Orientation of alpha-neurotoxin at the subunit interfaces of the nicotinic acetylcholine receptor

The alpha-neurotoxins are three-fingered peptide toxins that bind selectively at interfaces formed by the alpha subunit and its associating subunit partner, gamma, delta, or epsilon of the nicotinic acetylcholine receptor. Because the alpha-neurotoxin from Naja mossambica mossambica I shows an unusual selectivity for the alpha gamma and alpha delta over the alpha epsilon subunit interface, residue replacement and mutant cycle analysis of paired residues enabled us to identify the determinants in the gamma and delta sequences governing alpha-toxin recognition. To complement this approach, we have similarly analyzed residues on the alpha subunit face of the binding site dictating specificity for alpha-toxin. Analysis of the alpha gamma interface shows unique pairwise interactions between the charged residues on the alpha-toxin and three regions on the alpha subunit located around residue Asp(99), between residues Trp(149) and Val(153), and between residues Trp(187) and Asp(200). Substitutions of cationic residues at positions between Trp(149) and Val(153) markedly reduce the rate of alpha-toxin binding, and these cationic residues appear to be determinants in preventing alpha-toxin binding to alpha 2, alpha 3, and alpha 4 subunit containing receptors. Replacement of selected residues in the alpha-toxin shows that Ser(8) on loop I and Arg(33) and Arg(36) on the face of loop II, in apposition to loop I, are critical to the alpha-toxin for association with the alpha subunit. Pairwise mutant cycle analysis has enabled us to position residues on the concave face of the three alpha-toxin loops with respect to alpha and gamma subunit residues in the alpha-toxin binding site. Binding of NmmI alpha-toxin to the alpha gamma interface appears to have dominant electrostatic interactions not seen at the alpha delta interface.     

Residues in the epsilon subunit of the nicotinic acetylcholine receptor interact to confer selectivity of waglerin-1 for the alpha-epsilon subunit interface site

Waglerin-1 (Wtx-1) is a 22-amino acid peptide that competitively antagonizes muscle nicotinic acetylcholine receptors (nAChRs). Previous work demonstrated that Wtx-1 binds to mouse nAChRs with higher affinity than receptors from rats or humans, and distinguished residues in alpha and epsilon subunits that govern the species selectivity. These studies also showed that Wtx-1 binds selectively to the alpha-epsilon binding site with significantly higher affinity than to the alpha-delta binding site. Here we identify residues at equivalent positions in the epsilon, gamma, and delta subunits that govern Wtx-1 selectivity for one of the two binding sites on the nAChR pentamer. Using a series of chimeric and point mutant subunits, we show that residues Gly-57, Asp-59, Tyr-111, Tyr-115, and Asp-173 of the epsilon subunit account predominantly for the 3700-fold higher affinity of the alpha-epsilon site relative to that of the alpha-gamma site. Similarly, we find that residues Lys-34, Gly-57, Asp-59, and Asp-173 account predominantly for the high affinity of the alpha-epsilon site relative to that of the alpha-delta site. Analysis of combinations of point mutations reveals that Asp-173 in the epsilon subunit is required together with the remaining determinants in the epsilon subunit to achieve Wtx-1 selectivity. In particular, Lys-34 interacts with Asp-173 to confer high affinity, resulting in a DeltaDeltaG(INT) of -2.3 kcal/mol in the epsilon subunit and a DeltaDeltaG(INT) of -1.3 kcal/mol in the delta subunit. Asp-173 is part of a nonhomologous insertion not found in the acetylcholine binding protein structure. The key role of this insertion in Wtx-1 selectivity indicates that it is proximal to the ligand binding site. We use the binding and interaction energies for Wtx-1 to generate structural models of the alpha-epsilon, alpha-gamma, and alpha-delta binding sites containing the nonhomologous insertion.     

