We report a new, higher resolution NMR structure of ␣-bungarotoxin that defines the structure-determining disulfide core and ␤-sheet regions. We further report the NMR structure of the stoichiometric complex formed between ␣-bungarotoxin and a recombinantly expressed 19-mer peptide ( The nicotinic acetylcholine receptor (nAChR)1 (1) is a ligandgated ion channel that mediates excitatory transmission at the neuromuscular junction and at synapses in the central and peripheral nervous systems. It is the most intensely studied member of the ligand-gated ion channel superfamily and serves as a model for understanding the structure and function of related ion conducting channels including glycine, ␥-aminobutyric acid type A, ␥-aminobutyric acid type C, and type 3 serotonin receptors. nAChRs are pentameric complexes that assemble in the membrane with 5-fold symmetry. Each subunit contains an N-terminal extracellular domain about 200 amino acids long followed by four membrane-spanning segments (M1-M4) with an intracellular loop of variable length between M3 and M4. The second transmembrane region from each subunit contributes to the formation of the wall lining the channel pore. In muscle and Torpedo electric organ, the subunit composition is (␣1) 2 ␤␥␦ and (␣1) 2 ␤⑀␦ in embryonic and adult tissue, respectively (for review, see Ref.

The mitogen activated protein (MAP) kinase ERK2 contains recruitment sites that engage canonical and non-canonical motifs found in a variety of upstream kinases, regulating phosphatases and downstream targets. Interactions involving two of these sites, the D-recruitment site (DRS) and the F-recruitment site (FRS), have been shown to play a key role in signal transduction by ERK/MAP kinases. The dynamic nature of these recruitment events makes NMR uniquely suited to provide significant insight into these interactions. While NMR studies of kinases in general have been greatly hindered by their large size and complex dynamic behavior leading to the sub-optimal performance of standard methodologies, we have overcome these difficulties for inactive full-length ERK2 and obtained an acceptable level of backbone resonance assignments. This allowed a detailed investigation of the structural perturbations that accompany interactions involving both canonical and non-canonical recruitment events. No crystallographic information exists for the latter. We found that the chemical shift perturbations in inactive ERK2, indicative of structural changes in the presence of canonical and non-canonical motifs, are not restricted to the recruitment sites, but also involve the linker that connects the N- and C-lobes and, in most cases, a gatekeeper residue that is thought to exert allosteric control over catalytic activity. We also found that, even though the canonical motifs interact with the DRS utilizing both charge-charge and hydrophobic interactions, the non-canonical interactions primarily involve the latter. These results demonstrate the feasibility of solution NMR techniques for a comprehensive analysis of docking interactions in a full-length ERK/MAP kinase.

PDZ domains are ubiquitous peptide-binding modules that mediate protein-protein interactions in a wide variety of intracellular trafficking and localization processes. These include the pathways that regulate the membrane trafficking and endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel mutated in patients with cystic fibrosis. Correspondingly, a number of PDZ proteins have now been identified that directly or indirectly interact with the C terminus of CFTR. One of these is CAL, whose overexpression in heterologous cells directs the lysosomal degradation of WT-CFTR in a dose-dependent fashion and reduces the amount of CFTR found at the cell surface. Here, we show that RNA interference targeting endogenous CAL specifically increases cell-surface expression of the disease-associated ⌬F508-CFTR mutant and thus enhances transepithelial chloride currents in a polarized human patient bronchial epithelial cell line. We have reconstituted the CAL-CFTR interaction in vitro from purified components, demonstrating for the first time that the binding is direct and allowing us to characterize its components biochemically and biophysically. To test the hypothesis that inhibition of the binding site could also reverse CAL-mediated suppression of CFTR, a three-dimensional homology model of the CAL⅐CFTR complex was constructed and used to generate a CAL mutant whose binding pocket is correctly folded but has lost its ability to bind CFTR. Although produced at the same levels as wild-type protein, the mutant does not affect CFTR expression levels. Taken together, our data establish CAL as a candidate therapeutic target for correction of post-maturational trafficking defects in cystic fibrosis.