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The CFTR inhibitory factor (Cif) is an epoxide hydrolase (EH) virulence factor secreted by the bacterium Pseudomonas aeruginosa. Sequence alignments reveal a pattern of Cif-like substitutions that proved to be characteristic of a new subfamily of bacterial EHs. At the same time, crystallographic and mutagenetic data suggest that EH activity is required for virulence and that Cif's active site remains generally compatible with a canonical two-step EH mechanism. A hallmark of this mechanism is the formation of a covalent hydroxyalkyl-enzyme intermediate by nucleophilic attack. In several well-studied EHs, this intermediate has been captured at near stoichiometric levels, presumably reflecting rate-limiting hydrolysis. Here we show by mass spectrometry that only minimal levels of the expected intermediate can be trapped with WT Cif. In contrast, substantial amounts of intermediate are recovered from an active-site mutant (Cif-E153Q) that selectively targets the second, hydrolytic release step. Utilizing Cif-E153Q and a previously reported nucleophile mutant (Cif-D129S), we then captured Cif in the substrate-bound, hydroxyalkyl-intermediate, and product-bound states for 1,2-epoxyhexane, yielding the first crystallographic snapshots of an EH at these key stages along the reaction coordinate. Taken together, our data illuminate the proposed two-step hydrolytic mechanism of a new class of bacterial virulence factor. They also suggest that the failure of WT Cif to accumulate a covalent hydroxyalkyl-enzyme intermediate reflects an active-site chemistry in which hydrolysis is no longer the rate-limiting step, a noncanonical kinetic regime that may explain similar observations with a number of other EHs.

AMPA receptors are glutamate-gated ion channels that are essential mediators of synaptic signals in the central nervous system. They form tetramers that are assembled as combinations of subunits GluR1-4, each of which contains a ligand-binding domain (LBD). Crystal structures of the GluR2 LBD have revealed an agonist-binding cleft, which is located between two lobes and which acts like a Venus flytrap. In general, agonist efficacy is correlated with the extent of cleft closure. However, recent observations show that cleft closure is not the sole determinant of the relative efficacy for glutamate receptors. In addition, these studies have focused on the GluR2 subunit, which is the specific target of a physiologically important RNA-editing modification in vivo. We therefore sought to test the generality of the cleft closure-efficacy correlation for other AMPA-R subunits. Here, we present crystal structures of the GluR4(flip) LBD in complex with both full and partial agonists. As for GluR2, both agonists stabilize a closed-cleft conformation, and the partial agonist induces a smaller cleft closure than the full agonist. However, a detailed analysis of LBD-kainate interactions reveals the importance of subtle backbone conformational changes in the ligand-binding pocket in determining the magnitude of agonist-associated conformational changes. Furthermore, the GluR4 subunit exhibits a different correlation between receptor activation and LBD cleft closure than does GluR2.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial chloride channel mutated in patients with cystic fibrosis. Its expression and functional interactions in the apical membrane are regulated by several PDZ (PSD-95, discs large, zonula occludens-1) proteins, which mediate protein-protein interactions, typically by binding C-terminal recognition motifs. In particular, the CFTR-associated ligand (CAL) limits cell-surface levels of the most common disease-associated mutant DeltaF508-CFTR. CAL also mediates degradation of wild-type CFTR, targeting it to lysosomes following endocytosis. Nevertheless, wild-type CFTR survives numerous cycles of uptake and recycling. In doing so, how does it repeatedly avoid CAL-mediated degradation? One mechanism may involve competition between CAL and other PDZ proteins including Na (+)/H (+) exchanger-3 regulatory factors 1 and 2 (NHERF1 and NHERF2), which functionally stabilize cell-surface CFTR. Thus, to understand the biochemical basis of WT-CFTR persistence, we need to know the relative affinities of these partners. However, no quantitative binding data are available for CAL or the individual NHERF2 PDZ domains, and published estimates for the NHERF1 PDZ domains conflict. Here we demonstrate that the affinity of the CAL PDZ domain for the CFTR C-terminus is much weaker than those of NHERF1 and NHERF2 domains, enabling wild-type CFTR to avoid premature entrapment in the lysosomal pathway. At the same time, CAL's affinity is evidently sufficient to capture and degrade more rapidly cycling mutants, such as DeltaF508-CFTR. The relatively weak affinity of the CAL:CFTR interaction may provide a pharmacological window for stabilizing rescued DeltaF508-CFTR in patients with cystic fibrosis.

The association of the cystic fibrosis transmembrane regulator (CFTR) with two PDZ-containing molecular scaffolds (CAL and EBP50) plays an important role in CFTR trafficking and membrane maintenance. The CFTR-molecular scaffold interaction is mediated by the association of the C-terminus of the transmembrane regulator with the PDZ domains. Here, we characterize the structure and dynamics of the PDZ of CAL and the complex formed with CFTR employing high-resolution NMR. On the basis of NMR relaxation data, the alpha2 helix as well as the beta2-beta3 loop of CAL PDZ domain undergoes rapid dynamics. Molecular dynamics simulations suggest a concerted motion between the alpha2 helix and the beta1-beta2 and beta2-beta3 loops, elements which define the binding pocket, suggesting that dynamics may play a role in PDZ-ligand specificity. The C-terminus of CFTR binds to CAL with the final four residues (-D(-)(3)-T-R-L(0)) within the canonical PDZ-binding motif, between the beta2 strand and the alpha2 helix. The R(-)(1) and D(-)(3) side chains make a number of contacts with the PDZ domain; many of these interactions differ from those in the CFTR-EBP50 complex, suggesting sites that can be targeted in the development of PDZ-selective inhibitors that may help modulate CFTR function.