Pairwise electrostatic interactions between alpha-neurotoxins and gamma, delta, and epsilon subunits of the nicotinic acetylcholine receptor

alpha-Neurotoxins bind with high affinity to alpha-gamma and alpha-delta subunit interfaces of the nicotinic acetylcholine receptor. Since this high affinity complex likely involves a van der Waals surface area of approximately 1200 A(2) and 25-35 residues on the receptor surface, analysis of side chains should delineate major interactions and the orientation of bound alpha-neurotoxin. Three distinct regions on the gamma subunit, defined by Trp(55), Leu(119), Asp(174), and Glu(176), contribute to alpha-toxin affinity. Of six charge reversal mutations on the three loops of Naja mossambica mossambica alpha-toxin, Lys(27) --> Glu, Arg(33) --> Glu, and Arg(36) --> Glu in loop II reduce binding energy substantially, while mutations in loops I and III have little effect. Paired residues were analyzed by thermodynamic mutant cycles to delineate electrostatic linkages between the six alpha-toxin charge reversal mutations and three key residues on the gamma subunit. Large coupling energies were found between Arg(33) at the tip of loop II and gammaLeu(119) (-5.7 kcal/mol) and between Lys(27) and gammaGlu(176) (-5.9 kcal/mol). gammaTrp(55) couples strongly to both Arg(33) and Lys(27), whereas gammaAsp(174) couples minimally to charged alpha-toxin residues. Arg(36), despite strong energetic contributions, does not partner with any gamma subunit residues, perhaps indicating its proximity to the alpha subunit. By analyzing cationic, neutral and anionic residues in the mutant cycles, interactions at gamma176 and gamma119 can be distinguished from those at gamma55.     

Identification of residues at the alpha and epsilon subunit interfaces mediating species selectivity of Waglerin-1 for nicotinic acetylcholine receptors.

Waglerin-1 (Wtx-1) is a 22-amino acid peptide that is a competitive antagonist of the muscle nicotinic receptor (nAChR). We find that Wtx-1 binds 2100-fold more tightly to the alpha-epsilon than to the alpha-delta binding site interface of the mouse nAChR. Moreover, Wtx-1 binds 100-fold more tightly to the alpha-epsilon interface from mouse nAChR than that from rat or human sources. Site-directed mutagenesis of residues differing in the extracellular domains of rat and mouse epsilon subunits indicates that residues 59 and 115 mediate the species difference in Wtx-1 affinity. Mutation of residues 59 (Asp in mouse, Glu in rat epsilon) and 115 (Tyr in mouse, Ser in rat epsilon) converts Wtx-1 affinity for the alpha-epsilon interface of one species to that of the other species. Studies of different mutations at position 59 indicate both steric and electrostatic contributions to Wtx-1 affinity, whereas at position 115, both aromatic and polar groups contribute to affinity. The human nAChR also has lower affinity for Wtx-1 than mouse nAChR, but unlike rat nAChR, residues in both alpha and epsilon subunits mediate the affinity difference. In human nAChR, polar residues (Ser-187 and Thr-189) confer low affinity, whereas in mouse nAChR aromatic residues (Trp-187 and Phe-189) confer high affinity. The overall results show that non-conserved residues at the nAChR binding site, although not crucial for activation by ACh, govern the potency of neuromuscular toxins.     

The glutamate switch of bacteriophage T7 DNA helicase: role in coupling nucleotide triphosphate (NTP) and DNA binding to NTP hydrolysis

The DNA helicase encoded by gene 4 of bacteriophage T7 forms a hexameric ring in the presence of dTTP, allowing it to bind DNA in its central core. The oligomerization also creates nucleotide-binding sites located at the interfaces of the subunits. DNA binding stimulates the hydrolysis of dTTP but the mechanism for this two-step control is not clear. We have identified a glutamate switch, analogous to the glutamate switch found in AAA+ enzymes that couples dTTP hydrolysis to DNA binding. A crystal structure of T7 helicase shows that a glutamate residue (Glu-343), located at the subunit interface, is positioned to catalyze a nucleophilic attack on the γ-phosphate of a bound nucleoside 5'-triphosphate. However, in the absence of a nucleotide, Glu-343 changes orientation, interacting with Arg-493 on the adjacent subunit. This interaction interrupts the interaction of Arg-493 with Asn-468 of the central β-hairpin, which in turn disrupts DNA binding. When Glu-343 is replaced with glutamine the altered helicase, unlike the wild-type helicase, binds DNA in the presence of dTDP. When both Arg-493 and Asn-468 are replaced with alanine, dTTP hydrolysis is no longer stimulated in the presence of DNA. Taken together, these results suggest that the orientation of Glu-343 plays a key role in coupling nucleotide hydrolysis to the binding of DNA.     

