Determination of alpha-conotoxin binding modes on neuronal nicotinic acetylcholine receptors

alpha-Conotoxins, from cone snails, and alpha-neurotoxins, from snakes, are competitive inhibitors of nicotinic acetylcholine receptors (nAChRs) that have overlapping binding sites in the ACh binding pocket. These disulphide-rich peptides are used extensively as tools to localize and pharmacologically characterize specific nAChRs subtypes. Recently, a homology model based on the high-resolution structure of an ACh binding protein (AChBP) allowed the three-fingered alpha-neurotoxins to be docked onto the alpha7 nAChR. To investigate if alpha-conotoxins interact with the nAChR in a similar manner, we built homology models of human alpha7 and alpha3beta2 nAChRs, and performed docking simulations of alpha-conotoxins ImI, PnIB, PnIA and MII using the program GOLD. Docking revealed that alpha-conotoxins have a different mode of interaction compared with alpha-neurotoxins, with surprisingly few nAChR residues in common between their overlapping binding sites. These docking experiments show that ImI and PnIB bind to the ACh binding pocket via a small cavity located above the beta9/beta10 hairpin of the (+)alpha7 nAChR subunit. Interestingly, PnIB, PnIA and MII were found to bind in a similar location on alpha7 or alpha3beta2 receptors mostly through hydrophobic interactions, while ImI bound further from the ACh binding pocket, mostly through electrostatic interactions. These findings, which distinguish alpha-conotoxin and alpha-neurotoxin binding modes, have implications for the rational design of selective nAChR antagonists.     

Beta2 subunit contribution to 4/7 alpha-conotoxin binding to the nicotinic acetylcholine receptor

The structures of acetylcholine-binding protein (AChBP) and nicotinic acetylcholine receptor (nAChR) homology models have been used to interpret data from mutagenesis experiments at the nAChR. However, little is known about AChBP-derived structures as predictive tools. Molecular surface analysis of nAChR models has revealed a conserved cleft as the likely binding site for the 4/7 alpha-conotoxins. Here, we used an alpha3beta2 model to identify beta2 subunit residues in this cleft and investigated their influence on the binding of alpha-conotoxins MII, PnIA, and GID to the alpha3beta2 nAChR by two-electrode voltage clamp analysis. Although a beta2-L119Q mutation strongly reduced the affinity of all three alpha-conotoxins, beta2-F117A, beta2-V109A, and beta2-V109G mutations selectively enhanced the binding of MII and GID. An increased activity of alpha-conotoxins GID and MII was also observed when the beta2-F117A mutant was combined with the alpha4 instead of the alpha3 subunit. Investigation of A10L-PnIA indicated that high affinity binding to beta2-F117A, beta2-V109A, and beta2-V109G mutants was conferred by amino acids with a long side chain in position 10 (PnIA numbering). Docking simulations of 4/7 alpha-conotoxin binding to the alpha3beta2 model supported a direct interaction between mutated nAChR residues and alpha-conotoxin residues 6, 7, and 10. Taken together, these data provide evidence that the beta subunit contributes to alpha-conotoxin binding and selectivity and demonstrate that a small cleft leading to the agonist binding site is targeted by alpha-conotoxins to block the nAChR.     

Identification of functional segments within the beta2I-domain of integrin alphaMbeta2

The alpha(M)beta(2) integrin plays an important role in leukocyte biology through its interactions with a diverse set of ligands. Efficient ligand binding requires the involvement of both the alpha(M) and beta(2) subunits. Past ligand binding studies have focused mainly on the alpha(M) subunit, with the beta(2) subunit being largely unexplored. Therefore, in this study we conducted homolog-scanning mutagenesis on the I-domain (residues 125-385) within the beta(2) subunit. We identified four noncontiguous sequences (Arg(144)-Lys(148), Gln(199)-Ala(203), Leu(225)-Leu(230), and Gly(305)-His(309)) that are critical for fibrinogen and C3bi binding to alpha(M)beta(2). Molecular modeling revealed that these four sequences reside within a narrow region on the surface of the beta(2)I-domain, in close proximity to three potential cation-binding sites. Among these sequences, Gln(199)-Ala(203), Leu(225)-Leu(230), and Gly(305)-His(309) are important for the binding of both ligands, whereas Arg(144)-Lys(148) is more critical for fibrinogen than for C3bi binding. These sequences within the beta(2)I-domain are directly involved in ligand binding, since 1) switching these segments to their corresponding beta(1) sequences destroyed ligand binding; 2) loss of function was not due to a nonspecific gross conformational change, since the defective alpha(M)beta(2) mutants reacted well with a panel of conformation-dependent mAbs; 3) mutation of these functional sequences did not effect Ca(2+) binding; and 4) synthetic peptides corresponding to sequences Gln(199)-Ala(203) and Gly(305)-His(309) blocked ligand binding to alpha(M)beta(2), and the peptides interacted directly with fibrinogen and C3bi. Given the similarity among all integrin beta subunits, our results may help us to understand the underlying mechanism of integrin-ligand interactions in general.     

