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Toxins (Basel)
2019 Oct 16;1110:. doi: 10.3390/toxins11100603.
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Identification of Crucial Residues in α-Conotoxin EI Inhibiting Muscle Nicotinic Acetylcholine Receptor.
Ning J
,
Ren J
,
Xiong Y
,
Wu Y
,
Zhangsun M
,
Zhangsun D
,
Zhu X
,
Luo S
.
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α-Conotoxins (α-CTxs) are small disulfide-rich peptides from venom of Conus species that target nicotinic acetylcholine receptors (nAChRs). The muscle-type nAChRs have been recognized as a potential target for several diseases, such as myogenic disorders, muscle dystrophies, and myasthenia gravis. EI, an α4/7-CTx, mainly blocks α1β1δε nAChRs and has an extra N-terminal extension of three amino acids. In this study, the alanine scanning (Ala-scan) mutagenesis was applied in order to identify key residues of EI for binding with mouse α1β1δε nAChR. The Ala-substituted analogues were tested for their abilities of modulating muscle and neuronal nAChRs in Xenopus laevis oocytes using two-electrode voltage clamp (TEVC) recordings. Electrophysiological results indicated that the vital residues for functional activity of EI were His-7, Pro-8, Met-12, and Pro-15. These changes exhibited a significant decrease in potency of EI against mouse α1β1δε nAChR. Interestingly, replacing the critical serine (Ser) at position 13 with an alanine (Ala) residue resulted in a 2-fold increase in potency at the α1β1δε nAChR, and showed loss of activity on α3β2 and α3β4 nAChRs. Selectivity and potency of [S13A] EI was improved compared with wild-type EI (WT EI). In addition, the structure-activity relationship (SAR) of EI revealed that the "Arg1-Asn2-Hyp3" residues at the N-terminus conferred potency at the muscle-type nAChRs, and the deletion analogue △1-3 EI caused a total loss of activity at the α1β1δε nAChR. Circular dichroism (CD) spectroscopy studies demonstrated that activity loss of truncated analogue △1-3 EI for α1β1δε nAChR is attributed to disturbance of the secondary structure. In this report, an Ala-scan mutagenesis strategy is presented to identify crucial residues that are significantly affecting potency of E1 for mouse α1β1δε nAChR. It may also be important in remodeling of some novel ligands for inhibiting muscle-type nAChRs.
81872794 National Natural Science Foundation of China, 31760249 National Natural Science Foundation of China, 81660585 National Natural Science Foundation of China, 81420108028 Major International Joint Research Project of National Natural Science Foundation of China, hdkyx201725 Hainan University Youth foundation, IRT_15R15 Changjiang Scholars and Innovative Research Team in University Grant
Figure 1. The HPLC and ESI–MS profiles of α-CTx EI and [S13A] EI. The peptide EI was purified to a single compound using a reversed-phase analytical Vydac C18 column, eluted over a linear gradient 10–45% buffer B for 20 min, where buffer A = 0.075% TFA, remainder H2O and buffer B = 0.050% TFA, 90% acetonitrile, remainder H2O. (A) HPLC chromatogram of fully oxidized and folded peptide EI. (B) A monoisotopic mass of 2092.82 Da (calculated 2092.84 Da) for EI was observed in the ESI–MS spectrum. (C) HPLC chromatogram of fully oxidized peptide [S13A] EI. (D) ESI–MS data for [S13A] EI with observed monoisotopic mass of 2076.80 Da (Calculated 2076.82 Da).
Figure 2. [S13A] EI inhibition of α1β1δε, α3β2, and α3β4 nicotinic acetylcholine receptors (nAChRs) compared with WT EI inhibition of these receptors. Cloned mouse α1β1δε (A), rat α3β4 (B), rat α3β2 (C) nAChR subtypes heterologously expressed in Xenopus laevis oocytes were recorded by TEVC. Superimposed traces representative of ACh-evoked current inhibition of α1β1δε (A), α3β4 (B), and α3β2 (C) nAChR subtypes by EI (I) and [S13A] EI (II). All data represent mean ± S.E.M, n = 3–5.
Figure 3. Effect of α-CTx EI and analogues at the mouse α1β1δε nAChR. (A) Concentration–response analysis for inhibition of mouse α1β1δε nAChR by Ala-substituted analogues in N-terminal “tail” amino acids. (B) Concentration–response curves for the inhibitory of mouse α1β1δε nAChR by EI analogues with Ala substitutions in the loop1 region. (C) The inhibition of mouse α1β1δε nAChR by EI analogues with Ala substitutions in the loop2 region was analyzed by concentration–response studies. (D) Concentration–response analysis for inhibition of mouse α1β1δε nAChR by N-terminally truncated analogues in α-CTx EI. All data represent mean ± S.E.M., n = 6–8.
Figure 4. The effect on mouse α1β1δε expressed in Xenopus laevis oocytes by N-terminal truncated analogues. Mouse α1β1δε (A), rat α3β4 (B), rat α3β2 (C) nAChR subtypes expressed in Xenopus oocytes were activated by ACh. Superimposed traces representative of ACh-evoked current inhibition of α1β1δε (A), α3β4 (B), and α3β2 (C) nAChR subtypes by △1–2 EI (I) and △1–3 EI (II). All data represent mean ± S.E.M, n = 3–5.
Figure 5. Characteristics of EI and its analogues. Circular dichroism (CD) spectra of Ala substitutions in the sequence of α-CTx EI compared with native globular EI. (A). It revealed that [S13A] EI mutant had similar spectra to globular EI. (B). [P8A] EI, [P15A] EI, and △1–3 EI analogues do not display typically the α-helix characteristic and exhibit just a positive peak, indicating their secondary structures are disrupted.