Interaction of p72syk with the gamma and beta subunits of the high-affinity receptor for immunoglobulin E, Fc epsilon RI.

Activation of protein tyrosine kinases is one of the initial events following aggregation of the high-affinity receptor for immunoglobulin E (Fc epsilon RI) on RBL-2H3 cells, a model mast cell line. The protein tyrosine kinase p72syk (Syk), which contains two Src homology 2 (SH2) domains, is activated and associates with phosphorylated Fc epsilon RI subunits after receptor aggregation. In this report, we used Syk SH2 domains, expressed in tandem or individually, as fusion proteins to identify Syk-binding proteins in RBL-2H3 lysates. We show that the tandem Syk SH2 domains selectively associate with tyrosine-phosphorylated forms of the gamma and beta subunits of Fc epsilon RI. The isolated carboxy-proximal SH2 domain exhibited a significantly higher affinity for the Fc epsilon RI subunits than did the amino-proximal domain. When in tandem, the Syk SH2 domains showed enhanced binding to phosphorylated gamma and beta subunits. The conserved tyrosine-based activation motifs contained in the cytoplasmic domains of the gamma and beta subunits, characterized by two YXXL/I sequences in tandem, represent potential high-affinity binding sites for the dual SH2 domains of Syk. Peptide competition studies indicated that Syk exhibits a higher affinity for the phosphorylated tyrosine activation motif of the gamma subunit than for that of the beta subunit. In addition, we show that Syk is the major protein in RBL-2H3 cells that is affinity isolated with phosphorylated peptides corresponding to the phosphorylated gamma subunit motif. These data suggest that Syk associates with the gamma subunit of the high-affinity receptor for immunoglobulin E through an interaction between the tandem SH2 domains of SH2 domains of Syk and the phosphorylated tyrosine activation motif of the gamma subunit and that Syk may be the major signaling protein that binds to Fc epsilon RI tyrosine activation motif of the gamma subunit and that Syk may be the major signaling protein that binds to Dc epsilon tyrosine activation motifs in RBL-2H3 cells.     

Characterization of an adult muscle acetylcholine receptor subunit by expression in fibroblasts.

To study the functional and structural roles of the epsilon subunit in adult muscle acetylcholine receptor (AChR), we have co-expressed the alpha and epsilon subunits of the mouse receptor in transfected fibroblasts. Ligand binding studies suggest that association of epsilon with alpha subunit results in a lower association rate constant for 125I-labeled alpha-bungarotoxin binding than that of the unassembled alpha subunit, approaching that for toxin binding to the AChR. Furthermore, alpha epsilon complexes contain high affinity binding sites for competitive antagonists and agonists not present in the unassembled alpha subunit, but similar to one of the two nonequivalent binding sites in the adult AChR. Structural analysis of alpha epsilon complexes by sucrose gradient velocity centrifugation suggests that some of the complexes formed are trimers or tetramers of alpha and epsilon subunits. Comparison of these data with those previously obtained for alpha gamma complexes suggests that gamma and epsilon have homologous functional roles and identical structural positions in the fetal and adult AChRs, respectively.     

Binding of alpha-bungarotoxin to synthetic peptides corresponding to residues 173-204 of the alpha subunit of Torpedo, calf, and human acetylcholine receptor and restoration of high-affinity binding by sodium dodecyl sulfate

In order to investigate structure-function relationships of a segment of the acetylcholine receptor alpha subunit, binding of alpha-bungarotoxin to synthetic peptides corresponding to residues 173-204 of Torpedo, calf, and human alpha subunits was compared using a solid-phase radioassay. The affinities of 125I-alpha-bungarotoxin for the calf and human peptides were 15- and 150-fold less, respectively, than for the Torpedo peptide. On the basis of nonconservative substitutions in the calf and human sequences, aromatic residues (Tyr-181, Trp-187, and Tyr-189) are important for the higher affinity binding of the Torpedo peptide. Substitution of negatively charged Glu-180 with uncharged Gln in the calf peptide did not significantly affect toxin binding, indicating Glu-180 alone does not comprise the anionic subsite on the receptor to which the cationic quaternary ammonium groups of cholinergic agents bind. d-Tubocurarine competed toxin binding to the modified calf 32-mer which lacks Glu-180 and Asp-195 present in Torpedo. Thus, the negative subsite could be formed by another negatively charged residue or by more than one amino acid side chain. It is possible that the positive charges on cholinergic ligands are countered by a negative electrostatic potential provided by polar groups, such as the hydroxyl group of tyrosine, present on several residues in this region, and the negative charges present on any of residues 175, 180, 195, or 200. Equilibrium saturation binding of alpha-bungarotoxin to Torpedo peptide 173-204 revealed a minor binding component with an apparent KD of 4.2 nM and a major component with a KD of 63 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