The alpha 1 beta 1 integrin recognition site of the basement membrane collagen molecule [alpha 1(IV)]2 alpha 2(IV).

Cells interact with type IV collagen mainly via the integrins alpha 1 beta 1 and alpha 2 beta 1. A triple helical CNBr derived fragment CB3[IV], which contains the recognition sites for both integrins, was isolated from type IV collagen. Trypsin treatment of CB3[IV] gave rise to four smaller fragments, F1-F4, of which the smallest one, F4, contained the recognition site for alpha 1 beta 1. Further fragmentation of F4 by thermolysin treatment at 50 degrees C led to fragment TL1, which represents the C-terminal half of F4, and which was no longer able to interact with alpha 1 beta 1. Therefore the recognition site of alpha 1 beta 1 had to be located within the N-terminal half of F4, a position which was verified by electron micrographs of a crosslinked F2-alpha 1 beta 1 complex. Modification of the Arg and Asp residues, which abolished the binding activity of F4, led to the identification of Arg (461) within the alpha 2(IV) and Asp (461) within the alpha 1 (IV) chain as essential residues for the alpha 1 beta 1. The array of these two residues on the surface of the triple helix is discussed.     

NMR structure determination of alpha-conotoxin BuIA, a novel neuronal nicotinic acetylcholine receptor antagonist with an unusual 4/4 disulfide scaffold

We have determined a high-resolution three-dimensional structure of alpha-conotoxin BuIA, a 13-residue peptide toxin isolated from Conus bullatus. Despite its unusual 4/4 disulfide bond layout alpha-conotoxin BuIA exhibits strong antagonistic activity at alpha6/alpha3beta2beta3, alpha3beta2, and alpha3beta4 nAChR subtypes like some alpha4/7 conotoxins. alpha-Conotoxin BuIA lacks the C-terminal beta-turn present within the second disulfide loop of alpha4/7 conotoxins, having only a "pseudo omega-shaped" molecular topology. Nevertheless, it contains a functionally critical two-turn helix motif, a feature ubiquitously found in alpha4/7 conotoxins. Such an aspect seems mainly responsible for similarities in the receptor recognition profile of alpha-conotoxin BuIA to alpha4/7 conotoxins. Structural comparison of alpha-conotoxin BuIA with alpha4/7 conotoxins and alpha4/3 conotoxin ImI suggests that presence of the second helical turn portion of the two-turn helix motif in alpha4/7 and alpha4/4 conotoxins may be important for binding to the alpha3 and/or alpha6 subunit of nAChR.     

The fourth blade within the beta-propeller is involved specifically in C3bi recognition by integrin alpha M beta 2

Interactions between the complement degradation product C3bi and leukocyte integrin alpha M beta 2 are critical to phagocytosis of opsonized particles in host defense against foreign pathogens and certain malignant cells. Previous studies have mapped critical residues for C3bi binding to the I-domains of the alpha M and the beta2 subunits. However, the role of the alpha M beta-propeller in ligand binding remains less well defined, and the functional residues are still unknown. In the present study, we studied the function of the alpha M beta-propeller in specific ligand recognition by alpha M beta 2 using a number of different approaches, and we report four major findings. 1) Substitution of five individual segments (Asp398-Ala402, Leu412-Leu419, Tyr426-Met434, Phe435-Glu443, and Ser444-Thr451) within the W4 blade of the beta-propeller with their homologous counterparts in integrin alpha2 abrogated C3bi binding, whereas substitution of eight other segments outside this blade had no effect. 2) These five mutants defective in C3bi binding supported strong alpha M beta 2-mediated and cation-dependent cell adhesion to fibrinogen, suggesting that the conformations of these five defective mutants were intact. 3) Polyclonal antibodies recognizing sequences within the W4 blade significantly blocked C3bi binding by wild-type alpha M beta 2. 4) A synthetic peptide corresponding to Gln424-Gly440 within W4 interacted directly with C3bi. In conclusion, our data demonstrate that the W4 blade (residues Asp398 to Thr451) is involved specifically in C3bi but not fibrinogen binding to alpha M beta 2. Altogether, our study supports a model in which three separate domains of alpha M beta 2 (the alpha MI-domain, the alpha M beta-propeller, and the beta 2I-domain) function together and contribute to the formation of the C3bi-binding site.     

