High-resolution crystal structure of HA33 of botulinum neurotoxin type B progenitor toxin complex

Botulinum neurotoxins (BoNTs) are produced as progenitor toxin complexes (PTCs) by Clostridium botulinum. The PTCs are composed of BoNT and non-toxic neurotoxin-associated proteins (NAPs), which serve to protect and deliver BoNT through the gastrointestinal tract in food borne botulism. HA33 is a key NAP component that specifically recognizes host carbohydrates and helps enrich PTC on the intestinal lumen preceding its transport across the epithelial barriers. Here, we report the crystal structure of HA33 of type B PTC (HA33/B) in complex with lactose at 1.46 Å resolution. The structural comparisons among HA33 of serotypes A–D reveal two different HA33–glycan interaction modes. The glycan-binding pockets on HA33/A and B are more suitable to recognize galactose-containing glycans in comparison to the equivalent sites on HA33/C and D. On the contrary, HA33/C and D could potentially recognize Neu5Ac as an independent receptor, whereas HA33/A and B do not. These findings indicate that the different oral toxicity and host susceptibility observed among different BoNT serotypes could be partly determined by the serotype-specific interaction between HA33 and host carbohydrate receptors. Furthermore, we have identified a key structural water molecule that mediates the HA33/B–lactose interactions. It provides the structural basis for development of new receptor-mimicking compounds, which have enhanced binding affinity with HA33 through their water-displacing moiety.     

Molecular composition of progenitor toxin produced by Clostridium botulinum type C strain 6813

The molecular composition of the purified progenitor toxin produced by a Clostridium botulinum type C strain 6813 (C-6813) was analyzed. The strain produced two types of progenitor toxins (M and L). Purified L toxin is formed by conjugation of the M toxin (composed of a neurotoxin and a non-toxic nonhemagglutinin) with additional hemagglutinin (HA) components. The dual cleavage sites at loop region of the dichain structure neurotoxin were identified between Arg444-Ser445 and Lys449-Thr450 by the analyses of C-terminal of the light chain and N-terminal of the heavy chain. Analysis of partial amino acid sequences of fragments generated by limited proteolysis of the neurotoxin has shown to that the neurotoxin protein produced by C-6813 was a hybrid molecule composed of type C and D neurotoxins as previously reported. HA components consist of a mixture of several subcomponents with molecular weights of 70-, 55-, 33-, 26~21- and 17-kDa. The N-terminal amino acid sequences of 70-, 55-, and 26~21-kDa proteins indicated that the 70-kDa protein was intact HA-70 gene product, and other 55- and 26~21-kDa proteins were derived from the 70-kDa protein by modification with proteolysis after translation of HA-70 gene. Furthermore, several amino acid differences were exhibited in the amino acid sequence as compared with the deduced sequence from the nucleotide sequence of the HA-70 gene which was common among type C (strains C-St and C-468) and D progenitor toxins (strains D-CB16 and D-1873).     

Sugar-binding sites of the HA1 subcomponent of Clostridium botulinum type C progenitor toxin

Clostridium botulinum type C 16S progenitor toxin contains a hemagglutinin (HA) subcomponent, designated HA1, which appears to play an important role in the effective internalization of the toxin in gastrointestinal epithelial cells and in creating a broad specificity for the oligosaccharide structure that corresponds to various targets. In this study, using the recombinant protein fused to glutathione S-transferase, we investigated the binding specificity of the HA1 subcomponent to sugars and estimated the binding sites of HA1 based on X-ray crystallography and soaking experiments using various sugars. N-Acetylneuraminic acid, N-acetylgalactosamine, and galactose effectively inhibited the binding that occurs between glutathione S-transferase-HA1 and mucins, whereas N-acetylglucosamine and glucose did not inhibit it. The crystal structures of HA1 complex with N-acetylneuraminic acid, N-acetylgalactosamine, and galactose were also determined. There are two sugar-binding sites, sites I and II. Site I corresponds to the electron densities noted for all sugars and is located at the C-terminal β-trefoil domain, while site II corresponds to the electron densities noted only for galactose. An aromatic amino acid residue, Trp176, at site I has a stacking interaction with the hexose ring of the sugars. On the other hand, there is no aromatic residue at site II; thus, the interaction with galactose seems to be poor. The double mutant W176A at site I and D271F at site II has no avidity for N-acetylneuraminic acid but has avidity for galactose. In this report, the binding specificity of botulinum C16S toxin HA1 to various sugars is demonstrated based on its structural features.     

