Spectroscopic evidence of tetanus toxin translocation domain bilayer-induced refolding and insertion.

Tetanus neurotoxin (TeNT) is an A-B toxin with three functional domains: endopeptidase, translocation (HCT), and receptor binding. Endosomal acidification triggers HCT to interact with and insert into the membrane, translocating the endopeptidase across the bilayer. Although the function of HCT is well defined, the mechanism by which it accomplishes this task is unknown. To gain insight into the HCT membrane interaction on both local and global scales, we utilized an isolated, beltless HCT variant (bHCT), which retained the ability to release potassium ions from vesicles. To examine which bHCT residues interact with the membrane, we widely sampled the surface of bHCT using 47 single-cysteine variants labeled with the environmentally sensitive fluorophore NBD. At neutral pH, no interaction was observed for any variant. In contrast, all NBD-labeled positions reported environmental change in the presence of acidic pH and membranes containing anionic lipids. We then examined the conformation of inserted bHCT using circular dichroism and intrinsic fluorescence. Upon entering the membrane, bHCT retained predominantly α-helical secondary structure, whereas the tertiary structure exhibited substantial refolding. The use of lipid-attached quenchers revealed that at least one of the three tryptophan residues penetrated deep into the hydrocarbon core of the membrane, suggesting formation of a bHCT transmembrane conformation. The possible conformational topology was further explored with the hydropathy analysis webtool MPEx, which identified a large, potential α-helical transmembrane region. Altogether, the spectroscopic evidence supports a model in which, upon acidification, the majority of TeNT bHCT entered the membrane with a concurrent change in tertiary structure.

Transient kinetic analysis of oxidative dealkylation by the direct reversal DNA repair enzyme AlkB.

AlkB is a bacterial Fe(II)- and 2-oxoglutarate-dependent dioxygenase that repairs a wide range of alkylated nucleobases in DNA and RNA as part of the adaptive response to exogenous nucleic acid-alkylating agents. Although there has been longstanding interest in the structure and specificity of Escherichia coli AlkB and its homologs, difficulties in assaying their repair activities have limited our understanding of their substrate specificities and kinetic mechanisms. Here, we used quantitative kinetic approaches to determine the transient kinetics of recognition and repair of alkylated DNA by AlkB. These experiments revealed that AlkB is a much faster alkylation repair enzyme than previously reported and that it is significantly faster than DNA repair glycosylases that recognize and excise some of the same base lesions. We observed that whereas 1,N6-ethenoadenine can be repaired by AlkB with similar efficiencies in both single- and double-stranded DNA, 1-methyladenine is preferentially repaired in single-stranded DNA. Our results lay the groundwork for future studies of AlkB and its human homologs ALKBH2 and ALKBH3.

Human erythrocyte band 3 is a host receptor for Plasmodium falciparum glutamic acid-rich protein.

Malaria remains a major global threat to human health and economic development. Microvascular lesions caused by Plasmodium falciparum-infected human erythrocytes/red blood cells are hallmarks of severe pathogenesis contributing to high mortality, particularly in children from sub-Saharan Africa. In this study, we used a phage display complementary DNA library screening strategy to identify P falciparum glutamic acid-rich protein (PfGARP) as a secreted ligand that recognizes an ectodomain of human erythrocyte anion-exchanger, band 3/AE1, as a host receptor. Domain mapping of PfGARP revealed distinct nonoverlapping repeats encoding the immune response epitopes and core erythrocyte-binding activity. Synthetic peptides derived from the erythrocyte-binding repeats of PfGARP induced erythrocyte aggregation reminiscent of the rosetting phenomenon. Using peptides derived from the immunogenic repeats, a quantitative immunoassay was developed to detect a selective immune response against PfGARP in human plasma samples obtained from patients in rural Mali, suggesting the feasibility of PfGARP as a potential biomarker of disease progression. Collectively, our results suggest that PfGARP may play a functional role in enhancing the adhesive properties of human erythrocytes by engaging band 3 as a host receptor. We propose that immunological and pharmacological inhibition of PfGARP may unveil new therapeutic options for mitigating lesions in cerebral and pregnancy-associated malaria.

