Structure of the first transmembrane domain of the neuronal acetylcholine receptor beta2 subunit

Abstract

The recent cryoelectron microscopy structure of the Torpedo nicotinic acetylcholine receptor (nAChR) at 4-A resolution shows long helices for all transmembrane (TM) domains. This is in disagreement with several previous reports that the first TM domain of nAChR and other Cys-loop receptors are not entirely helical. In this study, we determined the structure and backbone dynamics of an extended segment encompassing the first TM domain (TM1e) of nAChR beta(2) subunit in dodecylphosphocholine micelles, using solution-state NMR and circular dichroism (CD) spectroscopy. Both CD and NMR results show less helicity in TM1e than in Torpedo nAChR structure (Protein Data Bank: 2BG9). The helical ending residues at the C-terminus are the same in the TM1e NMR structure and the Torpedo nAChR structure, but the helical starting residue (I-217) in TM1e is seven residues closer to the C-terminus. Interestingly, the helical starting residue is two residues before the highly conserved P-219, in accordance with the hypothesis that proline causes helical distortions at three residues preceding it. The NMR relaxation measurements show a dynamics pattern consistent with TM1e structure. The substantial nonhelical content adds greater flexibilities to TM1e, thereby implicating a different molecular basis for nAChR function compared to a longer and more rigid helical TM1.

FIGURE 1

Far-ultraviolet CD spectrum of TM1e of the human n-acetylcholine receptor β₂ subunit in DPC micelles, showing ∼49% helical content.

FIGURE 2

The backbone amide proton region of a ¹H-¹⁵N HSQC NMR spectrum of 0.83 mM TM1e of the human n-acetylcholine receptor β₂ subunit in aqueous DPC micelles at 30°C. The resonance assignments are indicated by the one-letter amino acid code and the relative sequence number.

FIGURE 3

Summary of hydrogen-binding (HB) restraints, NOE connectivity, and the CSI for H^α protons of TM1e of the human n-acetylcholine receptor β2 subunit in DPC micelles. The residues, which temperature coefficients were smaller than 0.004 ppm/K, were considered being involved in HB (•). Otherwise, the residues were considered without HB (○). The temperature coefficient of Leu-11 was not determined due to its weak NMR signal. The remaining residues were not ¹⁵N labeled, and their temperature coefficients were not determined. Sequential and midrange-range NOE connectivity is linked by line segments with widths proportional to the observed NOE intensities.

FIGURE 4

(A) One of the lowest energy NMR structures of TM1e and (B) the structure of the TM1 domain of the Torpedo nAChR β₁-subunit derived from the cryo-EM data (6) are shown for comparison. The highly conserved proline residue (Pro-14 in TM1e and Pro-227 in the β₁ subunits) is highlighted in yellow. Notice that the TM1e structure of the β₂ subunit has 15 residues in the helical region, compared to 20 residues in the structural model of the β₁ subunit derived from the 4-Å cryo-EM data.

FIGURE 5

The longitudinal and transverse relaxation rate constants, (A) R₁ and (B) R₂, (C) the ¹⁵N-{¹H} NOE values, and (D) the squared order parameter S² derived from the Tensor 2 analysis of the backbone amide ¹⁵N of TM1e in DPC micelles at 30°C. Errors were derived from the uncertainties of the least-squares fitting to the exponential decay function (for R₁ and R₂) or from the signal/noise ratios (for NOE). For S², error bars are standard deviation derived from 300 Monte Carlo simulations.