V-ATPases are multisubunit membrane protein complexes that use the energy provided by ATP hydrolysis to generate a proton gradient across various intracellular and plasma membranes. In doing so, they maintain an acidic pH in the lumen of intracellular organelles and acidify extracellular milieu to support specific cellular functions. V-ATPases are structurally similar to the F1F0-ATP synthase, with an intrinsic membrane domain (V0) and an extrinsic peripheral domain (V1) joined by several connecting elements. To gain a clear functional understanding of the catalytic mechanism, and of the stability requirements for regulatory processes in the enzyme, a clear topology of the enzyme has to be established. In particular, the composition and arrangement of the peripheral stator subunits must be firmly settled, as these play specific roles in catalysis and regulation. We have designed a strategy allowing us to coexpress different combinations of these subunits to delineate specific interactions. In this study, we report the interaction between the peripheral stator EG complex and subunits C and H of the V-ATPase from the yeast Saccharomyces cerevisae. A combination of analytical gel filtration, native gel electrophoresis, and ultracentrifugation analysis allowed us to ascertain the homogeneity and molar mass of the purified EGC complex as well as of the EG complex, supporting the formation of 1:1(:1) stoichiometric complexes. The EGC complex can be formed in vitro by combining equimolar amounts of subunit C and the EG subcomplex and results most likely from the initial interaction between subunits E and C.

Upon agonist binding, the bilobate ligand-binding domains of the ionotropic glutamate receptors (iGluR) undergo a cleft closure whose magnitude correlates broadly with the efficacy of the agonist. AMPA (alpha-amino-5-methyl-3-hydroxy-4-isoxazolepropionic acid) and kainate are nonphysiological agonists that distinguish between subsets of iGluR. Kainate acts with low efficacy at AMPA receptors. Here we report that the structure-based mutation L651V converts the GluR4 AMPA receptor into a dual-specificity AMPA/kainate receptor fully activated by both agonists. To probe the stereochemical basis of partial agonism, we have also investigated the correlation between agonist efficacy and a series of vibrational and fluorescence spectroscopic signals of agonist binding to the corresponding wild-type and mutant GluR4 ligand-binding domains. Two signals track the extent of channel activation: the maximal change in intrinsic tryptophan fluorescence and the environment of the single non-disulfide bonded C426, which appears to probe the strength of interactions with the ligand alpha-amino group. Both of these signals arise from functional groups that are poised to detect changes in the extent of channel cleft closure and thus provide additional information about the coupling between conformational changes in the ligand-binding domain and activation of the intact receptor.

We have expressed, purified, and characterized glutamate receptor ion channels (GluR) assembled as homomers of the subunit GluRB. For the first time, single-milligram quantities of biochemically homogeneous GluR have been obtained. The protein exhibits the expected pharmacological profile and a high specific activity for ligand binding. Density-gradient centrifugation reveals a uniform oligomeric assembly and a molecular mass suggesting that the channel is a tetramer. On the basis of electron microscopic images, the receptor appears to form an elongated structure that is visualized in several orientations. The molecular dimensions of the molecule are approximately 11 x 14 x 17 nm, and solvent-accessible features can be seen; these may contribute to formation of the ion-conducting pathway of the channel. The channel dimensions are consistent with an overall 2-fold symmetric assembly, suggesting that the tetrameric receptor may be a dimer of dimers.

Fourier transform infrared spectroscopy was used to investigate ligand-protein interactions in the ligand-binding domain of the GluR4 glutamate receptor subunit. Glutamate binding induces more extensive secondary structural changes in the ligand-binding domain than does kainate binding. Glutamate also alters the hydrogen bonding strength of the single free cysteine side chain in the domain, while kainate does not. On the other hand, the interaction of a binding site arginine residue with kainate appears to be stronger than that with glutamate. These results identify chemical and structural differences that may explain the different functional characteristics of the two agonists acting on ionotropic glutamate receptors. In doing so, they complement and extend recent crystallographic structures of the ligand-binding domain.

Solution scattering studies were performed on a ligand-binding domain (S1S2) of a glutamate receptor ion channel (GluR) in order to study GluR-binding and signal-transduction mechanisms. The core of the ligand-binding domain is homologous to prokaryotic periplasmic binding proteins (PBP), whose binding mechanism involves a dramatic cleft closure: the "Venus flytrap". Several models of GluR function have proposed that a similar cleft closure is induced by agonist binding. We have directly tested this putative functional homology by measuring the radius of gyration of S1S2 in the presence and absence of saturating concentrations of agonists. In contrast to the PBP, S1S2 shows no reduction in radius of gyration upon agonist binding, excluding a comparably large conformational change. Furthermore, we determined an ab initio molecular envelope for our S1S2 construct, which also contains the peptides that connect the PBP homology core to the three transmembrane domains and to an N-terminal domain. By fitting an atomic model of the ligand-binding domain core to the envelope of our extended construct, we were able to establish the likely position of these connecting peptides. Their positions relative to one another and to the expected sites of an agonist-induced conformational change suggest that ion channel gating and desensitization may involve more subtle and complex mechanisms than have been assumed based on the structural homology to the PBP.