Expansion of base excision repair compensates for a lack of DNA repair by oxidative dealkylation in budding yeast.

The Mag1 and Tpa1 proteins from budding yeast (Saccharomyces cerevisiae) have both been reported to repair alkylation damage in DNA. Mag1 initiates the base excision repair pathway by removing alkylated bases from DNA, and Tpa1 has been proposed to directly repair alkylated bases as does the prototypical oxidative dealkylase AlkB from Escherichia coli However, we found that in vivo repair of methyl methanesulfonate (MMS)-induced alkylation damage in DNA involves Mag1 but not Tpa1. We observed that yeast strains without tpa1 are no more sensitive to MMS than WT yeast, whereas mag1-deficient yeast are ∼500-fold more sensitive to MMS. We therefore investigated the substrate specificity of Mag1 and found that it excises alkylated bases that are known AlkB substrates. In contrast, purified recombinant Tpa1 did not repair these alkylated DNA substrates, but it did exhibit the prolyl hydroxylase activity that has also been ascribed to it. A comparison of several of the kinetic parameters of Mag1 and its E. coli homolog AlkA revealed that Mag1 catalyzes base excision from known AlkB substrates with greater efficiency than does AlkA, consistent with an expanded role of yeast Mag1 in repair of alkylation damage. Our results challenge the proposal that Tpa1 directly functions in DNA repair and suggest that Mag1-initiated base excision repair compensates for the absence of oxidative dealkylation of alkylated nucleobases in budding yeast. This expanded role of Mag1, as compared with alkylation repair glycosylases in other organisms, could explain the extreme sensitivity of Mag1-deficient S. cerevisiae toward alkylation damage.

HIV protease inhibitors block parasite signal peptide peptidases and prevent growth of Babesia microti parasites in erythrocytes.

Malaria and babesiosis are bloodborne protozoan infections for which the emergence of drug-resistant strains poses a threat. Our previous phage display cDNA screens established the essentiality of Plasmodium falciparum signal peptide peptidase (SPP) in asexual development at the blood stage of malaria infection. Given the structural similarities between SPP inhibitors and HIV protease inhibitors, we screened ten HIV protease inhibitors and selected Lopinavir and Atazanavir for their ability to inhibit PfSPP activity. Using a transcription-based assay, we observed that Lopinavir inhibits both parasite-and host-derived SPP activities whereas Atazanavir inhibited only parasite derived SPP activity. Consistent with their inhibitory effect on Plasmodium growth, both Lopinavir and Atazanavir strongly inhibited intraerythrocytic Babesia microti growth ex vivo. Moreover, Lopinavir prevented the steep rise in Babesia microti parasitemia typically observed in rag1-deficient mice. Our data provide first evidence that inhibition of parasite-derived SPPs by HIV protease inhibitors offers a promising therapeutic avenue for the treatment of severe babesiosis and infections caused by other Apicomplexa parasites.

Insights into the Mechanisms by Which Clostridial Neurotoxins Discriminate between Gangliosides.

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) are the causative agents of the paralytic diseases botulism and tetanus, respectively. Entry of toxins into neurons is mediated through initial interactions with gangliosides, followed by binding to a protein co-receptor. Herein, we aimed to understand the mechanism through which individual neurotoxins recognize the carbohydrate motif of gangliosides. Using cell-based and in vitro binding assays, in conjunction with structure-driven site-directed mutagenesis, a conserved hydrophobic residue within the BoNTs that contributes to both affinity and specificity toward Sia5-containing gangliosides was identified. We demonstrate that targeted mutations within the Sia5 binding pocket result in the generation of neurotoxins that either bind and enter cells more efficiently (BoNT/A1 and BoNT/B) or display altered ganglioside binding specificity (TeNT). These data support a model in which recognition of Sia5 is largely driven by hydrophobic interactions between the sugar and the Sia5 binding site.

