Validation of the single-stranded channel conformation of gramicidin A by solid-state NMR

Abstract

The monovalent cation selective channel formed by a dimer of the polypeptide gramicidin A has a single-stranded, right-handed helical motif with 6.5 residues per turn forming a 4-A diameter pore. The structure has been refined to high resolution against 120 orientational constraints obtained from samples in a liquid-crystalline phase lipid bilayer. These structural constraints from solid-state NMR reflect the orientation of spin interaction tensors with respect to a unique molecular axis. Because these tensors are fixed in the molecular frame and because the samples are uniformly aligned with respect to the magnetic field of the NMR spectrometer, each constraint restricts the orientation of internuclear vectors with respect to the laboratory frame of reference. The structural motif of this channel has been validated, and the high-resolution structure has led to precise models for cation binding, cation selectivity, and cation conductance efficiency. The structure is consistent with the electrophysiological data and numerous biophysical studies. Contrary to a recent claim [Burkhart, B. M., Li, N., Langs, D. A., Pangborn, W. A. & Duax, W. L. (1998) Proc. Natl. Acad. Sci. USA 95, 12950-12955], the solid-state NMR constraints for gramicidin A in a lipid bilayer are not consistent with an x-ray crystallographic structure for gramicidin having a double-stranded, right-handed helix with 7.2 residues per turn.

Figure 1

(A) The primary structural constraints for the polypeptide backbone are derived from the ¹⁵N–¹H and ¹⁵N–¹³C dipolar interactions, the C_α–²H quadrupolar interaction, and the anisotropic ¹⁵N chemical shift. The initial structure is developed by determining each peptide-plane orientation with respect to the magnetic field axis with two dipolar interactions. The relative orientations of the peptide planes is then determined (i.e., the φ and ψ angles) for a diplane structure. (B) The initial structure is assembled sequentially with overlapping diplanes. The initial structure is not a unique structure because of chirality ambiguities; however, the molecular fold, hydrogen-bonding pattern, helical sense, and residues per turn are uniquely defined.

Figure 2

Gramicidin A structures in different environments. In addition to the atomic ball-and-stick structures, a ribbon is added to accentuate the handedness and strandedness of the helix. The two monomers have different colored ribbons: one yellow and one orange. The backbone carbonyl oxygens that line the pore of the channel are highlighted in red, and the indole ¹⁵N sites, important in dictating the strandedness of the structure in a membrane environment, is shown in blue. For each structure the Ala³–Leu⁴ peptide-plane orientation is shown with respect to B. (A) The solid-state NMR-derived structure from a bilayer environment: single-stranded, right-handed, and 6.5 residues per turn (ref. ; PDB accession no. 1MAG). (B) An x-ray crystallographic structure of crystals prepared from Cs⁺/MeOH solution: double-stranded, right-handed, and 7.2 residues per turn (ref. ; PDB accession no. 1AV2). (C) A solution NMR structure from an SDS micellar environment: single-stranded, right-handed, and 6.3 residues per turn (ref. ; PDB accession no. 1GRM). (D) An x-ray crystallographic structure of crystals prepared from benzene/methanol solution: double-stranded, left-handed, and 5.6 residues per turn (ref. ; PDB accession no. 1ALZ).

Figure 3

Predicted NMR observables from the four structures shown in Fig. 2 are compared with the experimental values. The vertical scale is the difference in predicted and observed values of the NMR observables for the backbone divided by the error bar for each class of observables: 5 ppm for the ¹⁵N chemical shift, 100 Hz for the ¹⁵N–¹³C dipolar interaction; 2 kHz for the ¹⁵N–¹H dipolar interaction; and 5 kHz for the C_α–²H quadrupolar interaction. In addition to the deviations for the four structures shown in Fig. 2 (letters A–D here correspond to structures A–D in Fig. 2), the deviations are also shown for the solid-state NMR derived initial structure (A1). This structure is calculated based on the dipolar constraints and not on the ¹⁵N chemical shift or C_α–²H quadrupolar interactions; therefore, the deviations displayed for these data represent a validation of the initial structure that defines the structural motif.