The epsilon subunit of Escherichia coli coupling factor 1 is required for its binding to the cytoplasmic membrane.

The coupling factor, F1-ATPase of Escherichia coli (ECF1) contains five different subunits, alpha, beta, gamma, delta, and epsilon. Properties of delta-deficient ECF1 have previously been described. F1-ATPase containing only the alpha, beta, and gamma subunits was prepared from E. coli by passage of delta-deficient ECF1 through an affinity column containing immobilized antibodies to the epsilon subunit. The delta, epsilon-deficient enzyme has normal ATPase activity but cannot bind to ECF1-depleted membrane vesicles. Both the delta and epsilon subunits are required for the binding of delta, epsilon-deficient ECF1 to membranes and the restoration of oxidative phosphorylation. Either delta or epsilon will bind to the deficient enzyme to form a four-subunit complex. Neither four-subunit enzyme binds to depleted membranes. The epsilon subunit, does, however, slightly improve the binding affinity between delta and delta-deficient enzyme suggesting a possible interaction between the two subunits. Neither subunit binds to trypsin-treated ECF1, which contains only the alpha and beta subunits. A role for gamma in the binding of epsilon to F1 is suggested. epsilon does not bind to ECF1-depleted membranes. Therefore, the in vitro reconstitution of depleted membranes requires an initial complex formation between epsilon and the rest of ECF1 prior to membrane attachment. Reconstitution experiments indicate that only one epsilon is required per functional ECF1 molecule.     

Localization of agonist and competitive antagonist binding sites on nicotinic acetylcholine receptors

Identification of all residues involved in the recognition and binding of cholinergic ligands (e.g. agonists, competitive antagonists, and noncompetitive agonists) is a primary objective to understand which structural components are related to the physiological function of the nicotinic acetylcholine receptor (AChR). The picture for the localization of the agonist/competitive antagonist binding sites is now clearer in the light of newer and better experimental evidence. These sites are located mainly on both alpha subunits in a pocket approximately 30-35 A above the surface membrane. Since both alpha subunits are identical, the observed high and low affinity for different ligands on the receptor is conditioned by the interaction of the alpha subunit with other non-alpha subunits. This molecular interaction takes place at the interface formed by the different subunits. For example, the high-affinity acetylcholine (ACh) binding site of the muscle-type AChR is located on the alphadelta subunit interface, whereas the low-affinity ACh binding site is located on the alphagamma subunit interface. Regarding homomeric AChRs (e.g. alpha7, alpha8, and alpha9), up to five binding sites may be located on the alphaalpha subunit interfaces. From the point of view of subunit arrangement, the gamma subunit is in between both alpha subunits and the delta subunit follows the alpha aligned in a clockwise manner from the gamma. Although some competitive antagonists such as lophotoxin and alpha-bungarotoxin bind to the same high- and low-affinity sites as ACh, other cholinergic drugs may bind with opposite specificity. For instance, the location of the high- and the low-affinity binding site for curare-related drugs as well as for agonists such as the alkaloid nicotine and the potent analgesic epibatidine (only when the AChR is in the desensitized state) is determined by the alphagamma and the alphadelta subunit interface, respectively. The case of alpha-conotoxins (alpha-CoTxs) is unique since each alpha-CoTx from different species is recognized by a specific AChR type. In addition, the specificity of alpha-CoTxs for each subunit interface is species-dependent.In general terms we may state that both alpha subunits carry the principal component for the agonist/competitive antagonist binding sites, whereas the non-alpha subunits bear the complementary component. Concerning homomeric AChRs, both the principal and the complementary component exist on the alpha subunit. The principal component on the muscle-type AChR involves three loops-forming binding domains (loops A-C). Loop A (from mouse sequence) is mainly formed by residue Y(93), loop B is molded by amino acids W(149), Y(152), and probably G(153), while loop C is shaped by residues Y(190), C(192), C(193), and Y(198). The complementary component corresponding to each non-alpha subunit probably contributes with at least four loops. More specifically, the loops at the gamma subunit are: loop D which is formed by residue K(34), loop E that is designed by W(55) and E(57), loop F which is built by a stretch of amino acids comprising L(109), S(111), C(115), I(116), and Y(117), and finally loop G that is shaped by F(172) and by the negatively-charged amino acids D(174) and E(183). The complementary component on the delta subunit, which corresponds to the high-affinity ACh binding site, is formed by homologous loops. Regarding alpha-neurotoxins, several snake and alpha-CoTxs bear specific residues that are energetically coupled with their corresponding pairs on the AChR binding site. The principal component for snake alpha-neurotoxins is located on the residue sequence alpha1W(184)-D(200), which includes loop C. In addition, amino acid sequence 55-74 from the alpha1 subunit (which includes loop E), and residues gammaL(119) (close to loop F) and gammaE(176) (close to loop G) at the low-affinity binding site, or deltaL(121) (close to the homologous region of loop G) at the high-affinity binding site, are i     