Merozoite surface protein 1 recognition of host glycophorin A mediates malaria parasite invasion of red blood cells.

Plasmodium falciparum invasion of human red blood cells (RBCs) is an intricate process requiring a number of distinct ligand-receptor interactions at the merozoite-erythrocyte interface. Merozoite surface protein 1 (MSP1), a highly abundant ligand coating the merozoite surface in all species of malaria parasites, is essential for RBC invasion and considered a leading candidate for inclusion in a multiple-subunit vaccine against malaria. Our previous studies identified an interaction between the carboxyl-terminus of MSP1 and RBC band 3. Here, by employing phage display technology, we report a novel interaction between the amino-terminus of MSP1 and RBC glycophorin A (GPA). Mapping of the binding domains established a direct interaction between malaria MSP1 and human GPA within a region of MSP1 known to potently inhibit P falciparum invasion of human RBCs. Furthermore, a genetically modified mouse model lacking the GPA- band 3 complex in RBCs is completely resistant to malaria infection in vivo. These findings suggest an essential role of the MSP1-GPA-band 3 complex during the initial adhesion phase of malaria parasite invasion of RBCs.

Chaperonin-Based Biolayer Interferometry To Assess the Kinetic Stability of Metastable, Aggregation-Prone Proteins.

Stabilizing the folded state of metastable and/or aggregation-prone proteins through exogenous ligand binding is an appealing strategy for decreasing disease pathologies caused by protein folding defects or deleterious kinetic transitions. Current methods of examining binding of a ligand to these marginally stable native states are limited because protein aggregation typically interferes with analysis. Here, we describe a rapid method for assessing the kinetic stability of folded proteins and monitoring the effects of ligand stabilization for both intrinsically stable proteins (monomers, oligomers, and multidomain proteins) and metastable proteins (e.g., low Tm) that uses a new GroEL chaperonin-based biolayer interferometry (BLI) denaturant pulse platform. A kinetically controlled denaturation isotherm is generated by exposing a target protein, immobilized on a BLI biosensor, to increasing denaturant concentrations (urea or GuHCl) in a pulsatile manner to induce partial or complete unfolding of the attached protein population. Following the rapid removal of the denaturant, the extent of hydrophobic unfolded/partially folded species that remains is detected by an increased level of GroEL binding. Because this kinetic denaturant pulse is brief, the amplitude of binding of GroEL to the immobilized protein depends on the duration of the exposure to the denaturant, the concentration of the denaturant, wash times, and the underlying protein unfolding-refolding kinetics; fixing all other parameters and plotting the GroEL binding amplitude versus denaturant pulse concentration result in a kinetically controlled denaturation isotherm. When folding osmolytes or stabilizing ligands are added to the immobilized target proteins before and during the denaturant pulse, the diminished population of unfolded/partially folded protein manifests as a decreased level of GroEL binding and/or a marked shift in these kinetically controlled denaturation profiles to higher denaturant concentrations. This particular platform approach can be used to identify small molecules and/or solution conditions that can stabilize or destabilize thermally stable proteins, multidomain proteins, oligomeric proteins, and, most importantly, aggregation-prone metastable proteins.

Tetanus neurotoxin utilizes two sequential membrane interactions for channel formation.

Tetanus neurotoxin (TeNT) causes neuroparalytic disease by entering the neuronal soma to block the release of neurotransmitters. However, the mechanism by which TeNT translocates its enzymatic domain (light chain) across endosomal membranes remains unclear. We found that TeNT and a truncated protein devoid of the receptor binding domain (TeNT-LHN) associated with membranes enriched in acidic phospholipids in a pH-dependent manner. Thus, in contrast to diphtheria toxin, the formation of a membrane-competent state of TeNT requires the membrane interface and is modulated by the bilayer composition. Channel formation is further enhanced by tethering of TeNT to the membrane through ganglioside co-receptors prior to acidification. Thus, TeNT channel formation can be resolved into two sequential steps: 1) interaction of the receptor binding domain (heavy chain receptor binding domain) with ganglioside co-receptors orients the translocation domain (heavy chain translocation domain) as the lumen of the endosome is acidified and 2) low pH, in conjunction with acidic lipids within the membrane drives the conformational changes in TeNT necessary for channel formation.