Synthesis and nicotinic receptor activity of a hydroxylated tropane

(+/-)-3alpha-hydroxy homoepibatidine 4 has been synthesized from the alkaloid scopolamine 5 and its properties as a nicotinic agonist assessed. While still binding strongly, the compound showed reduced agonist potency for the alpha(4)beta(2) nAChR compared with the parent compound epibatidine 1. Compound 4 also displayed generally similar binding and selectivity profiles at alpha(4)beta(2), alpha(2)beta(4), alpha(3)beta(4), and alpha(4)beta(4) nAChR subtypes to those for nicotine.     

Cloning and expression of zebrafish neuronal nicotinic acetylcholine receptors

We propose to use the zebrafish (Danio rerio) as a vertebrate model to study the role of neuronal nicotinic acetylcholine receptors (nAChR) in development. As a first step toward using zebrafish as a model, we cloned three zebrafish cDNAs with a high degree of sequence similarity to nAChR beta3, alpha2 and alpha7 subunits expressed in other species. RT-PCR was used to show that the beta3 and alpha2 subunit RNAs were present in zebrafish embryos only 2-5hours post-fertilization (hpf) while alpha7 subunit RNA was not detected until 8hpf, supporting the differential regulation of nAChRs during development. In situ hybridization was used to localize zebrafish beta3, alpha2, and alpha7 RNA expression. nAChR binding techniques were used to detect the early expression of two high-affinity [3H]-epibatidine binding sites in 2 days post-fertilization (dpf) zebrafish embryos with IC(50) values of 28.6pM and 29.7nM and in 5dpf embryos with IC(50) values of 28.4pM and 8.9nM. These studies are consistent with the involvement of neuronal nAChRs in early zebrafish development.     

Identification of a novel binding site for platelet integrins alpha IIb beta 3 (GPIIbIIIa) and alpha 5 beta 1 in the gamma C-domain of fibrinogen

The interactions of platelets with fibrinogen mediate a variety of responses including adhesion, platelet aggregation, and fibrin clot retraction. Whereas it was assumed that interactions of the platelet integrin alpha IIb beta 3 with the AGDV sequence in the gamma C-domain of fibrinogen and/or RGD sites in the A alpha chains are involved in clot retraction and adhesion, recent data demonstrated that fibrinogen lacking these sites still supported clot retraction. These findings suggested that an unknown site in fibrinogen and/or other integrins participate in clot retraction. Here we have identified a sequence within gamma C that mediates binding of fibrinogen to platelets. Synthetic peptide duplicating the 365-383 sequence in gamma C, designated P3, efficiently inhibited clot retraction in a dose-dependent manner. Furthermore, P3 supported platelet adhesion and was an effective inhibitor of platelet adhesion to fibrinogen fragments. Analysis of overlapping peptides spanning P3 and mutant recombinant gamma C-domains demonstrated that the P3 activity is contained primarily within gamma 370-383. Integrins alpha IIb beta 3 and alpha 5 beta 1 were implicated in recognition of P3, since platelet adhesion to the peptide was blocked by function-blocking monoclonal antibodies against these receptors. Direct evidence that alpha IIb beta 3 and alpha 5 beta 1 bind P3 was obtained by selective capture of these integrins from platelet lysates using a P3 affinity matrix. Thus, these data suggest that the P3 sequence in the gamma C-domain of fibrinogen defines a previously unknown recognition specificity of alpha IIb beta 3 and alpha 5 beta 1 and may function as a binding site for these integrins.     

