Structure of dual receptor binding to botulinum neurotoxin B

Botulinum neurotoxins are highly toxic, and bind two receptors to achieve their high affinity and specificity for neurons. Here we present the first structure of a botulinum neurotoxin bound to both its receptors. We determine the 2.3 Å structure of a ternary complex of botulinum neurotoxin type B bound to both its protein receptor Synaptotagmin II and its ganglioside receptor GD1a. We show that there is no direct contact between the two receptors, and that the binding affinity towards Synaptotagmin II is not influenced by the presence of GD1a. The interactions of botulinum neurotoxin type B with the sialic acid 5 moiety of GD1a are important for the ganglioside selectivity. The structure demonstrates that the protein receptor and the ganglioside receptor occupy nearby but separate binding sites, thus providing two independent anchoring points

Structural divergence of paralogous S components from ECF-type ABC transporters

Energy coupling factor (ECF) proteins are ATP-binding cassette transporters involved in the import of micronutrients in prokaryotes. They consist of two nucleotide-binding subunits and the integral membrane subunit EcfT, which together form the ECF module and a second integral membrane subunit that captures the substrate (the S component). Different S components, unrelated in sequence and specific for different ligands, can interact with the same ECF module. Here, we present a high-resolution crystal structure at 2.1 Å of the biotin-specific S component BioY from Lactococcus lactis. BioY shares only 16% sequence identity with the thiamin-specific S component ThiT from the same organism, of which we recently solved a crystal structure. Consistent with the lack of sequence similarity, BioY and ThiT display large structural differences (rmsd = 5.1 Å), but the divergence is not equally distributed over the molecules: The S components contain a structurally conserved N-terminal domain that is involved in the interaction with the ECF module and a highly divergent C-terminal domain that binds the substrate. The domain structure explains howthe S components with large overall structural differences can interact with the same ECF module while at the same time specifically bind very different substrates with subnanomolar affinity. Solitary BioY (in the absence of the ECF module) is monomeric in detergent solution and binds D-biotin with a high affinity but does not transport the substrate across the membrane.

Ligand Binding and Crystal Structures of the Substrate-Binding Domain of the ABC Transporter OpuA

Background: The ABC transporter OpuA from Lactococcus lactis transports glycine betaine upon activation by threshold values of ionic strength. In this study, the ligand binding characteristics of purified OpuA in a detergent-solubilized state and of its substrate-binding domain produced as soluble protein (OpuAC) was characterized. Principal Findings: The binding of glycine betaine to purified OpuA and OpuAC (KD=4–6 µM) did not show any salt dependence or cooperative effects, in contrast to the transport activity. OpuAC is highly specific for glycine betaine and the related proline betaine. Other compatible solutes like proline and carnitine bound with affinities that were 3 to 4 orders of magnitude lower. The low affinity substrates were not noticeably transported by membrane-reconstituted OpuA. OpuAC was crystallized in an open (1.9 Å) and closed-liganded (2.3 Å) conformation. The binding pocket is formed by three tryptophans (Trp-prism) coordinating the quaternary ammonium group of glycine betaine in the closed-liganded structure. Even though the binding site of OpuAC is identical to that of its B. subtilis homolog, the affinity for glycine betaine is 4-fold higher. Conclusions: Ionic strength did not affect substrate binding to OpuA, indicating that regulation of transport is not at the level of substrate binding, but rather at the level of translocation. The overlap between the crystal structures of OpuAC from L.lactis and B.subtilis, comprising the classical Trp-prism, show that the differences observed in the binding affinities originate from outside of the ligand binding site.

Enterococcal PcfF Is a Ribbon-Helix-Helix Protein That Recruits the Relaxase PcfG Through Binding and Bending of the oriT Sequence