Tumor biomarker glycoproteins in the seminal plasma of healthy human males are endogenous ligands for DC-SIGN.

DC-SIGN is an immune C-type lectin that is expressed on both immature and mature dendritic cells associated with peripheral and lymphoid tissues in humans. It is a pattern recognition receptor that binds to several pathogens including HIV-1, Ebola virus, Mycobacterium tuberculosis, Candida albicans, Helicobacter pylori, and Schistosoma mansoni. Evidence is now mounting that DC-SIGN also recognizes endogenous glycoproteins, and that such interactions play a major role in maintaining immune homeostasis in humans and mice. Autoantigens (neoantigens) are produced for the first time in the human testes and other organs of the male urogenital tract under androgenic stimulus during puberty. Such antigens trigger autoimmune orchitis if the immune response is not tightly regulated within this system. Endogenous ligands for DC-SIGN could play a role in modulating such responses. Human seminal plasma glycoproteins express a high level of terminal Lewis(x) and Lewis(y) carbohydrate antigens. These epitopes react specifically with the lectin domains of DC-SIGN. However, because the expression of these sequences is necessary but not sufficient for interaction with DC-SIGN, this study was undertaken to determine if any seminal plasma glycoproteins are also endogenous ligands for DC-SIGN. Glycoproteins bearing terminal Lewis(x) and Lewis(y) sequences were initially isolated by lectin affinity chromatography. Protein sequencing established that three tumor biomarker glycoproteins (clusterin, galectin-3 binding glycoprotein, prostatic acid phosphatase) and protein C inhibitor were purified by using this affinity method. The binding of DC-SIGN to these seminal plasma glycoproteins was demonstrated in both Western blot and immunoprecipitation studies. These findings have confirmed that human seminal plasma contains endogenous glycoprotein ligands for DC-SIGN that could play a role in maintaining immune homeostasis both in the male urogenital tract and the vagina after coitus.

Defining the functional footprint for recognition and repair of deaminated DNA.

Spontaneous deamination of DNA is mutagenic, if it is not repaired by the base excision repair (BER) pathway. Crystallographic data suggest that each BER enzyme has a compact DNA binding site. However, these structures lack information about poorly ordered termini, and the energetic contributions of specific protein-DNA contacts cannot be inferred. Furthermore, these structures do not reveal how DNA repair intermediates are passed between enzyme active sites. We used a functional footprinting approach to define the binding sites of the first two enzymes of the human BER pathway for the repair of deaminated purines, alkyladenine DNA glycosylase (AAG) and AP endonuclease (APE1). Although the functional footprint for full-length AAG is explained by crystal structures of truncated AAG, the footprint for full-length APE1 indicates a much larger binding site than is observed in crystal structures. AAG turnover is stimulated in the presence of APE1, indicating rapid exchange of AAG and APE1 at the abasic site produced by the AAG reaction. The coordinated reaction does not require an extended footprint, suggesting that each enzyme engages the site independently. Functional footprinting provides unique information relative to traditional footprinting approaches and is generally applicable to any DNA modifying enzyme or system of enzymes.

Botulinum neurotoxin serotype C associates with dual ganglioside receptors to facilitate cell entry.