Identification of sites of incorporation in the nicotinic acetylcholine receptor of a photoactivatible general anesthetic

Most general anesthetics including long chain aliphatic alcohols act as noncompetitive antagonists of the nicotinic acetylcholine receptor (nAChR). To locate the sites of interaction of a long chain alcohol with the Torpedo nAChR, we have used the photoactivatible alcohol 3-[(3)H]azioctanol, which inhibits the nAChR and photoincorporates into nAChR subunits. At 1 and 275 microm, 3-[(3)H]azioctanol photoincorporated into nAChR subunits with increased incorporation in the alpha-subunit in the desensitized state. The incorporation into the alpha-subunit was mapped to two large proteolytic fragments. One fragment of approximately 20 kDa (alpha V8-20), containing the M1, M2, and M3 transmembrane segments, showed enhanced incorporation in the presence of agonist whereas the other of approximately 10 kDa (alpha V8-10), containing the M4 transmembrane segment, did not show agonist-induced incorporation of label. Within alpha V8-20, the primary site of incorporation was alpha Glu-262 at the C-terminal end of alpha M2, labeled preferentially in the desensitized state. The incorporation at alpha Glu-262 approached saturation between 1 microm, with approximately 6% labeled, and 275 microm, with approximately 30% labeled. Low level incorporation was seen in residues at the agonist binding site and the protein-lipid interface at approximately 1% of the levels in alpha Glu-262. Therefore, the primary binding site of 3-azioctanol is within the ion channel with additional lower affinity interactions within the agonist binding site and at the protein-lipid interface.     

Defining nicotinic agonist binding surfaces through photoaffinity labeling

Nicotinic acetylcholine (ACh) receptor (nAChR) agonists are potential therapeutic agents for neurological dysfunction. In the present study, the homopentameric mollusk ACh binding protein (AChBP), used as a surrogate for the extracellular ligand-binding domain of the nAChR, was specifically derivatized by the highly potent agonist azidoepibatidine (AzEPI) prepared as a photoaffinity probe and radioligand. One EPI-nitrene photoactivated molecule was incorporated in each subunit interface binding site based on analysis of the intact derivatized protein. Tryptic fragments of the modified AChBP were analyzed by collision-induced dissociation and Edman sequencing of radiolabeled peptides. Each specific EPI-nitrene-modified site involved either Tyr195 of loop C on the principal or (+)-face or Met116 of loop E on the complementary or (-)-face. The two derivatization sites were observed in similar frequency, providing evidence of the reactivity of the azido/nitrene probe substituent and close proximity to both residues. [3H]AzEPI binds to the alpha4beta2 nAChR at a single high-affinity site and photoaffinity-labels only the alpha4 subunit, presumably modifying Tyr225 spatially corresponding to Tyr195 of AChBP. Phe137 of the beta2 nAChR subunit, equivalent to Met116 of AChBP, conceivably lacks sufficient reactivity with the nitrene generated from the probe. The present photoaffinity labeling in a physiologically relevant condition combined with the crystal structure of AChBP allows development of precise structural models for the AzEPI interactions with AChBP and alpha4beta2 nAChR. These findings enabled us to use AChBP as a structural surrogate to define the nAChR agonist site.     

Synthesis and pharmacological characterization of bivalent ligands of epibatidine at neuronal nicotinic acetylcholine receptors

A series of bivalent ligands 6a-d of epibatidine were synthesized. All four ligands showed nanomolar binding affinities at six neuronal nicotinic acetylcholine receptor (nAChR) subtypes in competition binding assays. In contrast to epibatidine, these bivalent ligands are weak partial agonists at the alpha3beta4 nAChR as shown by functional assays.     

