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-Å 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 ref lect 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. Orientational constraints derived from solid-state NMR can be used to determine high-resolution three-dimensional structures. Such an approach has been used to define the structure of the ion channel, gramicidin A, in lamellar phase lipids (ref. 1; PDB accession no. 1MAG). Although a reasonable model of this structure has been extant for nearly 30 years (2) and a structure was determined by solution NMR spectroscopy in SDS micelles (3, 4), crystallographic and solution NMR methods have not been successful in a lipid environment. Recently, the validity of the solid-state NMR structure has been challenged (5). In this report, the structural fold of the channel is validated by comparing predicted and observed values for structural constraints not used quantitatively in solving for the structural fold. Furthermore, the NMR observables are compared with predicted values from several structures in the Protein Data Bank. The results establish the high resolution of the solid-state structure and the clear validity of this motif in a lipid environment.Gramicidin A is a polymorphic structure and the dominant sequence in gramicidin D, the biosynthetic product from Bacillus brevis: HCO-Val-Gly-Ala-DLeu-Ala-DVal-Val-ValTrp-DLeu-Trp-DLeu-Trp-DLeu-Trp-NHCH 2 CH 2 OH. In isotropic organic solvents, this peptide typically forms a doublestranded dimer that may be parallel or antiparallel, lefthanded or right-handed and has a range of residues per turn from 5.6 to 7.2 (5-10). In the heterogeneous anisotropic lipid environment, the structure is almost exclusively singlestranded. Because of the short helix length, a single-stranded monomer buries one of its termini in the bilayer. Exposure of the carboxyl terminus to the bilayer surface and lack of exposure for the amino terminus was documented by shift reagent NMR experiments (11). It has been observed that the native monovalent cation selective channel function is maintained when formal charges are introduced at the carboxyl termin...

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