BACKGROUND: How botulinum neurotoxin serotype C (BoNT/C) enters neurons is unclear. RESULTS: BoNT/C utilizes dual gangliosides as host cell receptors. CONCLUSION: BoNT/C accesses gangliosides on the plasma membrane. SIGNIFICANCE: Plasma membrane accessibility of the dual ganglioside receptors suggests synaptic vesicle exocytosis may not be necessary to expose BoNT/C receptors. Botulinum neurotoxins (BoNTs) cleave SNARE proteins in motor neurons that inhibits synaptic vesicle (SV) exocytosis, resulting in flaccid paralysis. There are seven BoNT serotypes (A-G). In current models, BoNTs initially bind gangliosides on resting neurons and upon SV exocytosis associate with the luminal domains of SV-associated proteins as a second receptor. The entry of BoNT/C is less clear. Characterizing the heavy chain receptor binding domain (HCR), BoNT/C was shown to utilize gangliosides as dual host receptors. Crystallographic and biochemical studies showed that the two ganglioside binding sites, termed GBP2 and Sia-1, were independent and utilized unique mechanisms to bind complex gangliosides. The GBP2 binding site recognized gangliosides that contained a sia5 sialic acid, whereas the Sia-1 binding site recognized gangliosides that contained a sia7 sialic acid and sugars within the backbone of the ganglioside. Utilizing gangliosides that uniquely recognized the GBP2 and Sia-1 binding sites, HCR/C entry into Neuro-2A cells required both functional ganglioside binding sites. HCR/C entered cells differently than the HCR of tetanus toxin, which also utilizes dual gangliosides as host receptors. A point-mutated HCR/C that lacked GBP2 binding potential retained the ability to bind and enter Neuro-2A cells. This showed that ganglioside binding at the Sia-1 site was accessible on the plasma membrane, suggesting that SV exocytosis may not be required to expose BoNT/C receptors. These studies highlight the utility of BoNT HCRs as probes to study the role of gangliosides in neurotransmission.

Sperm chromatin structure and reproductive fitness are altered by substitution of a single amino acid in mouse protamine 1.

Conventional dogma presumes that protamine-mediated DNA compaction in sperm is achieved by electrostatic interactions between DNA and the arginine-rich core of protamines. Phylogenetic analysis reveals several non-arginine residues conserved within, but not across species. The significance of these residues and their post-translational modifications are poorly understood. Here, we investigated the role of K49, a rodent-specific lysine residue in protamine 1 (P1) that is acetylated early in spermiogenesis and retained in sperm. In sperm, alanine substitution (P1(K49A)) decreases sperm motility and male fertility-defects that are not rescued by arginine substitution (P1(K49R)). In zygotes, P1(K49A) leads to premature male pronuclear decompaction, altered DNA replication, and embryonic arrest. In vitro, P1(K49A) decreases protamine-DNA binding and alters DNA compaction and decompaction kinetics. Hence, a single amino acid substitution outside the P1 arginine core is sufficient to profoundly alter protein function and developmental outcomes, suggesting that protamine non-arginine residues are essential for reproductive fitness.

Unique ganglioside recognition strategies for clostridial neurotoxins.

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin are the causative agents of the paralytic diseases botulism and tetanus, respectively. The potency of the clostridial neurotoxins (CNTs) relies primarily on their highly specific binding to nerve terminals and cleavage of SNARE proteins. Although individual CNTs utilize distinct proteins for entry, they share common ganglioside co-receptors. Here, we report the crystal structure of the BoNT/F receptor-binding domain in complex with the sugar moiety of ganglioside GD1a. GD1a binds in a shallow groove formed by the conserved peptide motif E … H … SXWY … G, with additional stabilizing interactions provided by two arginine residues. Comparative analysis of BoNT/F with other CNTs revealed several differences in the interactions of each toxin with ganglioside. Notably, exchange of BoNT/F His-1241 with the corresponding lysine residue of BoNT/E resulted in increased affinity for GD1a and conferred the ability to bind ganglioside GM1a. Conversely, BoNT/E was not able to bind GM1a, demonstrating a discrete mechanism of ganglioside recognition. These findings provide a structural basis for ganglioside binding among the CNTs and show that individual toxins utilize unique ganglioside recognition strategies.

Nonspecific DNA binding and coordination of the first two steps of base excision repair.