Alpha-conotoxins ImI and ImII target distinct regions of the human alpha7 nicotinic acetylcholine receptor and distinguish human nicotinic receptor subtypes

The Conus peptides alpha-conotoxin ImI (alpha-ImI) and ImII (alpha-ImII) differ by only three of 11 residues in their primary sequences and yet are shown to inhibit the human alpha7 nicotinic acetylcholine receptor (nAChR) by targeting different sites. Mutations at both faces of the classical ligand binding site of the alpha7 nAChR strongly affect antagonism by alpha-ImI but not alpha-ImII. The effects of the mutations on alpha-ImI binding and functional antagonism are explained by computational docking of the NMR structure of alpha-ImI to a homology model of the ligand binding domain of the alpha7 nAChR. A distinct binding site for alpha-ImII is further demonstrated by its weakened antagonism for a chimeric receptor in which the membrane-spanning domains and intervening linkers of the alpha7 nAChR are replaced with the corresponding sequence from the serotonin type-3 receptor (5HT(3)). The two toxins also discriminate between different subtypes of human nicotinic receptors; alpha-ImII most strongly blocks the human alpha7 and alpha1beta1deltaepsilon receptor subtypes, while alpha-ImI most potently blocks the human alpha3beta2 subtype. Collectively, the data show that while alpha-ImI targets the classical competitive ligand binding site in a subtype selective manner, alpha-ImII is a probe of a novel inhibitory site in homomeric alpha7 nAChRs.     

Expression of nicotinic acetylcholine receptors in human and rat adrenal medulla.

Neuronal nicotinic receptors (nAChRs) are expressed in the brain but also in the peripheral tissues including the adrenal medulla. However, it is unclear which nAChRs are present in the human adrenal medulla. In the study, receptor binding assay, Western blot and RT-PCR have been performed to investigate the expression of nAChRs in adrenal medulla from human, rat and mouse. The results showed that in human adult adrenal medulla, mRNAs for nAChR alpha3, alpha4, alpha5, alpha7, beta2, beta3, and beta4 subunits but not beta2 in the fetal human adrenal medulla were expressed. Saturation binding of [3H]epibatidine showed two binding sites in human aged adrenal medulla. The specific binding of [3H]epibatidine (0.1 nM) was significantly higher in human fetal compared to human aged adrenal medulla. mRNAs for the alpha3, alpha4, alpha5, alpha7, beta2, and beta4 subunits but not the beta3 were detectable in adult rat and mouse adrenal medulla. No differences in gene-expression of the nAChRs were observed between new born, adult and aged rat adrenal medulla. Saturation binding of [3H]epibatidine showed only one binding site in rat adrenal medulla. Lower protein levels for the nAChR subunits were observed in the rat adrenal medulla compared to rat brain. There was lower protein levels of the nAChRs in aged rat adrenal medulla compared to the young rats. Sub-chronic treatment of nicotine to rats did not influence level of the nAChRs in the adrenal medulla. In conclusion, the expression of nAChRs in adrenal medulla is age- related and species dependent.     

Synthesis of 3,6-diazabicyclo[3.1.1]heptanes as novel ligands for neuronal nicotinic acetylcholine receptors

Alpha series of novel 3,6-diazabicyclo[3.1.1]heptane derivatives 4a-f was synthesized and their affinity and selectivity towards alpha4beta2 and alpha7 nAChR subtypes were evaluated. The results of the current study revealed a number of compounds (4a, 4b and 4c) having a very high affinity for alpha4beta2 (K(i) at alpha4beta2 ranging from 0.023 to 0.056 nM) versus alpha7 nAChR subtypes; among these compounds, the 3-(6-bromopyridin-3-yl)-3,6-diazabicyclo[3.1.1]heptane 4c was found to be the most alpha7alpha4beta2 selective term in receptor binding assays (alpha7alpha4beta2=1295). Moreover, compound 4d also had high affinity for the alpha4beta2 nAChR subtype (K(i)=1.2 nM) with considerably high selectivity (alpha7/alpha4beta2=23300).     

Analysis of the roles of RGD-binding integrins, alpha(4)/alpha(9) integrins, alpha(6) integrins, and CD9 in the interaction of the fertilin beta (ADAM2) disintegrin domain with the mouse egg membrane