Figure S1. The HPLC chromatograms and mass spectrum of EI, [R1A] EI and [D2A] EI respectively.(A) HPLC chromatograms of EI and the retention of EI is 12.31 min; (B) electrospray ionization mass spectrometry (ESI-MS) data for EI with an observed monoisotopic mass of 2092.82 Da. (C) HPLC chromatograms of [R1A] EI and the retention of[R1A] EI is 12.82 min; (D) electrospray ionization mass spectrometry (ESI-MS) data for [R1A] EI with an observed monoisotopic mass of 2054.81 Da.(E) HPLC chromatograms of [D2A] EI and the retention of [D2A] EI is 12.62 min;(F) electrospray ionization mass spectrometry (ESI-MS) data for [D2A] EI with an observed monoisotopic mass of 2049.78 Da.
Figure S2. HPLC chromatograms and mass spectrum of [O3A] EI, [Y6A] EI and [H7A] EI respectively.
(A) HPLC chromatograms of [O3A] EI and the retention of [O3A] EI is 12.93 min; (B) electrospray ionization mass spectrometry (ESI-MS) data for [O3A] EI with an observed monoisotopic mass of 2051.62 Da. (C)HPLC chromatograms of [Y6A] EI and the retention of [Y6A] EI is 12.24 min; (D) electrospray ionization mass spectrometry (ESI-MS) data for [Y6A] EI with an observed monoisotopic mass of 2051.62 Da. (E) HPLC chromatograms of GI [H7A] EI and the retention of [H7A] EI is 12.70 min; (F) electrospray ionization mass spectrometry (ESI-MS) data for [H7A] EI with an observed monoisotopic mass of 2027.54 Da.
Figure S3. HPLC chromatograms and mass spectrum of [P8A] EI, [T9A] EI and [N11A] EI respectively. (A) HPLC chromatograms of [P8A] EI and the retention of [P8A] EI is 12.55 min; (B) electrospray ionization mass spectrometry (ESI-MS) data for [P8A] EIwith an observed monoisotopic mass of 2066.80 Da. (C) HPLC chromatograms of [T9A] EI and the retention of [T9A] EI is 12.18 min; (D) electrospray ionization mass spectrometry (ESI-MS) data for [T9A] EI with an observed monoisotopic mass of 2063.66 Da. (E) HPLC chromatograms of [N11A] EI and the retention of [N11A] EI is 14.22min; (F) electrospray ionization mass spectrometry (ESI-MS) data for [N11A] EI with an observed monoisotopic mass of 2050.50 Da.
Figure S4. HPLC chromatograms and mass spectrum of [M12A] EI, [S13A] EI and [N14A] EI respectively. (A) HPLC chromatograms of [M12A] EI and the retention of [M12A] EI is 11.05 min; (B) electrospray ionization mass spectrometry (ESI-MS) data for [M12A] EI with an observed monoisotopic mass of 2033.58 Da. (C) HPLC chromatograms of [S13A] EI and the retention of [S13A] EI is 12.91 min; (D) electrospray ionization mass spectrometry (ESI-MS) data for [S13A] EI with an
observed monoisotopic mass of 2076.80 Da. (E) HPLC chromatograms of [N14A] EI and the retention of [N14A] EI is 13.52 min; (F) electrospray ionization mass spectrometry (ESI-MS) data for [N14A] EI with an observed monoisotopic mass of 2050.50 Da.
Figure S5. HPLC chromatograms and mass spectrum of [P15A] EI, [Q16A] EI and [I17A] EI respectively. (A) HPLC chromatograms of [P15A] EI and the retention of [P15A] EI is 12.43 min; (B) electrospray ionization mass spectrometry (ESI-MS) data for [P15A] EI with an observed monoisotopic mass of 2066.80 Da. (C) HPLC chromatograms of [Q16A] EI and the retention of [Q16A] EI is 13.23 min; (D) electrospray ionization mass spectrometry (ESI-MS) data for [Q16A] EI with an observed monoisotopic mass of 2035.80 Da. (E) HPLC chromatograms of [I17A] EI and the retention of [I17A] EI is 11.76 min; (F) electrospray ionization mass spectrometry (ESI-MS) data for [I17A] EI with an observed monoisotopic mass of 2050.76 Da.
Figure S6. HPLC chromatograms and mass spectrum of △1EI, △1-2EI and △1-3EI respectively. (A) HPLC chromatograms of △1EIand the retention of △1EIis 12.63 min; (B) electrospray ionization mass spectrometry (ESI-MS) data for △1EIwith an observed monoisotopic mass of 1937.62 Da. (C) HPLC chromatograms of △1-2EIand the retention of △1-2EIis 12.18 min; (D) electrospray ionization mass spectrometry (ESI-MS) data for △1-2EIwith an observed monoisotopic mass of 1822.68 Da. (E) HPLC chromatograms of △1-3EIand the retention of △1-3EI is 12.59 min; (F) electrospray ionization mass
spectrometry (ESI-MS) data for △1-3EIwith an observed monoisotopic mass of 1709.28 Da
Figure S7. Three-dimensional structure of (A) EI (PDB: 1K64), (B) PIA (PDB: 1ZLC), (C) GID (PDB: 1MTQ), comparison of the sequences of α-Conotoxin EI (Reference 16), PIA (Reference 25) and GID (Reference 24).
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