The base excision repair (BER) pathway repairs a wide variety of damaged nucleobases in DNA. This pathway is initiated by a DNA repair glycosylase, which locates the site of damage and catalyzes the excision of the damaged nucleobase. The resulting abasic site is further processed by apurinic/apyrimidinic site endonuclease 1 (APE1) to create a single-strand nick with the 3'-hydroxyl that serves as a primer for DNA repair synthesis. Because an abasic site is highly mutagenic, it is critical that the steps of the BER pathway be coordinated. Most human glycosylases bind tightly to their abasic product. APE1 displaces the bound glycosylase, thereby stimulating multiple-turnover base excision. It has been proposed that direct protein-protein interactions are involved in the stimulation by APE1, but no common interaction motifs have been identified among the glycosylases that are stimulated by APE1. We characterized the APE1 stimulation of alkyladenine DNA glycosylase (AAG) using a variety of symmetric and asymmetric lesion-containing oligonucleotides. Efficient stimulation of a wide variety of substrates favors a model in which both AAG and APE1 can simultaneously bind to DNA but may not interact directly. Rather, nonspecific DNA binding by both AAG and APE1 enables APE1 to replace AAG at the abasic site. AAG is not displaced into solution but remains bound to an adjacent undamaged site. We propose that nonspecific DNA binding interactions allow transient exposure of the abasic site so that it can be captured by APE1.

Identification of a unique ganglioside binding loop within botulinum neurotoxins C and D-SA .

The botulinum neurotoxins (BoNTs) are the most potent protein toxins for humans. There are seven serotypes of BoNTs (A-G) based on a lack of cross antiserum neutralization. BoNTs utilize gangliosides as components of the host receptors for binding and entry into neurons. Members of BoNT/C and BoNT/D serotypes include mosaic toxins that are organized in D/C and C/D toxins. One D/C mosaic toxin, BoNT/D-South Africa (BoNT/D-SA), was not fully neutralized by immunization with BoNT serotype C or D, which stimulated this study. Here the crystal structures of the receptor binding domains of BoNT/C, BoNT/D, and BoNT/D-SA are presented. Biochemical and cell binding studies show that BoNT/C and BoNT/D-SA possess unique mechanisms for ganglioside binding. These studies provide new information about how the BoNTs can enter host cells as well as a basis for understanding the immunological diversity of these neurotoxins.

Gangliosides as high affinity receptors for tetanus neurotoxin.

Tetanus neurotoxin (TeNT) is an exotoxin produced by Clostridium tetani that causes paralytic death to hundreds of thousands of humans annually. TeNT cleaves vesicle-associated membrane protein-2, which inhibits neurotransmitter release in the central nervous system to elicit spastic paralysis, but the molecular basis for TeNT entry into neurons remains unclear. TeNT is a approximately 150-kDa protein that has AB structure-function properties; the A domain is a zinc metalloprotease, and the B domain encodes a translocation domain and C-terminal receptor-binding domain (HCR/T). Earlier studies showed that HCR/T bound gangliosides via two carbohydrate-binding sites, termed the lactose-binding site (the "W" pocket) and the sialic acid-binding site (the "R" pocket). Here we report that TeNT high affinity binding to neurons is mediated solely by gangliosides. Glycan array and solid phase binding analyses identified gangliosides that bound exclusively to either the W pocket or the R pocket of TeNT; GM1a bound to the W pocket, and GD3 bound to the R pocket. Using these gangliosides and mutated forms of HCR/T that lacked one or both carbohydrate-binding pocket, gangliosides binding to both of the W and R pockets were shown to be necessary for high affinity binding to neuronal and non-neuronal cells. The crystal structure of a ternary complex of HCR/T with sugar components of two gangliosides bound to the W and R supported the binding of gangliosides to both carbohydrate pockets. These data show that gangliosides are functional dual receptors for TeNT.

Toxins from bacteria.

