1 and 2 of mStau2 bind RNA with similar affinities and kinetics as dsRBDs 3 and 4. While RNA binding by these tandem domains is transient, all four dsRBDs recognize their target RNAs with high stability. Rescue experiments in Drosophila oocytes demonstrate that mStau2 partially rescues dmStau-dependent mRNA localization. In contrast, a rescue with mStau2 bearing RNA-binding mutations in dsRBD1-2 fails, confirming the physiological relevance of our findings. In summary, our data show that the dsRBDs 1-2 play essential roles in the mRNA recognition and function of Stau-family proteins of different species.

Defining and expanding the phenotype of QARS-associated developmental epileptic encephalopathy.

OBJECTIVE: The study is aimed at widening the clinical and genetic spectrum and at assessing genotype-phenotype associations in QARS encephalopathy. METHODS: Through diagnostic gene panel screening in an epilepsy cohort, and recruiting through GeneMatcher and our international network, we collected 10 patients with biallelic QARS variants. In addition, we collected data on 12 patients described in the literature to further delineate the associated phenotype in a total cohort of 22 patients. Computer modeling was used to assess changes on protein folding. RESULTS: Biallelic pathogenic variants in QARS cause a triad of progressive microcephaly, moderate to severe developmental delay, and early-onset epilepsy. Microcephaly was present at birth in 65%, and in all patients at follow-up. Moderate (14%) or severe (73%) developmental delay was characteristic, with no achievement of sitting (85%), walking (86%), or talking (90%). Additional features included irritability (91%), hypertonia/spasticity (75%), hypotonia (83%), stereotypic movements (75%), and short stature (56%). Seventy-nine percent had pharmacoresistant epilepsy with mainly neonatal onset. Characteristic cranial MRI findings include early-onset progressive atrophy of cerebral cortex (89%) and cerebellum (61%), enlargement of ventricles (95%), and age-dependent delayed myelination (88%). A small subset of patients displayed a less severe phenotype. CONCLUSIONS: These data revealed first genotype-phenotype associations and may serve for improved interpretation of new QARS variants and well-founded genetic counseling.

Crystal structure of a biliverdin-bound phycobiliprotein: Interdependence of oligomerization and chromophorylation.

Small, ultra-red fluorescence protein (smURFP) introduces the non-native biliverdin (BV) chromophore to phycobiliproteins (PBPs), allowing them to be used as transgenic labels for in vivo mammalian imaging. Presently, no structural information exists for PBPs bound to the non-native BV chromophore, which limits the further development of smURFP and related proteins as imaging labels or indicators. Here we describe the first crystal structure of a PBP bound to BV. The structures of smURFP-Y56R with BV and smURFP-Y56F without BV reveal unique oligomerization interfaces different from those in wild-type PBPs bound to native chromophores. Our structures suggest that the oligomerization interface affects the BV binding site, creating a link between oligomerization and chromophorylation that we confirmed through site-directed mutagenesis and that may help guide efforts to improve the notorious chromophorylation of smURFP and other PBPs engineered to bind BV.

Neutral metalloaminopeptidases APN and MetAP2 as newly discovered anticancer molecular targets of actinomycin D and its simple analogs.

The potent transcription inhibitor Actinomycin D is used with several cancers. Here, we report the discovery that this naturally occurring antibiotic inhibits two human neutral aminopeptidases, the cell-surface alanine aminopeptidase and intracellular methionine aminopeptidase type 2. These metallo-containing exopeptidases participate in tumor cell expansion and motility and are targets for anticancer therapies. We show that the peptide portions of Actinomycin D and Actinomycin X2 are not required for effective inhibition, but the loss of these regions changes the mechanism of interaction. Two structurally less complex Actinomycin D analogs containing the phenoxazone chromophores, Questiomycin A and Actinocin, appear to be competitive inhibitors of both aminopeptidases, with potencies similar to the non-competitive macrocyclic parent compound (Ki in the micromolar range). The mode of action for all four compounds and both enzymes was demonstrated by molecular modeling and docking in the corresponding active sites. This knowledge gives new perspectives to Actinomycin D's action on tumors and suggests new avenues and molecules for medical applications.

PURA syndrome: clinical delineation and genotype-phenotype study in 32 individuals with review of published literature.

BACKGROUND: De novo mutations in PURA have recently been described to cause PURA syndrome, a neurodevelopmental disorder characterised by severe intellectual disability (ID), epilepsy, feeding difficulties and neonatal hypotonia. OBJECTIVES: To delineate the clinical spectrum of PURA syndrome and study genotype-phenotype correlations. METHODS: Diagnostic or research-based exome or Sanger sequencing was performed in individuals with ID. We systematically collected clinical and mutation data on newly ascertained PURA syndrome individuals, evaluated data of previously reported individuals and performed a computational analysis of photographs. We classified mutations based on predicted effect using 3D in silico models of crystal structures of Drosophila-derived Pur-alpha homologues. Finally, we explored genotype-phenotype correlations by analysis of both recurrent mutations as well as mutation classes. RESULTS: We report mutations in PURA (purine-rich element binding protein A) in 32 individuals, the largest cohort described so far. Evaluation of clinical data, including 22 previously published cases, revealed that all have moderate to severe ID and neonatal-onset symptoms, including hypotonia (96%), respiratory problems (57%), feeding difficulties (77%), exaggerated startle response (44%), hypersomnolence (66%) and hypothermia (35%). Epilepsy (54%) and gastrointestinal (69%), ophthalmological (51%) and endocrine problems (42%) were observed frequently. Computational analysis of facial photographs showed subtle facial dysmorphism. No strong genotype-phenotype correlation was identified by subgrouping mutations into functional classes. CONCLUSION: We delineate the clinical spectrum of PURA syndrome with the identification of 32 additional individuals. The identification of one individual through targeted Sanger sequencing points towards the clinical recognisability of the syndrome. Genotype-phenotype analysis showed no significant correlation between mutation classes and disease severity.

Evidence for Complex Formation of the Bacillus cereus Haemolysin BL Components in Solution.

Haemolysin BL is an important virulence factor regarding the diarrheal type of food poisoning caused by Bacillus cereus. However, the pathogenic importance of this three-component enterotoxin is difficult to access, as nearly all natural B. cereus culture supernatants additionally contain the highly cytotoxic Nhe, the second three-component toxin involved in the aetiology of B. cereus-induced food-borne diseases. To better address the toxic properties of the Hbl complex, a system for overexpression and purification of functional, cytotoxic, recombinant (r)Hbl components L2, L1 and B from E. coli was established and an nheABC deletion mutant was constructed from B. cereus reference strain F837/76. Furthermore, 35 hybridoma cell lines producing monoclonal antibodies (mAbs) against Hbl L2, L1 and B were generated. While mAbs 1H9 and 1D8 neutralized Hbl toxicity and thus, represent important tools for future investigations of the mode-of-action of Hbl on the target cell surface, mAb 1D7, in contrast, even enhanced Hbl toxicity by supporting the binding of Hbl B to the cell surface. By using the specific mAbs in Dot blots, indirect and hybrid sandwich enzyme immuno assays (EIAs), complex formation between Hbl L1 and B, as well as L1 and L2 in solution could be shown for the first time. Surface plasmon resonance experiments with the rHbl components confirmed these results with KD values of 4.7 × 10-7 M and 1.5 × 10-7 M, respectively. These findings together with the newly created tools lay the foundation for the detailed elucidation of the molecular mode-of-action of the highly complex three-component Hbl toxin.