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Mature TGF-β isoforms, which are covalent dimers, signal by binding to three types of cell surface receptors, the type I, II and III TGF-β receptors. A complex composed of the TGF-β ligand and the type I and II receptors is required for signaling. The type II receptor is responsible for recruiting TGF-β into the heteromeric ligand/type I receptor/type II receptor complex. The purpose of this study was to test for the extent that avidity contributes to receptor affinity. Using a surface plasmon resonance (SPR)-based biosensor (the BIACORE), we captured the extracellular domain of the type II receptor (TβRIIED) at the biosensor surface in an oriented and stable manner by using a de novo designed coiled-coil (E/K coil) heterodimerizing system. We characterized the kinetics of binding of three TGF-β isoforms to this immobilized TβRIIED. The results demonstrate that the stoichiometry of TGF-β binding to TβRIIED was one dimeric ligand to two receptors. All three TGF-β isoforms had rapid and similar association rates, but different dissociation rates, which resulted in the equilibrium dissociation constants being approximately 5 pM for the TGF-β1 and -β3 isoforms, and 5 nM for the TGF-β2 isoform. Since these apparent affinities are at least four orders of magnitude higher than those determined when TGF-β was immobilized, and are close to those determined for TβRII at the cell surface, we suggest that avidity contributes significantly to high affinity receptor binding both at the biosensor and cell surfaces. Finally, we demonstrated that the coiled-coil immobilization approach does not require the purification of the captured protein, making it an attractive tool for the rapid study of any protein–protein interaction. [Copyright &y& Elsevier]

A member of the family of RTX toxins, Escherichia coli haemolysin A, is secreted from Gram-negative bacteria. It carries a C-terminal secretion signal of approximately 50 residues, targeting the protein to the secretion or translocation complex, in which the ABC-transporter HlyB is a central element. We have purified the nucleotide-binding domain of HlyB (HlyB–NBD) and a C-terminal 23 kDa fragment of HlyA plus the His-tag (HlyA1), which contains the secretion sequence. Employing surface plasmon resonance, we were able to demonstrate that the HlyB–NBD and HlyA1 interact with a KD of approximately 4 μM. No interaction was detected between the HlyA fragment and unrelated NBDs, OpuAA, involved in import of osmoprotectants, and human TAP1–NBD, involved in the export of antigenic peptides. Moreover, a truncated version of HlyA1, lacking the secretion signal, failed to interact with the HlyB–NBD. In addition, we showed that ATP accelerated the dissociation of the HlyB–NBD/HlyA1 complex. Taking these results together, we propose a model for an early stage of initiation of secretion in vivo, in which the NBD of HlyB, specifically recognizes the C terminus of the transport substrate, HlyA, and where secretion is initiated by subsequent displacement of HlyA from HlyB by ATP. [Copyright &y& Elsevier]

Crotoxin, a potent neurotoxin from the South American rattlesnake Crotalus durissus terrificus, is a heterodimeric phospholipase A2 (EC 3.1.1.4), which blocks the release of acetylcholine from peripheral neurons. We previously have suggested the existence of a 48 kDa crotoxin-binding protein in the presynaptic membranes of the electric organ of Torpedo marmorata. Here, we report the purification and characterization of this protein that we called the crotoxin acceptor protein from Torpedo (CAPT). The membranes of electric organs from Torpedo were solubilized with a detergent (4% (w/v) Triton X-100) and CAPT was isolated by affinity chromatography on a crotoxin column. SDS–PAGE showed that the purified protein was homogeneous and cross-linking studies with radioiodinated crotoxin confirmed that it had retained its toxin-binding properties. The purified CAPT has similar molecular mass as crocalbin, a crotoxin-binding protein isolated from porcine brains, yet anti-crocalbin antiserum failed to recognize CAPT. Surface plasmon resonance biosensor technology was used to measure the specific interaction between crotoxin and solubilized CAPT. Using this method, it was possible to follow CAPT throughout the purification procedure. As well, an apparent dissociation constant (Kdapp) of 3.4 nM was calculated for the interaction of pure CAPT and crotoxin from the dissociation rate constant (koff=1.2×10−2 s−1) and the association rate constant (kon=3.5×106 M−1s−1). [Copyright &y& Elsevier]