Characterization of Toxin Complex Produced by a Unique Strain of Clostridium botulinum Serotype D 4947

A unique strain of Clostridium botulinum, serotype D 4947 (D-4947), produces a considerable amount of a 650 kDa toxin complex (L-TC) and a small amount of a 280 kDa M-TC, a 540 kDa TC, and a 610 kDa TC. The complexes are composed of only un-nicked components, including neurotoxin (NT), nontoxic nonhemagglutinin (NTNHA) and hemagglutinin subcomponents (HA-70, HA-33 and HA-17). Unlike other NTs from all serotype strains, separation of D-4947 NT from L-TC, except for M-TC, during chromatography required highly alkaline conditions around pH 8.8. The separated NT and NTNHA/HAs complex can be reconstituted to L-TC that is indistinguishable from the parent L-TC with respect to toxicity, hemagglutination activity and gel filtration profile. The isoelectric points of NT and NTNHA/HAs were close together depending on the number of HA-33/17 molecules. We have established a new method to separate the unique D-4947 NT from the complex, which will yield valuable information on structure of botulinum toxin.     

Identification of RNA species in the RNA-toxin complex and structure of the complex in Clostridium botulinum type E

Clostridium botulinum type E toxin was isolated in the form of a complex with RNA(s) from bacterial cells. Characterization of the complexed RNA remains to be elucidated. The RNA is identified here as ribosomal RNA (rRNA) having 23S and 16S components. The RNA-toxin complexes were found to be made up of three types with different molecular sizes. The three types of RNA-toxin complex are toxin bound to both the 23S and 16S rRNA, toxin bound to the 16S rRNA and a small amount of 23S rRNA, and toxin bound only to the 16S rRNA.     

Similarity in Nucleotide Sequence of the Gene Encoding Nontoxic Component of Botulinum Toxin Produced by Toxigenic Clostridium butyricum Strain BL6340 and Clostridium botulinum Type E Strain Mashike

The complete nucleotide and deduced amino acid sequence of the nontoxic component of botulinum type E progenitor toxin is determined in recombinant plasmid pU9BUH containing about 6.0 kb HindIII fragment obtained from chromosomal DNA of Clostridium butyricum strain BL6340. The open reading frame (ORF) of this nontoxic component gene is composed of 3,486 nucleotide bases (1,162 amino acid residues). The molecular weight calculated from deduced amino acid residues is estimated 13,6810.1. The present study revealed that 33 nucleotide bases of 3,486 are different in the nontoxic component gene between C.butyricum strain BL6340 and C. botulinum type E strain Mashike. This corresponds to the difference of 17 amino acid residues in these nontoxic component.     

Characterization of nicking of the nontoxic-nonhemagglutinin components of Clostridium botulinum types C and D progenitor toxin

Clostridium botulinum C and D strains produce two types of progenitor toxins, M and L. Previously we reported that a 130-kDa nontoxic-nonhemagglutinin (NTNHA) component of the M toxin produced by type D strain CB16 was nicked at a unique site, leading to a 15-kDa N-terminal fragment and a 115-kDa C-terminal fragment. In this study, we identified the amino acid sequences around the nicking sites in the NTNHAs of the M toxins produced by C. botulinum type C and D strains by analysis of their C-terminal and N-terminal sequences and mass spectrometry. The C-terminus of the 15-kDa fragments was identified as Lys127 from these strains, indicating that a bacterial trypsin-like protease is responsible for the nicking. The 115-kDa fragment had mixtures of three different N-terminal amino acid sequences beginning with Leu135, Val139, and Ser141, indicating that 7–13 amino acid residues were deleted from the nicking site. The sequence beginning with Leu135 would also suggest cleavage by a trypsin-like protease, while the other two N-terminal amino acid sequences beginning with Val139 and Ser141 would imply proteolysis by an unknown protease. The nicked NTNHA forms a binary complex of two fragments that could not be separated without sodium dodecyl sulfate.     