Bacterial toxins damage the host at the site of bacterial infection or distant from the site. Bacterial toxins can be single proteins or oligomeric protein complexes that are organized with distinct AB structure-function properties. The A domain encodes a catalytic activity. ADP ribosylation of host proteins is the earliest post-translational modification determined to be performed by bacterial toxins; other modifications include glucosylation and proteolysis. Bacterial toxins also catalyze the non-covalent modification of host protein function or can modify host cell properties through direct protein-protein interactions. The B domain includes two functional domains: a receptor-binding domain, which defines the tropism of a toxin for a cell and a translocation domain that delivers the A domain across a lipid bilayer, either on the plasma membrane or the endosome. Bacterial toxins are often characterized based upon the secretion mechanism that delivers the toxin out of the bacterium, termed types I-VII. This review summarizes the major families of bacterial toxins and also describes the specific structure-function properties of the botulinum neurotoxins.

Human AP endonuclease 1 stimulates multiple-turnover base excision by alkyladenine DNA glycosylase.

Human alkyladenine DNA glycosylase (AAG) locates and excises a wide variety of damaged purine bases from DNA, including hypoxanthine that is formed by the oxidative deamination of adenine. We used steady state, pre-steady state, and single-turnover kinetic assays to show that the multiple-turnover excision of hypoxanthine in vitro is limited by release of the abasic DNA product. This suggests the possibility that the product release step is regulated in vivo by interactions with other base excision repair (BER) proteins. Such coordination of BER activities would protect the abasic DNA repair intermediate and ensure its correct processing. AP endonuclease 1 (APE1) is the predominant enzyme for processing abasic DNA sites in human cells. Therefore, we have investigated the functional effects of added APE1 on the base excision activity of AAG. We find that APE1 stimulates the multiple-turnover excision of hypoxanthine by AAG but has no effect on single-turnover excision. Since the amino terminus of AAG has been implicated in other protein-protein interactions, we also characterize the deletion mutant lacking the first 79 amino acids. We find that APE1 fully stimulates the multiple-turnover glycosylase activity of this mutant, demonstrating that the amino terminus of AAG is not strictly required for this functional interaction. These results are consistent with a model in which APE1 displaces AAG from the abasic site, thereby coordinating the first two steps of the base excision repair pathway.

Glycosylated SV2 and gangliosides as dual receptors for botulinum neurotoxin serotype F.

Botulinum neurotoxin causes rapid flaccid paralysis through the inhibition of acetylcholine release at the neuromuscular junction. The seven BoNT serotypes (A-G) have been proposed to bind motor neurons via ganglioside-protein dual receptors. To date, the structure-function properties of BoNT/F host receptor interactions have not been resolved. Here, we report the crystal structures of the receptor binding domains (HCR) of BoNT/A and BoNT/F and the characterization of the dual receptors for BoNT/F. The overall polypeptide fold of HCR/A is essentially identical to the receptor binding domain of the BoNT/A holotoxin, and the structure of HCR/F is very similar to that of HCR/A, except for two regions implicated in neuronal binding. Solid phase array analysis identified two HCR/F binding glycans: ganglioside GD1a and oligosaccharides containing an N-acetyllactosamine core. Using affinity chromatography, HCR/F bound native synaptic vesicle glycoproteins as part of a protein complex. Deglycosylation of glycoproteins using alpha(1-3,4)-fucosidase, endo-beta-galactosidase, and PNGase F disrupted the interaction with HCR/F, while the binding of HCR/B to its cognate receptor, synaptotagmin I, was unaffected. These data indicate that the HCR/F binds synaptic vesicle glycoproteins through the keratan sulfate moiety of SV2. The interaction of HCR/F with gangliosides was also investigated. HCR/F bound specifically to gangliosides that contain alpha2,3-linked sialic acid on the terminal galactose of a neutral saccharide core (binding order GT1b = GD1a >> GM3; no binding to GD1b and GM1a). Mutations within the putative ganglioside binding pocket of HCR/F decreased binding to gangliosides, synaptic vesicle protein complexes, and primary rat hippocampal neurons. Thus, BoNT/F neuronal discrimination involves the recognition of ganglioside and protein (glycosylated SV2) carbohydrate moieties, providing a structural basis for the high affinity and specificity of BoNT/F for neurons.