NMR Mapping and Secondary Structure Determination of the Major Acetylcholine Receptor R-Subunit Determinant Interacting with R-Bungarotoxin†,‡

The R-subunit of the nicotinic acetylcholine receptor (RAChR) contains a binding site for R-bungarotoxin (R-BTX), a snake-venom-derived R-neurotoxin. Previous studies have established that the segment comprising residues 173-204 of RAChR contains the major determinant interacting with the toxin, but the precise boundaries of this determinant have not been clearly defined to date. In this study, we applied NMR dynamic filtering to determine the exact sequence constituting the major RAChR determinant interacting with R-BTX. Two overlapping synthetic peptides corresponding to segments 179200 and 182-202 of the RAChR were complexed with R-BTX. HOHAHA and ROESY spectra of these complexes acquired with long mixing times highlight the residues of the peptide that do not interact with the toxin and retain considerable mobility upon binding to R-BTX. These results, together with changes in the chemical shifts of the peptide protons upon complex formation, suggest that residues 184-200 form the contact region. At pH 4, the molecular mass of the complex determined by dynamic light scattering (DLS) was found to be 11.2 kDa, in excellent agreement with the expected molecular mass of a 1:1 complex, while at pH >5 the DLS measurement of 20 kDa molecular mass indicated dimerization of the complex. These results were supported by T2 measurements. Complete resonance assignment of the 11.2 kDa complex of R-BTX bound to the RAChR peptide comprising residues 182-202 was obtained at pH 4 using homonuclear 2D NMR spectra measured at 800 MHz. The secondary structures of both R-BTX and the bound RAChR peptide were determined using 2D 1H NMR experiments. The peptide folds into a â-hairpin conformation, in which residues RH186-RV188 and RY198-RD200 form the two â-strands. Residues RY189-RT191 form an intermolecular â-sheet with residues BK38-BV40 of the second finger of R-BTX. These results accurately pinpoint the R-BTX-binding site on the RAChR and pave the way to structure determination of this important RAChR determinant involved in binding acetylcholine and cholinergic agonists and antagonists. The nicotinic acetylcholine receptor (AChR)1 is a ligandgated ion channel that is activated upon binding of acetylcholine. It is a 290 kDa membrane glycoprotein found in muscle and neuronal tissues consisting of five homologous subunits in the stoichiometry of R2âδγ or R2âδ (1-3). Located on the postsynaptic surface of the neuromuscular junction, the AChR translates the chemical signal of acetylcholine binding into an electrical one, leading to muscle contraction. The acetylcholine-binding site is formed by the Rδ and Rγ subunits (4). The R-subunit also contains a highaffinity-binding site for R-neurotoxin antagonists (5, 6), as well as the major immunogenic region (MIR), which is the main target of autoimmune antibodies in myasthenia gravis (7-9). R-Bungarotoxin (R-BTX) is a 74 amino acid, 8 kDa R-neurotoxin derived from the venom of Bungarus multicinctus. It binds to the postsynaptic muscle AChR with a KD of 10-11 M (10), competitively inhibiting acetylcholine binding, thereby preventing the depolarizing action on postsynaptic membranes and blocking neuromuscular transmission (11). Electron microscopy (12-14) and photolabeling experiments indicate that the binding site for R-BTX is restricted mostly to the R-subunit (15, 16), and partially overlaps that of acetylcholine (17-19). RAChR isolated from Torpedo californica binds R-BTX even after denaturation, indicating that a contiguous segment of this subunit forms a binding site for R-BTX (15). A major determinant of the toxin-binding site was mapped to residues 173-204 of RAChR (20). A 32 residue peptide corresponding to this segment binds R-BTX with a KD of 1.4 × 10-7 M, which is † This study was supported by a US-Israel Binational Science Foundation Grant 98-328 to J.A. ‡ The chemical shifts have been deposited in BioMagResBank under accession number 4838. * To whom correspondence should be addressed. J.A. is the Dr. Joseph and Ruth Owades Professor of Chemistry. Tel: 972 8 9343394, Fax: 972 8 9344136, E-mail: [email protected]. § Department of Structural Biology. | Chemical Services. 