Spontaneous nicking in the nontoxic-nonhemagglutinin component of the Clostridium botulinum toxin complex

The nontoxic-nonhemagglutinin (NTNHA) component, in both isolated form and the neurotoxin (NT)/NTNHA complexed form, was prepared protease-free from toxin complexes produced by Clostridium botulinum type D strain 4947. NTNHA in both preparations was found to be spontaneously converted to the nicked NTNHA form leading to 15- and 115-kDa fragments with the excision of several amino acid residues at specific sites on SDS-PAGE during long-term incubation, while that of the NT/NTNHA/hemagglutinin complexed form remained unnicked single-chain polypeptides under the same conditions. Considering that the NTNHA preparation contained small amounts of the nicked form of NTNHA and the addition of trypsin accelerated the cleavage, it is speculated that a nicked form of NTNHA remaining after the purification and/or NTNHA itself catalyzes the cleavage of intact NTNHA.     

Molecular Composition of the 16S Toxin Produced by a Clostridium botulinum Type D Strain, 1873

The 16S toxin was purified from a Clostridium botulinum type D strain 1873 (D-1873). Furthermore, the entire nucleotide sequences of the genes coding for the 16S toxin were determined. It became clear that the purified D-1873 16S toxin consists of neurotoxin, nontoxic nonhemagglutinin (NTNH), and hemagglutinin (HA), and that HA consists of four subcomponents, HA1, HA2, HA3a, and HA3b, the same as type D strain CB16 (D-CB16) 16S toxin. The nucleotide sequences of the nontoxic components of these two strains were also found to be identical except for several bases. However, the culture supernatant and the purified 16S toxin of D-1873 showed little HA activity, unlike D-CB16, though the fractions successively eluted after the D-1873 16S toxin peak from an SP-Toyopearl 650S column showed a low level of HA activity. The main difference between D-1873 and D-CB16 HA molecules was the mobility of the HA1 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Therefore it was presumed that the loss of HA activity of D-1873 16S toxin might be caused by the differences of processing HA after the translation.     

Gene arrangement in the upstream region of Clostridium botulinum type E and Clostridium butyricum BL6340 progenitor toxin genes is different from that of other types

The cluster of genes encoding the botulinum progenitor toxin and the upstream region including p21 and p47 were divided into three different gene arrangements (class I–III). To determine the gene similarity of the type E neurotoxin (BoNT/E) complex to other types, the gene organization in the upstream region of the nontoxic-nonhemagglutinin gene (ntnh) was investigated in chromosomal DNA from Clostridium botulinum type E strain Iwanai and C. butyricum strain BL6340. The gene cluster of type E progenitor toxin (Iwanai and BL6340) was similar to those of type F and type A (from infant botulism in Japan), but not to those of types A, B, and C. Though genes for the hemagglutinin component and P21 were not discovered, genes encoding P47, NTNH, and BoNT were found in type E strain Iwanai and C. butyricum strain BL6340. However, the genes of ORF-X₁ (435 bp) and ORF-X₂ (partially sequenced) were present just upstream of that of P47. The orientation of these genes was in inverted direction to that of p47. The gene cluster of type E progenitor toxin (Iwanai and BL6340) is, therefore, a specific arrangement (class IV) among the genes encoding components of the BoNT complex.     

The structure of the tetanus toxin reveals pH‐mediated domain dynamics

The tetanus neurotoxin (TeNT) is a highly potent toxin produced by Clostridium tetani that inhibits neurotransmission of inhibitory interneurons, causing spastic paralysis in the tetanus disease. TeNT differs from the other clostridial neurotoxins by its unique ability to target the central nervous system by retrograde axonal transport. The crystal structure of the tetanus toxin reveals a “closed” domain arrangement stabilised by two disulphide bridges, and the molecular details of the toxin's interaction with its polysaccharide receptor. An integrative analysis combining X‐ray crystallography, solution scattering and single particle electron cryo‐microscopy reveals pH‐mediated domain rearrangements that may give TeNT the ability to adapt to the multiple environments encountered during intoxication, and facilitate binding to distinct receptors.     

