Subunit Interface Selectivity of the α-Neurotoxins for the Nicotinic Acetylcholine Receptor
1999; Elsevier BV; Volume: 274; Issue: 14 Linguagem: Inglês
10.1074/jbc.274.14.9581
ISSN1083-351X
AutoresHitoshi Osaka, Siobhan Malany, Joan R. Kanter, Steven M. Sine, Palmer Taylor,
Tópico(s)Insect and Pesticide Research
ResumoPeptide toxins selective for particular subunit interfaces of the nicotinic acetylcholine receptor have proven invaluable in assigning candidate residues located in the two binding sites and for determining probable orientations of the bound peptide. We report here on a short α-neurotoxin from Naja mossambica mossambica (NmmI) that, similar to other α-neurotoxins, binds with high affinity to αγ and αδ subunit interfaces (K D∼100 pm) but binds with markedly reduced affinity to the αε interface (K D∼100 nm). By constructing chimeras composed of portions of the γ and ε subunits and coexpressing them with wild type α, β, and δ subunits in HEK 293 cells, we identify a region of the subunit sequence responsible for the difference in affinity. Within this region, γPro-175 and γGlu-176 confer high affinity, whereas Thr and Ala, found at homologous positions in ε, confer low affinity. To identify an interaction between γGlu-176 and residues in NmmI, we have examined cationic residues in the central loop of the toxin and measured binding of mutant toxin-receptor combinations. The data show strong pairwise interactions or coupling between γGlu-176 and Lys-27 of NmmI and progressively weaker interactions with Arg-33 and Arg-36 in loop II of this three-loop toxin. Thus, loop II of NmmI, and in particular the face of this loop closest to loop III, appears to come into close apposition with Glu-176 of the γ subunit surface of the binding site interface. Peptide toxins selective for particular subunit interfaces of the nicotinic acetylcholine receptor have proven invaluable in assigning candidate residues located in the two binding sites and for determining probable orientations of the bound peptide. We report here on a short α-neurotoxin from Naja mossambica mossambica (NmmI) that, similar to other α-neurotoxins, binds with high affinity to αγ and αδ subunit interfaces (K D∼100 pm) but binds with markedly reduced affinity to the αε interface (K D∼100 nm). By constructing chimeras composed of portions of the γ and ε subunits and coexpressing them with wild type α, β, and δ subunits in HEK 293 cells, we identify a region of the subunit sequence responsible for the difference in affinity. Within this region, γPro-175 and γGlu-176 confer high affinity, whereas Thr and Ala, found at homologous positions in ε, confer low affinity. To identify an interaction between γGlu-176 and residues in NmmI, we have examined cationic residues in the central loop of the toxin and measured binding of mutant toxin-receptor combinations. The data show strong pairwise interactions or coupling between γGlu-176 and Lys-27 of NmmI and progressively weaker interactions with Arg-33 and Arg-36 in loop II of this three-loop toxin. Thus, loop II of NmmI, and in particular the face of this loop closest to loop III, appears to come into close apposition with Glu-176 of the γ subunit surface of the binding site interface. nicotinic acetylcholine receptor Naja mossambica mossambica α-bungarotoxin wild type mutant The nicotinic acetylcholine receptor (nAChR)1 found in muscle is a pentamer composed of four homologous subunits present in the stoichiometry α2βγδ (fetal subtype) or α2βεδ (adult subtype). The subunits are arranged in a circular manner to surround a central channel in the order, αγαδβ or αεαδβ (1Unwin N. J. Mol. Biol. 1993; 229: 1101-1124Crossref PubMed Scopus (715) Google Scholar, 2Karlin A. Akabas M.H. Neuron. 1995; 15: 1231-1244Abstract Full Text PDF PubMed Scopus (563) Google Scholar, 3Hucho F. Tsetlin V.I. Machold J. Eur. J. Biochem. 1996; 239: 539-557Crossref PubMed Scopus (205) Google Scholar). The two binding sites for agonists, competitive antagonists, and the slowly dissociating α-neurotoxins are formed at interfaces between the αδ and αγ(ε) subunit pairs. The extracellular domain in each subunit is formed principally from the amino-terminal 210 amino acids, which is followed by four membrane-spanning domains. Residues within the amino-terminal 210 amino acids have been shown to be the major contributors to the ligand binding sites and for dictating the order of assembly of subunits.Three segments of the α subunit, well separated along the linear sequence, harbor major determinants for ligand binding; these segments contain the key residues around Tyr-93, between Trp-149 and Asp-152, and spanning the region from Val-188 through Asp-200 (see Refs. 3Hucho F. Tsetlin V.I. Machold J. Eur. J. Biochem. 1996; 239: 539-557Crossref PubMed Scopus (205) Google Scholar and 4Tsigelny I. Sugiyama N. Sine S.M. Taylor P. Biophys. J. 1997; 73: 52-66Abstract Full Text PDF PubMed Scopus (69) Google Scholarfor reviews). Similarly, four discontinuous segments of the non-α subunits, appearing on the opposite face of the subunit, contain major determinants for ligand selectivity; in the γ subunit these segments contain the key residues Lys-34, between Trp-55 and Gln-59, between Ser-111 and Tyr-117, and between Phe-172 and Asp-174.Since the early demonstration of irreversible neuromuscular blockade by the peptide from snake venom, α-bungarotoxin (5Chang C.C. Lee C.Y. Arch. Int. Pharmacodyn. Ther. 1963; 144: 241-257PubMed Google Scholar), and the use of labeled α-neurotoxins to identify the nAChR (6Changeux J.P. Kasai M. Lee C.Y. Proc. Natl. Acad. Sci. U. S. A. 1970; 67: 1241-1247Crossref PubMed Scopus (459) Google Scholar), these toxins have been the primary ligands employed for the identification and characterization of the muscle nAChR. Amino acid sequences are available for nearly 100 members of the α-neurotoxin family, which show a common basic structure consisting of three polypeptide loops emerging from a small globular core (7Endo T. Tamiya N. Pharmacol. Ther. 1987; 34: 403-451Crossref PubMed Scopus (122) Google Scholar). α-Neurotoxins can be divided into the short (4 disulfide bonds and 60–62 residues) and long neurotoxins (5 disulfide bonds and 66–74 residues). Crystal and solution structure determinations reveal similar tertiary structures. Although these structurally well defined toxins are known to bind at the subunit interfaces (αε or αγ and αδ), typically with aK D ≤ 100 pm, little is known about their precise orientation with respect to the subunits that form the interfaces.Points of attachment of α-neurotoxin within the nAChR binding sites have been examined by cross-linking chemically modified (8Witzemann V. Muchmore D. Raftery M.A. Biochemistry. 