Insights into the conformational equilibria of maltose-binding protein by analysis of high affinity mutants

The affinity of maltose-binding protein (MBP) for maltose and related carbohydrates was greatly increased by removal of groups in the interface opposite the ligand binding cleft. The wild-type protein has a KD of 1200 nM for maltose; mutation of residues Met-321 and Gln-325, both to alanine, resulted in a KD for maltose of 70 nM; deletion of 4 residues, Glu-172, Asn-173, Lys-175, and Tyr-176, which are part of a poorly ordered loop, results in a KD for maltose of 110 nM. Combining the mutations yields an increased affinity for maltodextrins and a KD of 6 nM for maltotriose. Comparison of ligand binding by the mutants, using surface plasmon resonance spectroscopy, indicates that decreases in the off-rate are responsible for the increased affinity. Small-angle x-ray scattering was used to demonstrate that the mutations do not significantly affect the solution conformation of MBP in either the presence or absence of maltose. The crystal structures of selected mutants showed that the mutations do not cause significant structural changes in either the closed or open conformation of MBP. These studies show that interactions in the interface opposite the ligand binding cleft, which we term the "balancing interface," are responsible for modulating the affinity of MBP for its ligand. Our results are consistent with a model in which the ligand-bound protein alternates between the closed and open conformations, and removal of interactions in the balancing interface decreases the stability of the open conformation, without affecting the closed conformation.     

The binding site of acetylcholine receptor as visualized in the X-Ray structure of a complex between alpha-bungarotoxin and a mimotope peptide.

We have determined the crystal structure at 1.8 A resolution of a complex of alpha-bungarotoxin with a high affinity 13-residue peptide that is homologous to the binding region of the alpha subunit of acetylcholine receptor. The peptide fits snugly to the toxin and adopts a beta hairpin conformation. The structures of the bound peptide and the homologous loop of acetylcholine binding protein, a soluble analog of the extracellular domain of acetylcholine receptor, are remarkably similar. Their superposition indicates that the toxin wraps around the receptor binding site loop, and in addition, binds tightly at the interface of two of the receptor subunits where it inserts a finger into the ligand binding site, thus blocking access to the acetylcholine binding site and explaining its strong antagonistic activity.     

Structural analysis of the regulatory dithiol-containing domain of the chloroplast ATP synthase gamma subunit