1 Abbreviations: AChR, nicotinic acetylcholine receptor; RAChR, R-subunit of AChR; R-BTX, R-bungarotoxin; NMR, nuclear magnetic resonance; 2D-NMR, two-dimensional NMR; HOHAHA, homonuclear Hartmann-Hahn spectroscopy; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser spectroscopy; ROESY, rotating-frame Overhauser spectroscopy; DQF-COSY, double-quantum-filtered correlation spectroscopy; T1F, spin-lattice relaxation time in the rotating frame; T2, transverse relaxation time constant; CD, circular dichroism; DLS, dynamic light scattering; FPLC, fast protein liquid chromatography; RP-HPLC, reverse-phase high-pressure liquid chromatography; IC50, concentration of competing inhibitor resulting in a 50% decrease in binding of the assayed ligand; R-BTX and RAChR peptide residues are designated by a superscript B (BX) and R (RX), respectively, before the one-letter amino acid code and position in the sequence. 5464 Biochemistry 2001, 40, 5464-5473 10.1021/bi0022689 CCC: $20.00 © 2001 American Chemical Society Published on Web 04/11/2001 comparable to the affinity of the intact denatured RAChR to the toxin (21, 22). Several studies have focused upon the R-BTX affinity to RAChR synthetic peptide analogues in an attempt to locate the R-BTX-binding domain on the receptor more precisely. These studies suggest various putative binding domains within the aforementioned 32 amino acid peptide such as residues 185-199 (23), 181200 (24), 179-196 (21, 22), 184-200 (25), and 185-196 (26). Concurrently, site-directed mutagenesis and affinity labeling studies have pinpointed residues within this domain critical to acetylcholine and antagonist binding (27, 28). Complexes of RAChR peptide analogues with R-BTX have also been the subject of structural studies. The solution structure of R-BTX in complex with a dodecapeptide, KHWVYYTCCPDT, corresponding to residues 185-196 of Torpedo RAChR, was solved by Basus and co-workers using NMR (29). They established interactions between R-BTX and the segment corresponding to residues 186-190 of the RAChR, yet no interactions were detected for the C-terminal half of the peptide. Thus, the short peptide studied by Basus and co-workers represents only a fraction of the R-BTXbinding domain in RAChR. Anglister, Katchalski-Katzir, and co-workers (30) recently determined the three-dimensional solution structure of R-BTX in complex with a 13 residue peptide (MRYYESSLKSYPD) selected from a phage-displayed peptide library. This complex is of special interest, since this tridecapeptide exhibits a 15-fold higher affinity to R-BTX in comparison to the dodecapeptide RAChR used by Basus et al. While the peptide corresponding to residues 185-196 of RAChR adopts an extended conformation when bound to the toxin (29), the toxin-bound library peptide is nearly globular and occupies a larger surface area of the R-BTX-binding site. In view of the larger number of interactions and the 15-fold higher binding constant for the library peptide, the globular conformation of this peptide seems to mimic a larger RAChR determinant, and provides a more detailed picture of the R-BTX-binding site for AChR. Despite these recent advances in the structural understanding of RAChR peptides in complex with R-BTX, the boundaries of the binding domain on the receptor are uncertain to date. While previous studies have been instrumental in locating this domain within the 32-mer segment, a systematic residue-by-residue truncation experiment has not been performed, and the precise boundaries of the binding domain remain undetermined. A comparison of the competitive inhibition of R-BTX binding to AChR by the aforementioned 12-mer (residues 185-196, IC50 ) 1.3 × 10-5 M), 18-mer (residues 181-198, IC50 ) 9.3 × 10-6 M), and 32-mer (residues 173-204, IC50 ) 1.4 × 10-7 M) (21) indicates