1979; 18: 5511-5518Crossref PubMed Scopus (43) Google Scholar, 9Hamilton S.L. Pratt D.R. Eaton D.C. Biochemistry. 1985; 24: 2210-2219Crossref PubMed Scopus (25) Google Scholar) or photoactivatable derivatives of α-neurotoxin (10Chatrenet B. Trémeau O. Bontems F. Goeldner M.P. Hirth C.G. Ménez A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3378-8211Crossref PubMed Scopus (30) Google Scholar, 11Kreienkamp H.J. Utkin Y.N. Weise C. Machold J. Tsetlin V.I. Hucho F. Biochemistry. 1992; 31: 8239-8244Crossref PubMed Scopus (37) Google Scholar, 12Machold J. Weise C. Utkin Y. Tsetlin V. Hucho F. Eur. J. Biochem. 1995; 234: 427-430Crossref PubMed Scopus (45) Google Scholar, 13Utkin Y.N. Krivoshein A.V. Davydov V.L. Kasheverov I.E. Franke P. Maslennikov I.V. Arseniev A.S. Hucho F. Tsetlin V.I. Eur. J. Biochem. 1998; 253: 229-235Crossref PubMed Scopus (20) Google Scholar) and by simple ultraviolet irradiation without chemical modification (14Oswald R.E. Changeux J.P. FEBS Lett. 1982; 139: 225-229Crossref PubMed Scopus (52) Google Scholar). These labeling studies have suggested contacts with both α and non-α subunits at the binding sites (see Refs. 2Karlin A. Akabas M.H. Neuron. 1995; 15: 1231-1244Abstract Full Text PDF PubMed Scopus (563) Google Scholar, 3Hucho F. Tsetlin V.I. Machold J. Eur. J. Biochem. 1996; 239: 539-557Crossref PubMed Scopus (205) Google Scholar, and 15Arias H.R. Brain Res. Rev. 1997; 25: 133-191Crossref PubMed Scopus (134) Google Scholar for reviews). Mutagenesis studies have also identified candidate residues in the principal loops of the α (16Kreienkamp H.-J. Sine S.M. Maeda R.K. Taylor P. J. Biol. Chem. 1994; 269: 8108-8114Abstract Full Text PDF PubMed Google Scholar) and non-α subunits (17Sine S.M. J. Biol. Chem. 1997; 272: 23521-23527Crossref PubMed Scopus (60) Google Scholar) that contribute to α-toxin binding. Although most α-toxins do not distinguish between the two sites on the receptor, an α-toxin from the venom of Naja mossambica mossambica (NmmI) distinguishes between the two sites of the Torpedo receptor (18Marchot P. Frachon P. Bougis P.E. Eur. J. Biochem. 1988; 174: 537-542Crossref PubMed Scopus (14) Google Scholar). Thus NmmI emerges as a potentially valuable ligand for determining regions of close approach between α-toxins and the non-α subunits at the binding site. Previous work showed that the αγ and αδ binding sites of the fetal mouse receptor exhibit similar affinities for NmmI (19Ackermann E.J. Taylor P. Biochemistry. 1997; 36: 12836-12844Crossref PubMed Scopus (39) Google Scholar). However, certain mutations in the NmmI toxin structure, and surprisingly also in the nAChR α subunit common to both sites, resulted in nonequivalent reductions in affinity at the αγ and αδ binding sites (19Ackermann E.J. Taylor P. Biochemistry. 1997; 36: 12836-12844Crossref PubMed Scopus (39) Google Scholar). Here we examine binding of recombinant NmmI α-toxin to fetal and adult mouse AChRs and find that the affinity of NmmI for the αε interface is 3 orders of magnitude lower than for the αγ and αδ interfaces. Using subunit chimeras and site-directed mutations in γ and ε subunits, we show that the enhanced affinity conferred by the γ over the ε subunit arises from Pro-175 and Glu-176 in the γ subunit. Mutant cycle analysis shows that Glu-176 interacts with cationic residues in loop II of the NmmI α-toxin.DISCUSSIONThe α-neurotoxins are a family of three-fingered peptide toxins found in venom of elapid snakes (7Endo T. Tamiya N. Pharmacol. Ther. 1987; 34: 403-451Crossref PubMed Scopus (122) Google Scholar). They have proven to be invaluable tools for the isolation and study of the nAChR because of their high affinities and slow rates of dissociation from the receptor (5Chang C.C. Lee C.Y. Arch. Int. Pharmacodyn. Ther. 1963; 144: 241-257PubMed Google Scholar, 6Changeux J.P. Kasai M. Lee C.Y. Proc. Natl. Acad. Sci. U. S. A. 1970; 67: 1241-1247Crossref PubMed Scopus (459) Google Scholar). Although the isolated α-subunit of the receptor retains the capacity to bind α-BgTx, whereas isolated β, γ, or δ subunits do not, the α-toxins bind with far lower affinity to the α subunit than to the intact receptor. Moreover, small agonists and antagonists do not compete with α-toxin binding to the isolated α subunit at expected concentrations (23Gershoni J.M. Hawrot E. Lentz T.L. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4973-4977Crossref PubMed Scopus (85) Google Scholar). These observations point to a predominant, but not sole, contribution to the α-neurotoxin binding coming from the α subunit. Our previous work showed that Val-188, Tyr-190, Pro-197, and Asp-200 of α subunit contribute to NmmI binding (19Ackermann E.J. Taylor P. Biochemistry. 1997; 36: 12836-12844Crossref PubMed Scopus (39) Google Scholar). Also glycosylation at positions 189 and 187, yielding oligosaccharides uniquely found in cobra and mongoose nAChR, reduced α-BgTx binding substantially (16Kreienkamp H.-J. Sine S.M. Maeda R.K. Taylor P. J. Biol. Chem. 1994; 269: 8108-8114Abstract Full Text PDF PubMed Google Scholar).Although the α subunit appears to be the predominant site of α-toxin binding, chemical cross-linking and mutagenesis studies show that non-α subunits are close to the site of α-neurotoxin binding (Refs. 8Witzemann V. Muchmore D. Raftery M.A. Biochemistry. 