The gamma subunit of the F1 portion of the chloroplast ATP synthase contains a critically placed dithiol that provides a redox switch converting the enzyme from a latent to an active ATPase. The switch prevents depletion of intracellular ATP pools in the dark when photophosphorylation is inactive. The dithiol is located in a special regulatory segment of about 40 amino acids that is absent from the gamma subunits of the eubacterial and mitochondrial enzymes. Site-directed mutagenesis was used to probe the relationship between the structure of the gamma regulatory segment and its function in ATPase regulation via its interaction with the inhibitory epsilon subunit. Mutations were designed using a homology model of the chloroplast gamma subunit based on the analogous structures of the bacterial and mitochondrial homologues. The mutations included (a) substituting both of the disulfide-forming cysteines (Cys199 and Cys205) for alanines, (b) deleting nine residues containing the dithiol, (c) deleting the region distal to the dithiol (residues 224-240), and (d) deleting the entire segment between residues 196 and 241 with the exception of a small spacer element, and (e) deleting pieces from a small loop segment predicted by the model to interact with the dithiol domain. Deletions within the dithiol domain and within parts of the loop segment resulted in loss of redox control of the ATPase activity of the F1 enzyme. Deleting the distal segment, the whole regulatory domain, or parts of the loop segment had the additional effect of reducing the maximum extent of inhibition obtained upon adding the epsilon subunit but did not abolish epsilon binding. The results suggest a mechanism by which the gamma and epsilon subunits interact with each other to induce the latent state of the enzyme.     

A loop involving catalytic chain residues 230-245 is essential for the stabilization of both allosteric forms of Escherichia coli aspartate transcarbamylase

The allosteric transition of Escherichia coli aspartate transcarbamylase involves significant alterations in structure at both the quaternary and tertiary levels. On the tertiary level, the 240s loop (residues 230-245 of the catalytic chain) repositions, influencing the conformation of Arg-229, a residue near the aspartate binding site. In the T state, Arg-229 is bent out of the active site and may be stabilized in this position by an interaction with Glu-272. In the R state, the conformation of Arg-229 changes, allowing it to interact with the beta-carboxylate of aspartate, and is stabilized in this position by a specific interaction with Glu-233. In order to ascertain the function of Arg-229, Glu-233, and Glu-272 in the catalytic and cooperative interactions of the enzyme, three mutant enzymes were created by site-specific mutagenesis. Arg-229 was replaced by Ala, while both Glu-233 and Glu-272 were replaced by Ser. The Arg-229----Ala and Glu-233----Ser enzymes exhibit 10,000-fold and 80-fold decreases in maximal activity, respectively, and they both exhibit a 2-fold increase in the aspartate concentration at half the maximal observed velocity, [S]0.5. The Arg-229----Ala enzyme still exhibits substantial homotropic cooperativity, but all cooperativity is lost in the Glu-233----Ser enzyme. The Glu-233----Ser enzyme also shows a 4-fold decrease in the carbamyl phosphate [S]0.5, while the Arg-229----Ala enzyme shows no change in the carbamyl phosphate [S]0.5 compared to the wild-type enzyme. The Glu-272 to Ser mutation results in a slight reduction in maximal activity, an increase in [S]0.5 for both aspartate and carbamyl phosphate, and reduced cooperativity. Analysis of the isolated catalytic subunits from these three mutant enzymes reveals that in each case the changes in the kinetic properties of the isolated catalytic subunit are similar to the changes caused by the mutation in the holoenzyme. PALA was able to activate the Glu-233----Ser enzyme, at low aspartate concentrations, even though the mutant holoenzyme did not exhibit any cooperativity, indicating that cooperative interactions still exist between the active sites in this enzyme. It is proposed that Glu-233 of the 240s loop helps create the high-activity-high-affinity R state by positioning the side chain of Arg-229 for aspartate binding while Glu-272 helps stabilize the low-activity-low-affinity T state by positioning the side chain of Arg-229 so that it cannot interact with aspartate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Conformation of the gamma subunit at the gamma-epsilon-c interface in the complete Escherichia coli F(1)-ATPase complex by site-directed spin labeling