Structure and action of the binary C2 toxin from Clostridium botulinum

C2 toxin from Clostridium botulinum is composed of the enzyme component C2-I, which ADP-ribosylates actin, and the binding and translocation component C2-II, responsible for the interaction with eukaryotic cell receptors and the following endocytosis. Three C2-I crystal structures at resolutions of up to 1.75 A are presented together with a crystal structure of C2-II at an appreciably lower resolution and a model of the prepore formed by fragment C2-IIa. The C2-I structure was determined at pH 3.0 and at pH 6.1. The structural differences are small, indicating that C2-I does not unfold, even at a pH value as low as 3.0. The ADP-ribosyl transferase activity of C2-I was determined for alpha and beta/gamma-actin and related to that of Iota toxin and of mutant S361R of C2-I that introduced the arginine observed in Iota toxin. The substantial activity differences between alpha and beta/gamma-actin cannot be explained by the protein structures currently available. The structure of the transport component C2-II at pH 4.3 was established by molecular replacement using a model of the protective antigen of anthrax toxin at pH 6.0. The C-terminal receptor-binding domain of C2-II could not be located but was present in the crystals. It may be mobile. The relative orientation and positions of the four other domains of C2-II do not differ much from those of the protective antigen, indicating that no large conformational changes occur between pH 4.3 and pH 6.0. A model of the C2-IIa prepore structure was constructed based on the corresponding assembly of the protective antigen. It revealed a surprisingly large number of asparagine residues lining the pore. The interaction between C2-I and C2-IIa and the translocation of C2-I into the target cell are discussed.     

The Sclerotinia sclerotiorum agglutinin represents a novel family of fungal lectins remotely related to the Clostridium botulinum non-toxin haemagglutinin HA33/A

Previous studies indicated that sclerotes of the phytopathogenic Ascomycete Sclerotinia sclerotiorum contain a lectin that based on its molecular structure, specificity and N-terminal amino acid sequence could not be classified yet into any lectin family. Using a combination of molecular cloning, frontal affinity chromatography and molecular modelling the identity of the S. sclerotiorum agglutinin (SSA) was analyzed. Molecular cloning demonstrated that SSA shares no sequence similarity with any known fungal lectin or protein. The lectin is synthesized as a 153 amino acid polypeptide without signal peptide and undergoes apart from the removal of the N-terminal methionine no further processing. Frontal affinity chromatography revealed that the binding site of SSA primarily accommodates a non-reducing terminal GalNAc with a preference for the alpha- over the beta-anomer. SSA also strongly interacts with both glycolipid type glycans with terminal non-reducing Gal or GalNAc and galactosylated N-glycans. SSA shares a residual sequence similarity with part of the non-toxin haemagglutinin HA33/A from Clostridium botulinum. Molecular modeling using the three-dimensional structure of HA33/A as a template indicated that SSA can fold into a similar beta-trefoil domain. Though these results should be interpreted with care it is tempting to speculate that the Sclerotiniaceae lectins thus appear to be structurally related to the ricin-B superfamily. All evidence suggests that SSA represents a novel family of fungal lectins with a unique sequence and sugar-binding properties. Taking into account that orthologues of SSA are fairly common within the family Sclerotiniaceae but could not be identified in any other fungal species one can reasonably conclude that SSA-type lectins are confined to a small taxonomic group of the Ascomycota.     