1979; 18: 5511-5518Crossref PubMed Scopus (43) Google Scholar, 9Hamilton S.L. Pratt D.R. Eaton D.C. Biochemistry. 1985; 24: 2210-2219Crossref PubMed Scopus (25) Google Scholar, 10Chatrenet B. Trémeau O. Bontems F. Goeldner M.P. Hirth C.G. Ménez A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3378-8211Crossref PubMed Scopus (30) Google Scholar, 11Kreienkamp H.J. Utkin Y.N. Weise C. Machold J. Tsetlin V.I. Hucho F. Biochemistry. 1992; 31: 8239-8244Crossref PubMed Scopus (37) Google Scholar, 12Machold J. Weise C. Utkin Y. Tsetlin V. Hucho F. Eur. J. Biochem. 1995; 234: 427-430Crossref PubMed Scopus (45) Google Scholar, 13Utkin Y.N. Krivoshein A.V. Davydov V.L. Kasheverov I.E. Franke P. Maslennikov I.V. Arseniev A.S. Hucho F. Tsetlin V.I. Eur. J. Biochem. 1998; 253: 229-235Crossref PubMed Scopus (20) Google Scholar, 14Oswald R.E. Changeux J.P. FEBS Lett. 1982; 139: 225-229Crossref PubMed Scopus (52) Google Scholar, 16Kreienkamp H.-J. Sine S.M. Maeda R.K. Taylor P. J. Biol. Chem. 1994; 269: 8108-8114Abstract Full Text PDF PubMed Google Scholar, 17Sine S.M. J. Biol. Chem. 1997; 272: 23521-23527Crossref PubMed Scopus (60) Google Scholar, and see Refs. 3Hucho F. Tsetlin V.I. Machold J. Eur. J. Biochem. 1996; 239: 539-557Crossref PubMed Scopus (205) Google Scholar and 15Arias H.R. Brain Res. Rev. 1997; 25: 133-191Crossref PubMed Scopus (134) Google Scholar for reviews). The results described here further illustrate the role of neighboring non-α subunits in contributing to high affinity α-toxin binding, as NmmI binds to αε interfaces of the adult type of nAChR (α2βεδ) with 3 orders of magnitude lower affinity than to the αγ and αδ interfaces of the fetal receptor (α2βγδ). Binding studies, initially using chimeras and subsequently point mutants, show that εThr-176/εAla-177 (Pro/Glu in γ/δ) contribute entirely to insensitivity of the αε interface to NmmI. The observation that α-bungarotoxin association is only slightly affected by the γ and ε sequence differences suggests that this region of the γ, ε, and δ subunits is not used equivalently for stabilization of the entire family of bound α-neurotoxins. At the present time, it is unclear whether stabilization from this region is unique to some of the short α-neurotoxins, or the long α-neurotoxins, such as α-bungarotoxin, acquire the bulk of their stabilization energy from other portions of the structure. Distinct differences in specificity between the short and long neurotoxins have been noted for the α7 subtype of nAChR (24Servent D. Winckler-Dietrich V. Hu H.Y. Kessler P. Drevet P. Bertrand D. Ménez A. J. Biol. Chem. 1997; 272: 24279-24286Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar).Residues at the γ175/176 positions were previously unrecognized as determinants of ligand binding. However, they are immediately adjacent to γAsp-174, which was shown by cross-linking to be ∼9 Å away from Cys-192/193 in the α subunit (25Czajkowski C. Karlin A. J. Biol. Chem. 1991; 266: 22603-22612Abstract Full Text PDF PubMed Google Scholar, 26Czajkowski C. Kaufmann C. Karlin A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6285-6289Crossref PubMed Scopus (106) Google Scholar) and was shown to influence the affinity of quaternary agonists and antagonists (27Martin M.D. Karlin A. Biochemistry. 1997; 36: 10742-10750Crossref PubMed Scopus (17) Google Scholar, 28Osaka H. Sugiyama N. Taylor P. J. Biol. Chem. 1998; 273: 12758-12765Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Moreover, the adjacent residues of γPhe-172 (δAsp-178, εIle-173) are known to confer site-selectivity to the smaller competitive peptide inhibitors such as α-conotoxin MI (29Sine S.M. Kreienkamp H.-J. Bren N. Maeda R. Taylor P. Neuron. 1995; 15: 205-211Abstract Full Text PDF PubMed Scopus (175) Google Scholar) and waglerin (30Molles B.E. Kline E.F. Sine S.M. McArdle J.J. Taylor P. J. Physiol. (paris). 1998; 92 (abstr.): 470Crossref Google Scholar). The equivalent region of the α7 subunit (Asp-163, Ile-164, and Ser-165), which presumably forms a homomeric pentamer of subunits, constitutes part of a putative Ca2+ binding region that faces the ligand binding site (31Galzi J.L. Bertrand S. Corringer P.J. Changeux J.P. Bertrand D. EMBO J. 1996; 15: 5824-5832Crossref PubMed Scopus (133) Google Scholar). At the α subunit interface of the binding site, both aromatic (Tyr-190, Tyr-198) and anionic (Asp-200) residues were mapped to the α-toxin binding surface (19Ackermann E.J. Taylor P. Biochemistry. 1997; 36: 12836-12844Crossref PubMed Scopus (39) Google Scholar). Here, we identify another anionic residue in the γ subunit, Glu-176, perhaps restricted in its position by a neighboring secondary amino acid Pro-175, on the γ subunit, as a crucial residue for binding.The significant linkage between Glu-176 and cationic residues in loop II of the toxin suggests that an electrostatic interaction contributes to the tight binding of the NmmI/nAChR complex. Because the linkage obtained from charge reversal is greater for Lys-27 than for Arg-33 and Arg-36, one would predict that the portion of loop II proximal to loop III of the toxin, is closest to the γ subunit (cf. Fig.5). In this analysis, the loss of free energy (ΔΔG) associated with a single charge mutation results from all interactions between the charged residue and its multiple neighboring residues. The pairwise interactions (ΔΔG INT) resulting in charge reversal of specified residues in the interacting molecules should then highlight the strength of interaction coming from the paired charged residues. In the absence of significant changes in conformation or hydration, ΔΔG INT from Equation 2 should largely reflect the Coulombic interaction between the respective paired residues (32Faiman G.A. Horovitz A. Protein Science. 