Water inside the selectivity filter of a K+ ion channel: structural heterogeneity, picosecond dynamics, and hydrogen-bonding

Abstract

Water inside biological ion channels regulates the key properties of these proteins such as selectivity, ion conductance, and gating. In this Article we measure the picosecond spectral diffusion of amide I vibrations of an isotope labeled KcsA potassium channel using two-dimensional infrared (2D IR) spectroscopy. By combining waiting time (100 - 2000 fs) 2D IR measurements of the KcsA channel including ¹³C¹⁸O isotope labeled Val76 and Gly77 residues with molecular dynamics simulations, we elucidated the site-specific dynamics of water and K⁺ ions inside the selectivity filter of KcsA. We observe inhomogeneous 2D lineshapes with extremely slow spectral diffusion. Our simulations quantitatively reproduce the experiments and show that water is the only component with any appreciable dynamics, whereas K⁺ ions and the protein are essentially static on a picosecond timescale. By analyzing simulated and experimental vibrational frequencies, we find that water in the selectivity filter can be oriented to form hydrogen bonds with adjacent, or non-adjacent carbonyl groups with the reorientation timescales being three times slower and comparable to that of water molecules in liquid, respectively. Water molecules can reside in the cavity sufficiently far from carbonyls and behave essentially like "free" gas-phase-like water with fast reorientation times. Remarkably, no interconversion between these configurations were observed on a picosecond timescale. These dynamics are in stark contrast with liquid water that remains highly dynamic even in the presence of ions at high concentrations.

Figure 1.

(A) Schematic illustration of the pore domain of the KcsA channel with closed intracellular gate and a conductive selectivity filter. Note that only two of the four protein chains are shown for clarity. Shown are the K⁺ ions (brown), crystallographic water coordinating K⁺ ion in the central cavity (dark blue spheres) and on the extracellular side (dark green sphere), behind the selectivity filter (purple spheres), mobile water (light blue spheres), and the five binding sites inside the selectivity filter (gray spheres) each accommodating either a water molecule or K⁺ ion. (B) The selectivity filter. Isotope-labeled residues Val76 and Gly77 whose 2D IR spectra measured and analyzed in this work are highlighted in blue and orange, respectively. (C) The two configurations in the selectivity filter considered in this work. K⁺ ions shown in purple.

Figure 2.

Experimental (A-D, J-M) and simulated (E-H, N-Q) 2D IR spectra of the KcsA channel with Val76 (A-H) and Gly77 (J-Q) isotope-labeled residues. Simulated spectra correspond to 60% of [S1, W,S3, W] and 40% of [W,S2, W,S4] ion configurations. Green lines show the center lines. Lower panels (I, R) show the center line slope of the experimental and simulated 2D line shapes as a function of time.

Figure 3.

(A) Amide I frequency-frequency correlation functions (FFCFs) for Val76 and Gly77 residues and [W,S2,W,S4] [S1,W,S3,W] configurations. Each FFCF is an average over four FFCFs calculated for each amide I chromophore in a protein chain. (B-D) Distribution of amide I frequency shifts of Val76 (green) and Gly77 (pink) caused by water molecules and the structural fragments of the selectivity filter that are consistent with the frequency shifts and FFCFs. (E) Distribution of amide I frequency shifts caused by K⁺ ions and the schematic illustration of the magnitude of fluctuation of K⁺ ions that causes the observed frequency shifts. (F) Rotational time-correlation functions for water molecules at various sites in the selectivity filter compared to bulk liquid TIP4P water at 300 K (pink). (G) Zoom-in of the first 600 fs of rotational time-correlation functions showing the initial inertial decay. (H) Hydrogen-bond number fluctuation time-correlation functions.

Figure 4.

Schematic illustration of all possible ways a water molecule can be oriented inside a binding site cavity demarked by the two sets of four carbonyl groups colored in green and orange. Water’s oxygen atoms are shown in red, hydrogen atoms in white. Possible hydrogen bonds are indicated by blue dashed lines.