Molecular properties of a hemagglutinin purified from type A Clostridium botulinum

Clostridium botulinum causes the food poisoning disease botulism by producing botulinum neurotoxin, the most potent toxin known. The neurotoxin is produced along with a group of neurotoxin-associated proteins, or NAPs, which protect it from the low pH and proteases of the gastrointestinal tract. Recently, we isolated one of the major components of NAPs, a 33-kDa hemagglutinin (Hn-33) [Fu et al. (1998), J. Protein Chem. 17, 53–60]. In this study, we present molecular properties of Hn-33 derived from several biochemical and biophysical techniques. Hn-33 in pure form requires a 66-fold lower concentration of sugar inhibition of its hemagglutination activity than in its complexed form with the neurotoxin and other NAPs. However, its protease resistance is not affected by sugar binding. Based on FT-IR and circular dichroism (CD) analysis, Hn-33 is a predominantly -sheet protein (74–77%). Hn-33 analysis by laser desorption mass spectrometry and size exclusion column chromatography reveals that it exists predominantly in a dimeric form in the aqueous solution. Even a very low concentration of SDS (0.05%) irreversibly destroyed the biological activity of Hn-33 by changing its secondary structure as revealed by far-UV CD analysis.     

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.     

Crystal structure of KD93, a novel protein expressed in human hematopoietic stem/progenitor cells

The crystal structure of a novel hypothetical protein, KD93, expressed in human hematopoietic stem/progenitor cells, was determined at 1.9A resolution using the multiple-wavelength anomalous dispersion (MAD) method. The protein KD93, which is encoded by the open reading frame HSPC031, is a NIP7 homologue and belongs to the UPF0113 family. The structural and functional information for the group of homologues has not yet been determined. Crystallographic analysis revealed that the overall fold of KD93 consists of two interlinked alpha/beta domains. Structure-based homology analysis with DALI revealed that the C domain of KD93 matches the PUA domain of some RNA modification enzymes, especially that of archaeosine tRNA-ribosyltransferase (ArcTGT), which suggests that its possible molecular function is related to RNA binding. The difference between the RNA binding regions of KD93 and ArcTGT in amino acid constitution and surface electrostatic potential indicate that they may have different RNA binding modes. The N domain of KD93 is a unique structure with no obvious similarity to other proteins with known three-dimensional structures. The high-resolution structure of KD93 provides a first view of a member of the family of hypothetical proteins. And the structure provides a framework to deduce and assay the molecular function of other proteins of the UPF0113 family.     

Solution structure of APETx1 from the sea anemone Anthopleura elegantissima: a new fold for an HERG toxin

APETx1 is a 42-amino acid toxin purified from the venom of the sea anemone Anthopleura elegantissima. This cysteine-rich peptide possesses three disulfide bridges (C4-C37, C6-C30, and C20-C38). Its pharmacological target is the Ether-a-gogo potassium channel. We herein determine the solution structure of APETx1 by use of conventional two-dimensional 1H-NMR techniques followed by torsion angle dynamics and refinement protocols. The calculated structure of APETx1 belongs to the disulfide-rich all-beta structural family, in which a three-stranded anti-parallel beta-sheet is the only secondary structure. APETx1 is the first Ether-a-gogo effector discovered to fold in this way. We therefore compare the structure of APETx1 to those of the two other known effectors of the Ether-a-gogo potassium channel, CnErg1 and BeKm-1, and analyze the topological disposition of key functional residues proposed by analysis of the electrostatic anisotropy. The interacting surface is made of a patch of aromatic residues (Y5, Y32, and F33) together with two basic residues (K8 and K18) at the periphery of the surface. We pinpoint the absence of the central lysine present in the functional surface of the two other Ether-a-gogo effectors.     

The role of histidine residues in the alpha toxin of Clostridium perfringens

The α -toxin (phospholipase C) of Clostridium perfringens has been reported to contain catalytically essential zinc ions We report here that histidine residues are essential for the co-ordination of these ion(s). Incubation of alpha toxin with diethylpyrocarbonate, a histidine modifying reagent, did not result in the loss of phospholipase C activity unless the protein was first incubated with EDTA, suggesting that zinc ions normally protect the susceptible histidine residues. When the amino acid sequences of three phospholipase C's were aligned, essential zinc binding histidine residues in the non-toxic B. cereus phospholipase C were found in similar positions in the toxic C. perfringens enzyme and the weakly toxic C. bifermentans phospholipase C.