that significant contact area is contributed by residues outside the shorter segments. These findings underline the need for structural studies of R-BTX complexes with longer peptide analogues of RAChR, capable of addressing the relevant structural questions at atomic resolution. A complete high-resolution structure of AChR has not been solved yet due to the difficulties in crystallizing this membrane protein. Different models of RAChR disagree on the presence of secondary structure in the R-BTX-binding domain (32-34). Tritium hydrogen exchange kinetics of the AChR have been analyzed, and retardation in the exchange rate was observed in the presence of R-BTX (35). It was suggested that R-BTX shields the AChR by forming an intermolecular â-sheet, thereby decreasing solvent accessibility. Circular dichroism (CD) measurements of a peptide corresponding to residues 185-196 of the RAChR indicated an increase of â-structure upon R-BTX binding (36). In this study, we use the sensitivity of the homonuclear Hartmann-Hahn (HOHAHA) and rotating-frame Overhauser spectroscopy (ROESY) NMR experiments to the T1F relaxation time of the detected protons. Peptide protons interacting with the toxin are immobilized upon binding and assume a T1F relaxation time comparable to that of the toxin protons. Peptide protons with no interaction with the toxin retain some mobility and have considerably longer T1F relaxation time. The mixing period in the HOHAHA and ROESY experiments is tuned to discriminate between the immobilized and flexible peptide protons, thus enabling us to accurately locate the R-BTX-binding domain on the RAChR. Standard 2DNMR techniques are applied to determine the secondary structure of the RAChR peptide in the binding site of R-BTX. Identification of the complete linear segment in RAChR recognized by R-BTX and determination of its secondary structure are a prerequisite for detailed structural studies by NMR or X-ray crystallography. In this study, we map the determinant of the RAChR involved in strong R-BTX binding, and elucidate the secondary structure of the RAChR peptide bound to R-BTX. EXPERIMENTAL PROCEDURES Peptide Synthesis and Complex Formation. The peptides Rp22 (KEARGWKHWVFYSCCPTTPYLD) and Rp25 (EERGWKHWVYYTCCPDTPYLDITEE), comprising residues 179-200 of mouse and residues 182-202 of Torpedo AChR, respectively, were synthesized on an AMS422 automated multiple peptide synthesizer (Gilson) and purified by RPHPLC. Rp25 was elongated by two glutamic residues at each terminus to increase peptide solubility. Formation of the RC192-RC193 disulfide bond was ensured for both peptides, emulating their oxidation state in the native AChR (37), although this state has little effect on R-BTX binding (38). In Rp25, this disulfide bond was formed by air-oxidation in a dilute peptide solution (10 mg/250 mL) to avoid oligomerization (39). The Ellman reagent was used to monitor the completion of the reaction (40). Rp22 was not treated, but its mass spectrum suggested it had oxidized during purification. The oxidized peptides were lyophilized and purified by RP-HPLC with an acetonitrile gradient. The composition of the peptides was verified by amino acid analysis, and their molecular mass and oxidation state were confirmed by mass spectrometry. R-BTX was purchased from Sigma. The R-BTX/Rp22 complex was prepared by mixing the peptide and R-BTX at a molar ratio of 0.75:1, respectively. The R-BTX/Rp25 complex was prepared by addition of excess oxidized Rp25 to R-BTX. To obtain a 1:1 complex of R-BTX and Rp25, this complex was purified by gel-filtration FPLC on a Pharamcia S-75 gel filtration column, using 250 mM NH4HCO3 as running buffer, followed by lyophilization. The formation of the complexes was verified by polyacrylamide gel electrophoresis, transverse NMR relaxation time (T2) measurements, and dynamic light scattering. Complex formation was also monitored by the disappearance of the BH4(Hδ) and the BW28(HN) NMR of the AChR Determinant Interacting with R-BTX Biochemistry, Vol. 40, No. 18, 2001 5465