Functional stability of water wire-carbonyl interactions in an ion channel

Abstract

Water wires are critical for the functioning of many membrane proteins, as in channels that conduct water, protons, and other ions. Here, in liquid crystalline lipid bilayers under symmetric environmental conditions, the selective hydrogen bonding interactions between eight waters comprising a water wire and a subset of 26 carbonyl oxygens lining the antiparallel dimeric gramicidin A channel are characterized by ¹⁷O NMR spectroscopy at 35.2 T (or 1,500 MHz for ¹H) and computational studies. While backbone ¹⁵N spectra clearly indicate structural symmetry between the two subunits, single site ¹⁷O labels of the pore-lining carbonyls report two resonances, implying a break in dimer symmetry caused by the selective interactions with the water wire. The ¹⁷O shifts document selective water hydrogen bonding with carbonyl oxygens that are stable on the millisecond timescale. Such interactions are supported by density functional theory calculations on snapshots taken from molecular dynamics simulations. Water hydrogen bonding in the pore is restricted to just three simultaneous interactions, unlike bulk water environs. The stability of the water wire orientation and its electric dipole leads to opposite charge-dipole interactions for K⁺ ions bound at the two ends of the pore, thereby providing a simple explanation for an ∼20-fold difference in K⁺ affinity between two binding sites that are ∼24 Å apart. The ¹⁷O NMR spectroscopy reported here represents a breakthrough in high field NMR technology that will have applications throughout molecular biophysics, because of the acute sensitivity of the ¹⁷O nucleus to its chemical environment.

Keywords: 17O NMR; gramicidin A; molecular dynamics; ultra-high field NMR; water wire.

Fig. 1.

Slices through the ¹H-¹⁵N dipolar and ¹⁵N anisotropic chemical shift dimensions of the 2D OS ssNMR (SAMPI4) spectra of ¹⁵N G2- and ¹⁵N A3-labeled gA at 35.2 T (36), showing excellent alignment of gA in liquid-crystalline lipid bilayers of DMPC and precise dimeric symmetry of the structure. The gramicidin structure is a dimer (upper monomer carbon is light blue and nitrogen dark blue; lower monomer carbon is gold and nitrogen dark blue). Oxygen is red and hydrogen is white. This illustrates the two ¹⁵N-¹H sites in each subunit (dark blue and white spheres) that were ¹⁵N-labeled for this OS ssNMR sample in the liquid-crystalline preparation. The spectrum of the two A3 sites is shown on the left and the two G2 sites on the right.

Fig. 2.

¹⁷O OS ssNMR of gA dimer in liquid-crystalline lipid bilayers at 35.2 T. G2, L4, L10, L12, and L14 were individually labeled with ¹⁷O such that each sample was labeled in both gA subunits with the same isotopically labeled amino acid residue as shown in the structural model. The two subunits are distinguished here with residue labels primed in one subunit and unprimed in the other subunit. The ¹H decoupled spectra show single resonances for L12 and L14 and two resonances for each of the other three sites: G2, L4, and L10. The colors in the structure are explained in Fig. 1.

Fig. 3.

(A) MD snapshot of a water wire, placed in the 1MAG structure and optimized by DFT, showing hydrogen bonding between the water wire and L10 and G2 carbonyls of the upper subunit (bright green) as well as hydrogen bonding between water molecules (pale green). Colors of atoms as in Fig. 1 except that the ¹⁷O-labeled peptide planes are colored in turquoise. L4 does not receive a hydrogen bond while L4′ appears to be in a position to receive a weak hydrogen bond. (B–D) Observed ¹⁷O spectral resonances for the L10 (B), L4 (C), and G2 (D) ¹⁷O (blue). The resonances marked (*) are shifted upfield by hydrogen bonding with water. Also shown are DFT calculations of the ¹⁷O frequency shift difference between the two subunits (red bars) compared to the observed shift difference (blue bars). Water interactions with these six carbonyl oxygens in the DFT optimized structure are shown with individual carbonyl oxygens (deep red) interacting or not interacting with water molecules..

Fig. 4.

¹⁷O OS ssNMR spectroscopy of ¹⁷O single site labeled gA at G2, L4, L10, L12, and L14 in liquid-crystalline lipid bilayers with 0.0 M K⁺, 0.07 M K⁺, and 2.4 M K⁺ (spectra for L4 and L14 at 0.07 M K⁺ [green] were not recorded as only small differences were observed for their spectra at 0.0 [blue] and 2.4 M K⁺ [black] ions). The high-affinity bound K⁺ ion is illustrated as a purple sphere and the low-affinity K⁺ ion as a yellow dotted sphere. The ion positions are based on previously published data (21) and the current study.