1996; 9: 315-316Google Scholar).Extensive studies on a related short neurotoxin, erabutoxin a, involving mutations at 36 toxin positions clearly revealed the importance of the tips of loops situated on the concave face of the toxin (33Pillet L. Trémeau O. Ducancel F. Drevet P. Zinn-Justin S. Pinkasfeld S. Boulain J.-C. Ménez A. J. Biol. Chem. 1993; 268: 909-916Abstract Full Text PDF PubMed Google Scholar, 34Trémeau O. Lemaire C. Drevet P. Pinkasfeld S. Ducancel F. Boulain J.-C. Ménez A. J. Biol. Chem. 1995; 270: 9362-9369Crossref PubMed Scopus (131) Google Scholar). These investigations showed that the K27E mutation of erabutoxin a decreases its affinity more than 100-fold forTorpedo nAChRs. Photo-activable p-azidobenzoyl and p-azidosalicyl groups attached to Lys-26 (analogous position at Lys-27 of NmmI) of neurotoxin II labeled γ and δ subunits of the receptor upon photolysis (11Kreienkamp H.J. Utkin Y.N. Weise C. Machold J. Tsetlin V.I. Hucho F. Biochemistry. 1992; 31: 8239-8244Crossref PubMed Scopus (37) Google Scholar, 12Machold J. Weise C. Utkin Y. Tsetlin V. Hucho F. Eur. J. Biochem. 1995; 234: 427-430Crossref PubMed Scopus (45) Google Scholar). Three different photoactivatable groups attached to the equivalent residue Lys-23 of a long neurotoxin, toxin 3, also labeled predominantly the γ and δ subunits in preference to the α subunit (13Utkin Y.N. Krivoshein A.V. Davydov V.L. Kasheverov I.E. Franke P. Maslennikov I.V. Arseniev A.S. Hucho F. Tsetlin V.I. Eur. J. Biochem. 1998; 253: 229-235Crossref PubMed Scopus (20) Google Scholar). Thus mutagenesis and chemical labeling studies showed a crucial role of lysine at position 27 and its proximity to γ and δ subunits. Here, our mutant cycle studies delineate the interaction between Glu-176 of the γ subunit and Lys-27 of NmmI toxin. The largest linkage in loop II between K27E and γE176K (ΔΔG = −5.9kcal/mol) and smaller linkages (ΔΔG INT = −1.6 to −3.0 kcal/mol) found previously between K27E and α subunit residues of Val-188, Tyr-190, Pro-197, and Asp-200 (35Ackermann E.J. Ang E.T.-H. Kanter R.J. Tsigelny I. Taylor P. J. Biol. Chem. 1998; 273: 10958-10964Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) correlate well with the labeling studies. A more complete elucidation of α-toxin-receptor interactions should enable us to orient a docked α-toxin with respect to the subunit interfaces, as well as refine existing models of the structure of the extracellular domain of the receptor (4Tsigelny I. Sugiyama N. Sine S.M. Taylor P. Biophys. J. 1997; 73: 52-66Abstract Full Text PDF PubMed Scopus (69) Google Scholar). The nicotinic acetylcholine receptor (nAChR)1 found in muscle is a pentamer composed of four homologous subunits present in the stoichiometry α2βγδ (fetal subtype) or α2βεδ (adult subtype). The subunits are arranged in a circular manner to surround a central channel in the order, αγαδβ or αεαδβ (1Unwin N. J. Mol. Biol. 1993; 229: 1101-1124Crossref PubMed Scopus (715) Google Scholar, 2Karlin A. Akabas M.H. Neuron. 1995; 15: 1231-1244Abstract Full Text PDF PubMed Scopus (563) Google Scholar, 3Hucho F. Tsetlin V.I. Machold J. Eur. J. Biochem. 1996; 239: 539-557Crossref PubMed Scopus (205) Google Scholar). The two binding sites for agonists, competitive antagonists, and the slowly dissociating α-neurotoxins are formed at interfaces between the αδ and αγ(ε) subunit pairs. The extracellular domain in each subunit is formed principally from the amino-terminal 210 amino acids, which is followed by four membrane-spanning domains. Residues within the amino-terminal 210 amino acids have been shown to be the major contributors to the ligand binding sites and for dictating the order of assembly of subunits. Three segments of the α subunit, well separated along the linear sequence, harbor major determinants for ligand binding; these segments contain the key residues around Tyr-93, between Trp-149 and Asp-152, and spanning the region from Val-188 through Asp-200 (see Refs. 3Hucho F. Tsetlin V.I. Machold J. Eur. J. Biochem. 1996; 239: 539-557Crossref PubMed Scopus (205) Google Scholar and 4Tsigelny I. Sugiyama N. Sine S.M. Taylor P. Biophys. J. 1997; 73: 52-66Abstract Full Text PDF PubMed Scopus (69) Google Scholarfor reviews). Similarly, four discontinuous segments of the non-α subunits, appearing on the opposite face of the subunit, contain major determinants for ligand selectivity; in the γ subunit these segments contain the key residues Lys-34, between Trp-55 and Gln-59, between Ser-111 and Tyr-117, and between Phe-172 and Asp-174. Since the early demonstration of irreversible neuromuscular blockade by the peptide from snake venom, α-bungarotoxin (5Chang C.C. Lee C.Y. Arch. Int. Pharmacodyn. Ther. 1963; 144: 241-257PubMed Google Scholar), and the use of labeled α-neurotoxins to identify the nAChR (6Changeux J.P. Kasai M. Lee C.Y. Proc. Natl. Acad. Sci. U. S. A. 1970; 67: 1241-1247Crossref PubMed Scopus (459) Google Scholar), these toxins have been the primary ligands employed for the identification and characterization of the muscle nAChR. Amino acid sequences are available for nearly 100 members of the α-neurotoxin family, which show a common basic structure consisting of three polypeptide loops emerging from a small globular core (7Endo T. Tamiya N. Pharmacol. Ther. 1987; 34: 403-451Crossref PubMed Scopus (122) Google Scholar). α-Neurotoxins can be divided into the short (4 disulfide bonds and 60–62 residues) and long neurotoxins (5 disulfide bonds and 66–74 residues). Crystal and solution structure determinations reveal similar tertiary structures. Although these structurally well defined toxins are known to bind at the subunit interfaces (αε or αγ and αδ), typically with aK D ≤ 100 pm, little is known about their precise orientation with respect to the subunits that form the interfaces. Points of attachment of α-neurotoxin within the nAChR binding sites have been examined by cross-linking chemically modified (8Witzemann V. Muchmore D. Raftery M.A. Biochemistry. 1979; 18: 5511-5518Crossref PubMed Scopus (43) Google Scholar, 9Hamilton S.L. Pratt D.R. Eaton D.C. Biochemistry. 