Structure-function relationships of the gamma-epsilon-c subunit interface of F(O)F(1) ATP synthase, a region of subunit interactions important in coupling between catalysis and transport, were investigated by site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. The EPR line widths and collision accessibilities of 18 spin-labeled, unique cysteine F(1) mutants from gammaLeu198 to gammaLeu215 indicate an alternating pattern in the mobility and accessibility parameters for positions gamma201-209, which is reminiscent of a beta-strand. Labels at positions gamma204 and gamma210 show tertiary contact upon F(1) binding to F(O) and gammaD210C has reduced coupling efficiency. gammaE208C could not be spin labeled, but the uncoupling effects of gammaE208K are suppressed by second-site mutations in the polar loop of subunit c [Ketchum, C. J. and Nakamoto, R. K. (1998) J. Biol. Chem. 273, 22292-22297]. The restricted mobility and accessibility of spin labels in the odd-numbered positions between gamma201 and gamma207 plus the 2-4-fold higher values in k(cat) for ATP hydrolysis of these same mutant F(1) indicate that the interactions of these residues with the epsilon subunit mediate its inhibitory activity. Disrupted interactions with epsilon subunit also cause reduced coupling efficiency. We propose a model for the gamma-epsilon-c interface of Escherichia coli F(O)F(1) ATP synthase in which side chains from the odd-numbered residues of the gammaLys201-gammaTyr207 beta-strand directly and functionally interact with the epsilon subunit, while the even-numbered, acidic residues gammaAsp204, gammaGlu208, and gammaAsp210 interact with the F(O) sector, probably with subunit c. gamma Subunit interactions with both subunits in this region are important for coupling efficiency.     

Critical residues influence the affinity and selectivity of alpha-conotoxin MI for nicotinic acetylcholine receptors.

The mammalian skeletal muscle acetylcholine receptor contains two nonequivalent acetylcholine binding sites, one each at the alpha/delta and alpha/gamma subunit interfaces. Alpha-Conotoxin MI, a 14-amino acid competitive antagonist, binds at both interfaces but has approximately 10(4) higher affinity for the alpha/delta site. We performed an "alanine walk" to identify the residues in alpha-MI that contribute to this selective interaction with the alpha/delta site. Electrophysiological measurements with Xenopus oocytes expressing normal receptors or receptors lacking either the gamma or delta subunit were made to assay toxin-receptor interaction. Alanine substitutions in most amino acid positions had only modest effects on toxin potency at either binding site. However, substitutions in two positions, proline-6 and tyrosine-12, dramatically reduced toxin potency at the high-affinity alpha/delta site while having comparatively little effect on low-affinity alpha/gamma binding. When tyrosine-12 was replaced by alanine, the toxin's selectivity for the high-affinity site (relative to that for the low-affinity site) was reduced from 45,000- to 30-fold. A series of additional amino acid substitutions in this position showed that increasing side chain size/hydrophobicity increases toxin potency at the alpha/delta site without affecting alpha/gamma binding. In contrast, when tyrosine-12 is diiodinated, toxin binding is nearly irreversible at the alpha/delta site but also increases by approximately 500-fold at the alpha/gamma site. The effects of position 12 substitutions are accounted for almost entirely by changes in the rate of toxin dissociation from the high-affinity alpha/delta binding site.     

The alpha subunit of the human IgE receptor (FcERI) is sufficient for high affinity IgE binding

The alpha subunit of the FcERI binds IgE with high affinity. Previous studies have demonstrated that alpha subunit expression requires the presence of beta and/or gamma subunits, and it is not known how these two subunits contribute to the ability of the alpha subunit to bind IgE. In this report, we describe the expression and characterization of a human chimeric alpha subunit. The data demonstrate that high affinity IgE binding does not require the presence of the beta and/or gamma subunits and that this activity is localized to the extracellular domain (residues 26-201) of the human alpha subunit. Permanent cell lines expressing the chimeric receptor were used to characterize the binding parameters of the alpha subunit. These cell lines provide a means of identifying therapeutic agents which may be effective in the treatment/management of allergic diseases.     

Role of Asn-16 and Ser-19 in anthopleurin B binding. Implications for the electrostatic nature of Na(V) site 3

