Internal hydration increases during activation of the G-protein-coupled receptor rhodopsin

Abstract

Rhodopsin, the membrane protein responsible for dim-light vision, until recently was the only G-protein-coupled receptor (GPCR) with a known crystal structure. As a result, there is enormous interest in studying its structure, dynamics, and function. Here we report the results of three all-atom molecular dynamics simulations, each at least 1.5 micros, which predict that substantial changes in internal hydration play a functional role in rhodopsin activation. We confirm with (1)H magic angle spinning NMR that the increased hydration is specific to the metarhodopsin-I intermediate. The internal water molecules interact with several conserved residues, suggesting that changes in internal hydration may be important during the activation of other GPCRs. The results serve to illustrate the synergism of long-time-scale molecular dynamics simulations and NMR in enhancing our understanding of GPCR function.

Figure 1

Proton transfer mechanisms in the formation of MI. We test two separate models for proton transfer in the retinal binding pocket after photoactivation. The complex counterion model begins with Glu-181 protonated and Glu-113 deprotonated; after retinal isomerizes, a proton is transferred from Glu-181 to Glu-113, leading to the counterion switch in MI. In the complex counterion model), Glu-181 and Glu-113 are both charged until MII formation.

Figure 2

The counterion switch and internal hydration. Parts A and C track the time evolution of the distances between the ionone ring and Glu-113 and -181, for the counterion switch and complex counterion models respectively. Parts B and D show the number of water molecules inside the protein cavity for the counterion switch and complex counterion simulations, respectively. Part D also contains the equivalent time series for a control simulation of dark state rhodopsin. The number of internal waters at t=0 differs in the two simulations because the number of waters fluctuated during dark state equilibration.

Figure 3

Internal water molecules solvate retinal during MI formation. A snapshot from the 1 μs point of the complex counterion simulation is shown, depicting the large number of waters inside the protein cavity and their interaction with the retinal. Trp-265 (green) has moved away from the retinal (yellow) head group. The image was created using PYMOL.

Figure 4

¹H NMR magnetization transfer experiment. The top portion shows the pulse sequence used in the experiment. The middle portion illustrates the route of magnetization transfer, from the water through the protein to the lipid. The lower panel shows the ¹H MAS NMR spectrum of rod outer segment disk membranes before (solid line) and immediately after photoactivation of rhodopsin (dashed line) as described below. Immediately after photoactivation a significant increase (up to 11%) in the saturation of lipid proton resonances was observed. Reproducibility of signal intensities was 1%. The effect was recorded as a function of time by recording intensity of the well-resolved =CH-CH2-CH= resonance (2.8 ppm) of polyunsaturated hydrocarbon chains (see insert, and Fig. 4). Attenuation increased also for other resonances, in particular those with contributions to intensity from rhodopsin protons. See Figure S1 in supplemental information for an example difference spectrum.

Figure 5

Time dependence of magnetization transfer from water to DHA via rhodopsin. The data shows the attenuation of the polyunsaturated resonance at 2.8 ppm. The open squares were measured at pH 7, where the MI/MII equilibrium strongly favors MII, while the filled circles were measured at pH 8 and T=20° C, where [MI]/[MII] ≈ 0.71. Photoactivation occurs at t=0.