Fertilin beta (also known as ADAM2), a mammalian sperm protein that mediates gamete cell adhesion during fertilization, is a member of the ADAM protein family whose members have disintegrin domains with homology to integrin ligands found in snake venoms. Fertilin beta utilizes an ECD sequence within its disintegrin domain to interact with the egg plasma membrane; the Asp is especially critical. Based on what is known about different integrin subfamilies and their ligands, we sought to characterize fertilin beta binding sites on mouse eggs, focusing on integrin subfamilies that recognize short peptide sequences that include an Asp residue: the alpha(5)/alpha(8)/alpha(v)/alpha(IIb) or RGD-binding subfamily (alpha(5)beta(1), alpha(8)beta(1), alpha(V)beta(1), alpha(V)beta(3), alpha(V)beta(5), alpha(V)beta(6), alpha(V)beta(8), and alpha(IIb)beta(3)) and the alpha(4)/alpha(9) subfamily (alpha(4)beta(1), alpha(9)beta(1), and alpha(4)beta(7)). We tested peptide sequences known to perturb interactions mediated by these integrins in two different assays for fertilin beta binding. Peptides with the sequence MLDG, which perturb alpha(4)/alpha(9) integrin-mediated interactions, significantly inhibit fertilin beta binding to eggs, which suggests a role for a member of this integrin subfamily as a fertilin beta receptor. RGD peptides, which perturb alpha(5)/alpha(8)/alpha(v)/alpha(IIb) integrin-mediated interactions, have partial inhibitory activity. The anti-alpha(6) antibody GoH3 has little or no inhibitory activity. An antibody to the integrin-associated tetraspanin protein CD9 inhibits the binding of a multivalent presentation of fertilin beta (immobilized on beads) but not soluble fertilin beta, which we speculate has implications for the role of CD9 in the strengthening of fertilin beta-mediated cell adhesion but not in initial ligand binding.     

Iodo-alpha-conotoxin MI selectively binds the alpha/delta subunit interface of muscle nicotinic acetylcholine receptors

The embryonic mouse muscle nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel formed by alpha1, beta1, delta, and gamma subunits. The receptor contains two ligand binding sites at alpha/delta and alpha/gamma subunit interfaces. [(3)H]Curare preferentially binds the alpha/gamma interface. We describe the synthesis and properties of a high-affinity iodinated ligand that selectively binds the alpha/delta interface. An analogue of alpha-conotoxin MI was synthesized with an iodine attached to Tyr-12 (iodo-alpha-MI). The analogue potently blocks the fetal mouse muscle subtype of nAChR expressed in Xenopus oocytes. It failed, however, to block alpha3beta4, alpha4beta2, or alpha7 nAChRs. Iodo-alpha-MI potently blocks the alpha1beta1delta but not the alpha1beta1gamma subunit combination expressed in Xenopus oocytes indicating selectivity for the alpha/delta subunit interface. Alpha-conotoxin MI was subsequently radioiodinated, and its properties were further evaluated. Saturation experiments indicate that radioiodinated alpha-conotoxin MI binds to TE671 cell homogenates with a Hill slope of 0.95 +/- 0.0094. Kinetic studies indicate that the binding of [(125)I]alpha-conotoxin MI is reversible (k(off) = 0.084 +/- 0.0045 min(-1)); k(on) is 8.5 x 10(7) min(-1) M(-1). The calculated k(d) is 0.98 nM. This potency is approximately 20-fold higher than the unmodified alpha-MI peptide. Unlike [(125)I]alpha-bungarotoxin, [(125)I]alpha-conotoxin MI binding to TE671 cell homogenates is fully displaceable by the small molecule antagonist d-tubocurarine.     

Synthesis and electrophysiological studies of a novel epibatidine analogue

The new epibatidine analogue exo-2-(2-pyridyl)-7-azabicyclo[2.2.1]heptane (2PABH) was synthesised. Separation of enantiomers was performed on chiral HPLC chromatography in polar-organic phase mode at 0 degree C. Enantiomeric purity was greater than 99.8%ee for the (-)- and 90.5%ee for the (+)-enantiomer respectively. Optical rotation was determined to be [alpha]23D = +/- 13 degrees. Electrophysiological studies of 2PABH were carried out on alpha 4 beta 2, alpha 3 beta 4 and alpha 7 nAChR subtypes cloned from rat and reconstituted in Xenopus oocytes. Both enantiomers could not significantly activate the heteromeric subtypes. The homomeric alpha 7 nAChR displays a high sensitivity only towards (-)-2PABH. The EC50 for (-)-2PABH and ACh were determined (32.5 +/- 9.5 microM, 137.3 +/- 16.5 microM). (-)-2PABH was shown to be a partial agonist (80% of ACh). Thus the efficacy of 2PABH differs markedly from that of epibatidine. The intramolecular N-N-distance and the spatial pyridine nitrogen orientation play a central role in nAChR recognition.