1985; 24: 2210-2219Crossref PubMed Scopus (25) Google Scholar) or photoactivatable derivatives of α-neurotoxin (10Chatrenet B. Trémeau O. Bontems F. Goeldner M.P. Hirth C.G. Ménez A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3378-8211Crossref PubMed Scopus (30) Google Scholar, 11Kreienkamp H.J. Utkin Y.N. Weise C. Machold J. Tsetlin V.I. Hucho F. Biochemistry. 1992; 31: 8239-8244Crossref PubMed Scopus (37) Google Scholar, 12Machold J. Weise C. Utkin Y. Tsetlin V. Hucho F. Eur. J. Biochem. 1995; 234: 427-430Crossref PubMed Scopus (45) Google Scholar, 13Utkin Y.N. Krivoshein A.V. Davydov V.L. Kasheverov I.E. Franke P. Maslennikov I.V. Arseniev A.S. Hucho F. Tsetlin V.I. Eur. J. Biochem. 1998; 253: 229-235Crossref PubMed Scopus (20) Google Scholar) and by simple ultraviolet irradiation without chemical modification (14Oswald R.E. Changeux J.P. FEBS Lett. 1982; 139: 225-229Crossref PubMed Scopus (52) Google Scholar). These labeling studies have suggested contacts with both α and non-α subunits at the binding sites (see Refs. 2Karlin A. Akabas M.H. Neuron. 1995; 15: 1231-1244Abstract Full Text PDF PubMed Scopus (563) Google Scholar, 3Hucho F. Tsetlin V.I. Machold J. Eur. J. Biochem. 1996; 239: 539-557Crossref PubMed Scopus (205) Google Scholar, and 15Arias H.R. Brain Res. Rev. 1997; 25: 133-191Crossref PubMed Scopus (134) Google Scholar for reviews). Mutagenesis studies have also identified candidate residues in the principal loops of the α (16Kreienkamp H.-J. Sine S.M. Maeda R.K. Taylor P. J. Biol. Chem. 1994; 269: 8108-8114Abstract Full Text PDF PubMed Google Scholar) and non-α subunits (17Sine S.M. J. Biol. Chem. 1997; 272: 23521-23527Crossref PubMed Scopus (60) Google Scholar) that contribute to α-toxin binding. Although most α-toxins do not distinguish between the two sites on the receptor, an α-toxin from the venom of Naja mossambica mossambica (NmmI) distinguishes between the two sites of the Torpedo receptor (18Marchot P. Frachon P. Bougis P.E. Eur. J. Biochem. 1988; 174: 537-542Crossref PubMed Scopus (14) Google Scholar). Thus NmmI emerges as a potentially valuable ligand for determining regions of close approach between α-toxins and the non-α subunits at the binding site. Previous work showed that the αγ and αδ binding sites of the fetal mouse receptor exhibit similar affinities for NmmI (19Ackermann E.J. Taylor P. Biochemistry. 1997; 36: 12836-12844Crossref PubMed Scopus (39) Google Scholar). However, certain mutations in the NmmI toxin structure, and surprisingly also in the nAChR α subunit common to both sites, resulted in nonequivalent reductions in affinity at the αγ and αδ binding sites (19Ackermann E.J. Taylor P. Biochemistry. 1997; 36: 12836-12844Crossref PubMed Scopus (39) Google Scholar). Here we examine binding of recombinant NmmI α-toxin to fetal and adult mouse AChRs and find that the affinity of NmmI for the αε interface is 3 orders of magnitude lower than for the αγ and αδ interfaces. Using subunit chimeras and site-directed mutations in γ and ε subunits, we show that the enhanced affinity conferred by the γ over the ε subunit arises from Pro-175 and Glu-176 in the γ subunit. Mutant cycle analysis shows that Glu-176 interacts with cationic residues in loop II of the NmmI α-toxin. DISCUSSIONThe α-neurotoxins are a family of three-fingered peptide toxins found in venom of elapid snakes (7Endo T. Tamiya N. Pharmacol. Ther. 1987; 34: 403-451Crossref PubMed Scopus (122) Google Scholar). They have proven to be invaluable tools for the isolation and study of the nAChR because of their high affinities and slow rates of dissociation from the receptor (5Chang C.C. Lee C.Y. Arch. Int. Pharmacodyn. Ther. 1963; 144: 241-257PubMed Google Scholar, 6Changeux J.P. Kasai M. Lee C.Y. Proc. Natl. Acad. Sci. U. S. A. 1970; 67: 1241-1247Crossref PubMed Scopus (459) Google Scholar). Although the isolated α-subunit of the receptor retains the capacity to bind α-BgTx, whereas isolated β, γ, or δ subunits do not, the α-toxins bind with far lower affinity to the α subunit than to the intact receptor. Moreover, small agonists and antagonists do not compete with α-toxin binding to the isolated α subunit at expected concentrations (23Gershoni J.M. Hawrot E. Lentz T.L. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4973-4977Crossref PubMed Scopus (85) Google Scholar). These observations point to a predominant, but not sole, contribution to the α-neurotoxin binding coming from the α subunit. Our previous work showed that Val-188, Tyr-190, Pro-197, and Asp-200 of α subunit contribute to NmmI binding (19Ackermann E.J. Taylor P. Biochemistry. 1997; 36: 12836-12844Crossref PubMed Scopus (39) Google Scholar). Also glycosylation at positions 189 and 187, yielding oligosaccharides uniquely found in cobra and mongoose nAChR, reduced α-BgTx binding substantially (16Kreienkamp H.-J. Sine S.M. Maeda R.K. Taylor P. J. Biol. Chem. 1994; 269: 8108-8114Abstract Full Text PDF PubMed Google Scholar).Although the α subunit appears to be the predominant site of α-toxin binding, chemical cross-linking and mutagenesis studies show that non-α subunits are close to the site of α-neurotoxin binding (Refs. 8Witzemann V. Muchmore D. Raftery M.A. Biochemistry. 1979; 18: 5511-5518Crossref PubMed Scopus (43) Google Scholar, 9Hamilton S.L. Pratt D.R. Eaton D.C. Biochemistry. 1985; 24: 2210-2219Crossref PubMed Scopus (25) Google Scholar, 10Chatrenet B. Trémeau O. Bontems F. Goeldner M.P. Hirth C.G. Ménez A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3378-8211Crossref PubMed Scopus (30) Google Scholar, 11Kreienkamp H.J. Utkin Y.N. Weise C. Machold J. Tsetlin V.I. Hucho F. Biochemistry. 1992; 31: 8239-8244Crossref PubMed Scopus (37) Google Scholar, 12Machold J. Weise C. Utkin Y. Tsetlin V. Hucho F. Eur. J. Biochem. 1995; 234: 427-430Crossref PubMed Scopus (45) Google Scholar, 13Utkin Y.N. Krivoshein A.V. Davydov V.L. Kasheverov I.E. Franke P. Maslennikov I.V. Arseniev A.S. Hucho F. Tsetlin V.I. Eur. J. Biochem. 