Retinal Ligand Mobility Explains Internal Hydration and Reconciles Active Rhodopsin Structures

Abstract

Rhodopsin, the mammalian dim-light receptor, is one of the best-characterized G-protein-coupled receptors, a pharmaceutically important class of membrane proteins that has garnered a great deal of attention because of the recent availability of structural information. Yet the mechanism of rhodopsin activation is not fully understood. Here, we use microsecond-scale all-atom molecular dynamics simulations, validated by solid-state 2H nuclear magnetic resonance spectroscopy, to understand the transition between the dark and metarhodopsin I (Meta I) states. Our analysis of these simulations reveals striking differences in ligand flexibility between the two states. Retinal is much more dynamic in Meta I, adopting an elongated conformation similar to that seen in the recent activelike crystal structures. Surprisingly, this elongation corresponds to both a dramatic influx of bulk water into the hydrophobic core of the protein and a concerted transition in the highly conserved Trp2656.48 residue. In addition, enhanced ligand flexibility upon light activation provides an explanation for the different retinal orientations observed in X-ray crystal structures of active rhodopsin.

Retinal Flip in Rhodopsin Activation?

“…By starting from the crystal structure and observing a stable 180 rotation about the long axis of the polyene chain, our simulations provide a plausible explanation for these differences. Furthermore, our results provide compelling support for our proposal that the increased ligand flexibility observed in Meta-I could lead to multiple retinal conformations in the active state (12).…”

“…It has been shown that the 6-s-trans conformation raises the pK a of the SB, favoring the protonated state (13), as in Meta-I. Moreover, the pathway that occurs from cis to trans is very similar to the one observed in previous longtimescale MD studies that examined the dark / Meta-I transition (11,12).…”

“…In particular, MD studies on the ms-timescale have reproduced solid-state 2 H NMR spectra of the Meta-I state (11), and showed an increase in both hydration and retinal flexibility versus the dark state (12). In this study, the most remarkable event occurs at roughly 2.5 ms in two of the three Meta-II to Meta-I simulations: the polyene chain in retinal undergoes a 180 rotation about its long axis, in which the C9-and C13-methyl groups shift orientation from the cytoplasmic side toward the extracellular side of rhodopsin (Fig.…”

“…Hydration plays a crucial role in rhodopsin function, in which water is necessary for rearrangement of the chromophore (12,14). The retinal flip is accompanied by a decrease in hydration of the binding pocket (Fig.…”

Area per Lipid and Cholesterol Interactions in Membranes from Separated Local-Field 13 C NMR Spectroscopy

“…Moreover, in vitro studies of integral membrane proteins or peptides often require that they be reconstituted with membrane lipids (19,(28)(29)(30), where bilayer structural dimensions involving hydrophobic matching and area per lipid are significant factors (1,14,31). The constraints imposed by the area per lipid at the membrane aqueous interface (31) also play an important part in validating molecular dynamics (MD) simulations of lipid bilayers (32) and biomembranes (13,33,34). In such cases, it is essential that investigators have a solid understanding of the membrane bilayer structure itself (35).…”