Anthopleurin B (ApB) is a type 1 sea anemone toxin, which binds to voltage-sensitive sodium channels (Na(V)'s), thereby delaying channel inactivation. Previous work from our laboratories has demonstrated that the structurally unconstrained region involving residues 8-17 of this polypeptide, designated the Arg-14 loop, is important for full toxin affinity (Seibert et al., (2003) Biochemistry 42, 14515). Within this region, important contributions are made by residues Arg-12 and Leu-18 (Gallagher and Blumenthal, (1994) J. Biol. Chem. 269, 254; Dias-Kadambi et al., (1996) J. Biol. Chem. 271, 23828). Moreover, replacement of glycine residues found at positions 10 or 15 of the loop by alanine has been shown to have profound, isoform-selective effects on toxin-binding kinetics (Seibert et al., (2003)Biochemistry 42, 14515). To thoroughly understand the importance of this entire region, the work described here investigates the contribution of ApB residues Asn-16, Thr-17, and Ser-19 to toxin affinity and isoform selectivity. Our results demonstrate that residues within and proximal to the C terminus of the Arg-14 loop are important modulators of ApB affinity for Na(V) channels, indicating that the loop and channel site 3 are likely in close contact. A comparison of the effects of multiple replacements at each position reveals that Asn-16 and Ser-19 are involved in binding, whereas Thr-17 is not. The fact that anionic replacements for Asn-16 or Ser-19 are highly deleterious for toxin binding strongly suggests that site 3 contains either formal anionic residues or regions of high electron density, which could be formed by aromatic clusters. These data represent the first indication of the presence of such residues or regions within Na(V) site 3.     

Determination of the 1-ethyl-3-[(3-dimethylamino)propyl]-carbodiimide- induced cross-link between the beta and epsilon subunits of Escherichia coli F1-ATPase.

The zero-length cross-link between the inhibitory epsilon subunit and one of three catalytic beta subunits of Escherichia coli F1-ATPase (alpha 3 beta 3 gamma delta epsilon), induced by a water-soluble carbodiimide, 1-ethyl-3-[(3-dimethylamino) propyl]-carbodiimide (EDC), has been determined at the amino acid level. Lability of cross-linked beta-epsilon to base suggested an ester cross-link rather than the expected amide. A 10-kDa cross-linked CNBr fragment derived from beta-epsilon was identified by electrophoresis on high percentage polyacrylamide gels. Sequence analysis of this peptide revealed the constituent peptides to be Asp-380 to Met-431 of beta and Glu-96 to Met-138 of epsilon. Glu-381 of beta was absent from cycle 2 indicating that it was one of the cross-linked residues, but no potential cross-linked residue in epsilon was identified in this analysis. A form of epsilon containing a methionine residue in place of Val-112 (epsilon V112M) was produced by site-directed mutagenesis. epsilon V112M was incorporated into F1-ATPase which was then cross-linked with EDC. An 8-kDa cross-linked CNBr fragment of beta-epsilon V112M was shown to contain the peptide of epsilon between residues Glu-96 and Met-112 and the peptide of beta between residues Asp-380 and Met-431. Again residue Glu-381 of beta was notably reduced and no missing residue from the epsilon peptide could be identified, but the peptide sequence limited the possible choices to Ser-106, Ser-107, or Ser-108. Furthermore, an epsilon mutant in which Ser-108 was replaced by cysteine could no longer be cross-linked to a beta subunit in F1-ATPase by EDC. Both mutant forms of epsilon supported growth of an uncC-deficient E. coli strain and inhibited F1-ATPase. These results indicate that the EDC-induced cross-link between the beta and epsilon subunits of F1-ATPase is an ester linkage between beta-Glu-381 and, likely, epsilon-Ser-108. As these residues must be located immediately adjacent to one another in F1-ATPase, our results define a site of subunit-subunit contact between beta and epsilon.     

Role of the intramolecular hydrogen bond network in the inhibitory power of chymotrypsin inhibitor 2

A series of mutants of chymotrypsin inhibitor 2 (CI2), at residues involved in intramolecular interactions that shape and constrain the binding loop, were studied to determine their relative importance for inhibition of the serine protease subtilisin BPN', and for resistance of the inhibitor to proteolysis. These functional properties were investigated in tandem with the crystal structures of the mutant inhibitor-enzyme complexes. A dense hydrogen bonding network that supports the binding loop in the vicinity of the scissile bond was found to be important both for enzyme affinity and for stability to proteolysis. Structural analysis, in combination with biochemical measurements, allows differentiation of the structural components most important for resistance to proteolysis and/or binding. The most critical participating residues in the network were found to be Thr-58, Glu-60, Arg-65, and Gly-83. Glu-60 is more important for resistance to proteolysis than for binding, while Arg-65 and two other Arg residues play a greater role in binding than in resistance to proteolysis. Structural comparisons reveal a wide variety of subtle conformational changes in response to mutation, with built-in robustness in the hydrogen bond network, such that loss of one contact is compensated by other new contacts.