1998; 253: 229-235Crossref PubMed Scopus (20) Google Scholar, 14Oswald R.E. Changeux J.P. FEBS Lett. 1982; 139: 225-229Crossref PubMed Scopus (52) Google Scholar, 16Kreienkamp H.-J. Sine S.M. Maeda R.K. Taylor P. J. Biol. Chem. 1994; 269: 8108-8114Abstract Full Text PDF PubMed Google Scholar, 17Sine S.M. J. Biol. Chem. 1997; 272: 23521-23527Crossref PubMed Scopus (60) Google Scholar, and see Refs. 3Hucho F. Tsetlin V.I. Machold J. Eur. J. Biochem. 1996; 239: 539-557Crossref PubMed Scopus (205) Google Scholar and 15Arias H.R. Brain Res. Rev. 1997; 25: 133-191Crossref PubMed Scopus (134) Google Scholar for reviews). The results described here further illustrate the role of neighboring non-α subunits in contributing to high affinity α-toxin binding, as NmmI binds to αε interfaces of the adult type of nAChR (α2βεδ) with 3 orders of magnitude lower affinity than to the αγ and αδ interfaces of the fetal receptor (α2βγδ). Binding studies, initially using chimeras and subsequently point mutants, show that εThr-176/εAla-177 (Pro/Glu in γ/δ) contribute entirely to insensitivity of the αε interface to NmmI. The observation that α-bungarotoxin association is only slightly affected by the γ and ε sequence differences suggests that this region of the γ, ε, and δ subunits is not used equivalently for stabilization of the entire family of bound α-neurotoxins. At the present time, it is unclear whether stabilization from this region is unique to some of the short α-neurotoxins, or the long α-neurotoxins, such as α-bungarotoxin, acquire the bulk of their stabilization energy from other portions of the structure. Distinct differences in specificity between the short and long neurotoxins have been noted for the α7 subtype of nAChR (24Servent D. Winckler-Dietrich V. Hu H.Y. Kessler P. Drevet P. Bertrand D. Ménez A. J. Biol. Chem. 1997; 272: 24279-24286Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar).Residues at the γ175/176 positions were previously unrecognized as determinants of ligand binding. However, they are immediately adjacent to γAsp-174, which was shown by cross-linking to be ∼9 Å away from Cys-192/193 in the α subunit (25Czajkowski C. Karlin A. J. Biol. Chem. 1991; 266: 22603-22612Abstract Full Text PDF PubMed Google Scholar, 26Czajkowski C. Kaufmann C. Karlin A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6285-6289Crossref PubMed Scopus (106) Google Scholar) and was shown to influence the affinity of quaternary agonists and antagonists (27Martin M.D. Karlin A. Biochemistry. 1997; 36: 10742-10750Crossref PubMed Scopus (17) Google Scholar, 28Osaka H. Sugiyama N. Taylor P. J. Biol. Chem. 1998; 273: 12758-12765Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Moreover, the adjacent residues of γPhe-172 (δAsp-178, εIle-173) are known to confer site-selectivity to the smaller competitive peptide inhibitors such as α-conotoxin MI (29Sine S.M. Kreienkamp H.-J. Bren N. Maeda R. Taylor P. Neuron. 1995; 15: 205-211Abstract Full Text PDF PubMed Scopus (175) Google Scholar) and waglerin (30Molles B.E. Kline E.F. Sine S.M. McArdle J.J. Taylor P. J. Physiol. (paris). 1998; 92 (abstr.): 470Crossref Google Scholar). The equivalent region of the α7 subunit (Asp-163, Ile-164, and Ser-165), which presumably forms a homomeric pentamer of subunits, constitutes part of a putative Ca2+ binding region that faces the ligand binding site (31Galzi J.L. Bertrand S. Corringer P.J. Changeux J.P. Bertrand D. EMBO J. 1996; 15: 5824-5832Crossref PubMed Scopus (133) Google Scholar). At the α subunit interface of the binding site, both aromatic (Tyr-190, Tyr-198) and anionic (Asp-200) residues were mapped to the α-toxin binding surface (19Ackermann E.J. Taylor P. Biochemistry. 1997; 36: 12836-12844Crossref PubMed Scopus (39) Google Scholar). Here, we identify another anionic residue in the γ subunit, Glu-176, perhaps restricted in its position by a neighboring secondary amino acid Pro-175, on the γ subunit, as a crucial residue for binding.The significant linkage between Glu-176 and cationic residues in loop II of the toxin suggests that an electrostatic interaction contributes to the tight binding of the NmmI/nAChR complex. Because the linkage obtained from charge reversal is greater for Lys-27 than for Arg-33 and Arg-36, one would predict that the portion of loop II proximal to loop III of the toxin, is closest to the γ subunit (cf. Fig.5). In this analysis, the loss of free energy (ΔΔG) associated with a single charge mutation results from all interactions between the charged residue and its multiple neighboring residues. The pairwise interactions (ΔΔG INT) resulting in charge reversal of specified residues in the interacting molecules should then highlight the strength of interaction coming from the paired charged residues. In the absence of significant changes in conformation or hydration, ΔΔG INT from Equation 2 should largely reflect the Coulombic interaction between the respective paired residues (32Faiman G.A. Horovitz A. Protein Science. 1996; 9: 315-316Google Scholar).Extensive studies on a related short neurotoxin, erabutoxin a, involving mutations at 36 toxin positions clearly revealed the importance of the tips of loops situated on the concave face of the toxin (33Pillet L. Trémeau O. Ducancel F. Drevet P. Zinn-Justin S. Pinkasfeld S. Boulain J.-C. Ménez A. J. Biol. Chem. 1993; 268: 909-916Abstract Full Text PDF PubMed Google Scholar, 34Trémeau O. Lemaire C. Drevet P. Pinkasfeld S. Ducancel F. Boulain J.-C. Ménez A. J. Biol. Chem. 1995; 270: 9362-9369Crossref PubMed Scopus (131) Google Scholar). These investigations showed that the K27E mutation of erabutoxin a decreases its affinity more than 100-fold forTorpedo nAChRs. Photo-activable p-azidobenzoyl and p-azidosalicyl groups attached to Lys-26 (analogous position at Lys-27 of NmmI) of neurotoxin II labeled γ and δ subunits of the receptor upon photolysis (11Kreienkamp H.J. Utkin Y.N. Weise C. Machold J. Tsetlin V.I. Hucho F. Biochemistry. 