The conjugative plasmid pCF10 from Enterococcus faecalis encodes a Type 4 Secretion System required for plasmid transfer. The accessory factor PcfF and relaxase PcfG initiate pCF10 transfer by forming the catalytically active relaxosome at the plasmid’s origin-of-transfer (oriT) sequence. Here, we report the crystal structure of the homodimeric PcfF, composed of an N-terminal DNA binding Ribbon-Helix-Helix (RHH) domain and a C-terminal stalk domain. We identified key residues in the RHH domain that are responsible for binding pCF10’s oriT sequence in vitro, and further showed that PcfF bends the DNA upon oriT binding. By mutational analysis and pull-down experiments, we identified residues in the stalk domain that contribute to interaction with PcfG. PcfF variant proteins defective in oriT or PcfG binding attenuated plasmid transfer in vivo, but also suggested that intrinsic or extrinsic factors might modulate relaxosome assembly. We propose that PcfF initiates relaxosome assembly by binding oriT and inducing DNA bending, which serves to recruit PcfG as well as extrinsic factors necessary for optimal plasmid processing and engagement with the pCF10 transfer machine

Germination and seed traits variations among West African provenances of Moringa oleifera Lam. (Burkina Faso)

Moringa oleifera is a fruit species of economic interest to West African small holder growers. To generate benefits to poor rural communities, improved knowledge is needed on plantation management and selection of the most reliable seed sources. The aim of the study was to investigate variation in seed traits and germination rates among 12 provenances of M. oleifera from West Africa in the women gardening center named “Amicale des Forestières du Burkina Faso (AMIFOB)” located at Ouagadougou, Burkina Faso (12°7’32’’N, 01°40’24’’W). The authors conducted an analysis of variance, a principal component analysis on seed traits variables (length, thickness and weight) and germination rates at 5 and 12 days after seed sowing, and finally ascending hierarchical classification based on similarity indices. The results showed significant variations (P<0.05) among provenances in seed traits: Ouahigouya provenance in the Sahelian area of Burkina Faso had the largest and heaviest seeds. Germination rate was significantly different after 5 days for Ouahigouya, Ouagadougou, Koudougou, Dano and Tamale provenances (P<0.05). Five days after seed sowing, the Sahelian provenance (Ouahigouya) recorded the greatest and fastest germination rate of 63%. Correlation analyses revealed no significant links between germination rate in 5 and 12 days after sowing and seed sizes. Seed traits and germination rates did not show clear cut distinct groups between Sahelian, Sudanian and sub Equatorial provenances. This research output provides an evidence of the genetic variability among M. oleifera provenances and hence the potential for future tree improvement programme

A structural classification of substrate-binding proteins

Substrate-binding proteins (SBP) are associated with a wide variety of protein complexes. The proteins are part of ATP-binding cassette transporters for substrate uptake, ion gradient driven transporters, DNA-binding proteins, as well as channels and receptors from both pro- and eukaryotes. A wealth of structural and functional data is available on SBPs, with over 120 unique entries in the Protein Data Bank (PDB). Over a decade ago these proteins were divided into three structural classes, but based on the currently available wealth of structural data, we propose a new classification into six clusters, based on features of their three-dimensional structure.

Addendum to "A structural classification of substrate-binding proteins" [FEBS Lett. 584 (2010) 2606-2617]

Shortly after our publication, Dr. Gavin Thomas informed usthat four proteins (BugD, BugE, Bug27 and TT1099), belonging tothe TAXI and TTT families of TRAP-transporters[1–4], were miss-ing from our analysis. We then performed a DALI search[5] inaddition to the FFAS server and PSI-BLAST and detected 7 new pro-tein structures. Those are: BugD (PDB code: 2F5X), BugE (PDBcode: 2DVZ), Bug27 (PDB code: 2QPQ), TT1099 (PDB code: 1US5),a maltose-binding protein (TMBP (PDB code: 1EU8)) and two glu-tamate-binding proteins, GluR0 and DEBP (PDB codes: 1II5 and2VHA, respectively). The structural similarity analysis was redonewith in total 114 unique entries in the PDB, but the overall out-come was the same. The newly included proteins fall within thepreviously determined clusters: TMBP clustered with the other oli-gosaccharide-binding proteins in Cluster D, and the other six pro-teins fell in Cluster F

Structure and Mode of Peptide Binding of Pheromone Receptor PrgZ

We present the crystal structure of the pheromone receptor protein PrgZ from Enterococcus faecalis in complex with the heptapeptide cCF10 (LVTLVFV), which is used in signaling between conjugative recipient and donor cells. Comparison of PrgZ with homologous oligopeptide-binding proteins (AppA and OppA) explains the high specificity of PrgZ for hydrophobic heptapeptides versus the promiscuity of peptide binding in the homologous proteins.