1992; 31: 8239-8244Crossref PubMed Scopus (37) Google Scholar, 12Machold J. Weise C. Utkin Y. Tsetlin V. Hucho F. Eur. J. Biochem. 1995; 234: 427-430Crossref PubMed Scopus (45) Google Scholar). Three different photoactivatable groups attached to the equivalent residue Lys-23 of a long neurotoxin, toxin 3, also labeled predominantly the γ and δ subunits in preference to the α subunit (13Utkin Y.N. Krivoshein A.V. Davydov V.L. Kasheverov I.E. Franke P. Maslennikov I.V. Arseniev A.S. Hucho F. Tsetlin V.I. Eur. J. Biochem. 1998; 253: 229-235Crossref PubMed Scopus (20) Google Scholar). Thus mutagenesis and chemical labeling studies showed a crucial role of lysine at position 27 and its proximity to γ and δ subunits. Here, our mutant cycle studies delineate the interaction between Glu-176 of the γ subunit and Lys-27 of NmmI toxin. The largest linkage in loop II between K27E and γE176K (ΔΔG = −5.9kcal/mol) and smaller linkages (ΔΔG INT = −1.6 to −3.0 kcal/mol) found previously between K27E and α subunit residues of Val-188, Tyr-190, Pro-197, and Asp-200 (35Ackermann E.J. Ang E.T.-H. Kanter R.J. Tsigelny I. Taylor P. J. Biol. Chem. 1998; 273: 10958-10964Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) correlate well with the labeling studies. A more complete elucidation of α-toxin-receptor interactions should enable us to orient a docked α-toxin with respect to the subunit interfaces, as well as refine existing models of the structure of the extracellular domain of the receptor (4Tsigelny I. Sugiyama N. Sine S.M. Taylor P. Biophys. J. 1997; 73: 52-66Abstract Full Text PDF PubMed Scopus (69) Google Scholar). The α-neurotoxins are a family of three-fingered peptide toxins found in venom of elapid snakes (7Endo T. Tamiya N. Pharmacol. Ther. 1987; 34: 403-451Crossref PubMed Scopus (122) Google Scholar). They have proven to be invaluable tools for the isolation and study of the nAChR because of their high affinities and slow rates of dissociation from the receptor (5Chang C.C. Lee C.Y. Arch. Int. Pharmacodyn. Ther. 1963; 144: 241-257PubMed Google Scholar, 6Changeux J.P. Kasai M. Lee C.Y. Proc. Natl. Acad. Sci. U. S. A. 1970; 67: 1241-1247Crossref PubMed Scopus (459) Google Scholar). Although the isolated α-subunit of the receptor retains the capacity to bind α-BgTx, whereas isolated β, γ, or δ subunits do not, the α-toxins bind with far lower affinity to the α subunit than to the intact receptor. Moreover, small agonists and antagonists do not compete with α-toxin binding to the isolated α subunit at expected concentrations (23Gershoni J.M. Hawrot E. Lentz T.L. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4973-4977Crossref PubMed Scopus (85) Google Scholar). These observations point to a predominant, but not sole, contribution to the α-neurotoxin binding coming from the α subunit. Our previous work showed that Val-188, Tyr-190, Pro-197, and Asp-200 of α subunit contribute to NmmI binding (19Ackermann E.J. Taylor P. Biochemistry. 1997; 36: 12836-12844Crossref PubMed Scopus (39) Google Scholar). Also glycosylation at positions 189 and 187, yielding oligosaccharides uniquely found in cobra and mongoose nAChR, reduced α-BgTx binding substantially (16Kreienkamp H.-J. Sine S.M. Maeda R.K. Taylor P. J. Biol. Chem. 1994; 269: 8108-8114Abstract Full Text PDF PubMed Google Scholar). Although the α subunit appears to be the predominant site of α-toxin binding, chemical cross-linking and mutagenesis studies show that non-α subunits are close to the site of α-neurotoxin binding (Refs. 8Witzemann V. Muchmore D. Raftery M.A. Biochemistry. 1979; 18: 5511-5518Crossref PubMed Scopus (43) Google Scholar, 9Hamilton S.L. Pratt D.R. Eaton D.C. Biochemistry. 1985; 24: 2210-2219Crossref PubMed Scopus (25) Google Scholar, 10Chatrenet B. Trémeau O. Bontems F. Goeldner M.P. Hirth C.G. Ménez A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3378-8211Crossref PubMed Scopus (30) Google Scholar, 11Kreienkamp H.J. Utkin Y.N. Weise C. Machold J. Tsetlin V.I. Hucho F. Biochemistry. 1992; 31: 8239-8244Crossref PubMed Scopus (37) Google Scholar, 12Machold J. Weise C. Utkin Y. Tsetlin V. Hucho F. Eur. J. Biochem. 1995; 234: 427-430Crossref PubMed Scopus (45) Google Scholar, 13Utkin Y.N. Krivoshein A.V. Davydov V.L. Kasheverov I.E. Franke P. Maslennikov I.V. Arseniev A.S. Hucho F. Tsetlin V.I. Eur. J. Biochem. 1998; 253: 229-235Crossref PubMed Scopus (20) Google Scholar, 14Oswald R.E. Changeux J.P. FEBS Lett. 1982; 139: 225-229Crossref PubMed Scopus (52) Google Scholar, 16Kreienkamp H.-J. Sine S.M. Maeda R.K. Taylor P. J. Biol. Chem. 1994; 269: 8108-8114Abstract Full Text PDF PubMed Google Scholar, 17Sine S.M. J. Biol. Chem. 1997; 272: 23521-23527Crossref PubMed Scopus (60) Google Scholar, and see Refs. 3Hucho F. Tsetlin V.I. Machold J. Eur. J. Biochem. 1996; 239: 539-557Crossref PubMed Scopus (205) Google Scholar and 15Arias H.R. Brain Res. Rev. 1997; 25: 133-191Crossref PubMed Scopus (134) Google Scholar for reviews). The results described here further illustrate the role of neighboring non-α subunits in contributing to high affinity α-toxin binding, as NmmI binds to αε interfaces of the adult type of nAChR (α2βεδ) with 3 orders of magnitude lower affinity than to the αγ and αδ interfaces of the fetal receptor (α2βγδ). Binding studies, initially using chimeras and subsequently point mutants, show that εThr-176/εAla-177 (Pro/Glu in γ/δ) contribute entirely to insensitivity of the αε interface to NmmI. The observation that α-bungarotoxin association is only slightly affected by the γ and ε sequence differences suggests that this region of the γ, ε, and δ subunits is not used equivalently for stabilization of the entire family of bound α-neurotoxins. At the present time, it is unclear whether stabilization from this region is unique to some of the short α-neurotoxins, or the long α-neurotoxins, such as α-bungarotoxin, acquire the bulk of their stabilization energy from other portions of the structure. Distinct differences in specificity between the short and long neurotoxins have been noted for the α7 subtype of nAChR (24Servent D. Winckler-Dietrich V. Hu H.Y. Kessler P. Drevet P. Bertrand D. Ménez A. J. Biol. Chem. 1997; 272: 24279-24286Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Residues at the γ175/176 positions were previously unrecognized as determinants of ligand binding. However, they are immediately adjacent to γAsp-174, which was shown by cross-linking to be ∼9 Å away from Cys-192/193 in the α subunit (25Czajkowski C. Karlin A. J. Biol. Chem. 1991; 266: 22603-22612Abstract Full Text PDF PubMed Google Scholar, 26Czajkowski C. Kaufmann C. Karlin A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6285-6289Crossref PubMed Scopus (106) Google Scholar) and was shown to influence the affinity of quaternary agonists and antagonists (27Martin M.D. Karlin A. Biochemistry. 1997; 36: 10742-10750Crossref PubMed Scopus (17) Google Scholar, 28Osaka H. Sugiyama N. Taylor P. J. Biol. Chem. 1998; 273: 12758-12765Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Moreover, the adjacent residues of γPhe-172 (δAsp-178, εIle-173) are known to confer site-selectivity to the smaller competitive peptide inhibitors such as α-conotoxin MI (29Sine S.M. Kreienkamp H.-J. Bren N. Maeda R. Taylor P. Neuron. 1995; 15: 205-211Abstract Full Text PDF PubMed Scopus (175) Google Scholar) and waglerin (30Molles B.E. Kline E.F. Sine S.M. McArdle J.J. Taylor P. J. Physiol. (paris). 1998; 92 (abstr.): 470Crossref Google Scholar). The equivalent region of the α7 subunit (Asp-163, Ile-164, and Ser-165), which presumably forms a homomeric pentamer of subunits, constitutes part of a putative Ca2+ binding region that faces the ligand binding site (31Galzi J.L. Bertrand S. Corringer P.J. Changeux J.P. Bertrand D. EMBO J. 1996; 15: 5824-5832Crossref PubMed Scopus (133) Google Scholar). At the α subunit interface of the binding site, both aromatic (Tyr-190, Tyr-198) and anionic (Asp-200) residues were mapped to the α-toxin binding surface (19Ackermann E.J. Taylor P. Biochemistry. 1997; 36: 12836-12844Crossref PubMed Scopus (39) Google Scholar). Here, we identify another anionic residue in the γ subunit, Glu-176, perhaps restricted in its position by a neighboring secondary amino acid Pro-175, on the γ subunit, as a crucial residue for binding. The significant linkage between Glu-176 and cationic residues in loop II of the toxin suggests that an electrostatic interaction contributes to the tight binding of the NmmI/nAChR complex. Because the linkage obtained from charge reversal is greater for Lys-27 than for Arg-33 and Arg-36, one would predict that the portion of loop II proximal to loop III of the toxin, is closest to the γ subunit (cf. Fig.5). In this analysis, the loss of free energy (ΔΔG) associated with a single charge mutation results from all interactions between the charged residue and its multiple neighboring residues. The pairwise interactions (ΔΔG INT) resulting in charge reversal of specified residues in the interacting molecules should then highlight the strength of interaction coming from the paired charged residues. In the absence of significant changes in conformation or hydration, ΔΔG INT from Equation 2 should largely reflect the Coulombic interaction between the respective paired residues (32Faiman G.A. Horovitz A. Protein Science. 1996; 9: 315-316Google Scholar). Extensive studies on a related short neurotoxin, erabutoxin a, involving mutations at 36 toxin positions clearly revealed the importance of the tips of loops situated on the concave face of the toxin (33Pillet L. Trémeau O. Ducancel F. Drevet P. Zinn-Justin S. Pinkasfeld S. Boulain J.-C. Ménez A. J. Biol. Chem. 1993; 268: 909-916Abstract Full Text PDF PubMed Google Scholar, 34Trémeau O. Lemaire C. Drevet P. Pinkasfeld S. Ducancel F. Boulain J.-C. Ménez A. J. Biol. Chem. 1995; 270: 9362-9369Crossref PubMed Scopus (131) Google Scholar). These investigations showed that the K27E mutation of erabutoxin a decreases its affinity more than 100-fold forTorpedo nAChRs. Photo-activable p-azidobenzoyl and p-azidosalicyl groups attached to Lys-26 (analogous position at Lys-27 of NmmI) of neurotoxin II labeled γ and δ subunits of the receptor upon photolysis (11Kreienkamp H.J. Utkin Y.N. Weise C. Machold J. Tsetlin V.I. Hucho F. Biochemistry. 1992; 31: 8239-8244Crossref PubMed Scopus (37) Google Scholar, 12Machold J. Weise C. Utkin Y. Tsetlin V. Hucho F. Eur. J. Biochem. 1995; 234: 427-430Crossref PubMed Scopus (45) Google Scholar). Three different photoactivatable groups attached to the equivalent residue Lys-23 of a long neurotoxin, toxin 3, also labeled predominantly the γ and δ subunits in preference to the α subunit (13Utkin Y.N. Krivoshein A.V. Davydov V.L. Kasheverov I.E. Franke P. Maslennikov I.V. Arseniev A.S. Hucho F. Tsetlin V.I. Eur. J. Biochem. 1998; 253: 229-235Crossref PubMed Scopus (20) Google Scholar). Thus mutagenesis and chemical labeling studies showed a crucial role of lysine at position 27 and its proximity to γ and δ subunits. Here, our mutant cycle studies delineate the interaction between Glu-176 of the γ subunit and Lys-27 of NmmI toxin. The largest linkage in loop II between K27E and γE176K (ΔΔG = −5.9kcal/mol) and smaller linkages (ΔΔG INT = −1.6 to −3.0 kcal/mol) found previously between K27E and α subunit residues of Val-188, Tyr-190, Pro-197, and Asp-200 (35Ackermann E.J. Ang E.T.-H. Kanter R.J. Tsigelny I. Taylor P. J. Biol. Chem. 1998; 273: 10958-10964Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) correlate well with the labeling studies. A more complete elucidation of α-toxin-receptor interactions should enable us to orient a docked α-toxin with respect to the subunit interfaces, as well as refine existing models of the structure of the extracellular domain of the receptor (4Tsigelny I. Sugiyama N. Sine S.M. Taylor P. Biophys. J. 1997; 73: 52-66Abstract Full Text PDF PubMed Scopus (69) Google Scholar).
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