Vibrational resonance, allostery, and activation in rhodopsin-like G protein-coupled receptors

Abstract

G protein-coupled receptors are a large family of membrane proteins activated by a variety of structurally diverse ligands making them highly adaptable signaling molecules. Despite recent advances in the structural biology of this protein family, the mechanism by which ligands induce allosteric changes in protein structure and dynamics for its signaling function remains a mystery. Here, we propose the use of terahertz spectroscopy combined with molecular dynamics simulation and protein evolutionary network modeling to address the mechanism of activation by directly probing the concerted fluctuations of retinal ligand and transmembrane helices in rhodopsin. This approach allows us to examine the role of conformational heterogeneity in the selection and stabilization of specific signaling pathways in the photo-activation of the receptor. We demonstrate that ligand-induced shifts in the conformational equilibrium prompt vibrational resonances in the protein structure that link the dynamics of conserved interactions with fluctuations of the active-state ligand. The connection of vibrational modes creates an allosteric association of coupled fluctuations that forms a coherent signaling pathway from the receptor ligand-binding pocket to the G-protein activation region. Our evolutionary analysis of rhodopsin-like GPCRs suggest that specific allosteric sites play a pivotal role in activating structural fluctuations that allosterically modulate functional signals.

Figure 1. Overview of the methods used in this investigation.

The black arrows indicate the MD simulation vibrational analyses methods that were used to assign the vibrational modes detected experimentally. The red arrows indicate the MD simulation methods that were directly used to interpret the experimental data. The blue arrows demonstrate how the computational methods relate to one another. In the chart PCA = principal component analysis, GSF = global structural fluctuations, FDA = force distribution analysis, and LSF = localized structural fluctuations.

Figure 2

(a) Localized structural fluctuation (LSF) network of interactions from a MD simulation of rhodopsin in the inactive-state. In the network representation of the LSFs the nodes represent amino acid residues and the links between the nodes signify a localized interaction. The size of the nodes denote the number of interactions (connections) that a given node possesses. In the network representation of the LSFs we find that the connections of the inactive receptor are grouped into two separate communities that represent both inactive (blue) and active-like (orange) protein conformational fluctuations. (b) The corresponding fluctuations are mapped onto a 3-D cartoon representation of rhodopsin.

Figure 3

(a) The experimental THz spectrum of rhodopsin in the dark-state (black line) and in the light-activated state (magenta, dashed line) in the 20–100 cm⁻¹ spectral region. The inset shows the 45–90 cm⁻¹ spectral region of the dark-state protein. (b) The torsional spectrum of the retinal from rhodopsin MD simulations in the dark-state (blue line) and in the Meta II activated-state (orange line). The torsion of the retinal is defined by the angle created by the C5-, C9-, and C13- methyl groups. The inset on the right shows the mobility of the retinal motion from the (R) dark-state MD simulation and (L) Meta II simulation where blue represents less mobility and red more mobility. The inset on the left side shows the 110–165 cm⁻¹ region of the calculated retinal torsional spectrum. (c) The Fourier Transform of the velocity autocorrelation function of protein residues surrounding the ligand-binding pocket in rhodopsin in the dark-state MD simulation. (d) Shows the residues used for the calculation of the velocity autocorrelation function spectrum in (c).

Figure 4

(a) The experimental THz spectrum of rhodopsin in the dark-state (black line) and light-activated state (magenta, dashed line) in the 100–160 cm⁻¹ spectral region. The inset shows the entire 100–200 cm⁻¹ spectral region of the experimental spectrum. (b) Cα− representation of rhodopsin showing the conformational mobility from the MD simulation of (R) dark-state and (L) Meta II activated-state of rhodopsin. Regions in blue represent areas of less mobility and regions in red more mobility.

Figure 5. Analysis of the MD simulation of Meta II.

(a) Conserved networks of H-bonds involving water molecules in rhodopsin taken from the literature, highlighting the D(E)RY, CWxP, Schiff base-counter ion and NPxxY network motifs are shown in red, blue, green, and magenta licorice representation, respectively. The licorice representation of residues in the blue, rectangular box show the amino acids comprising the CWxP motif that is believed to convert between two different rotamer conformations in the inactive and active states. (b) Cartoon representation of Meta II showing the mapping of the LSFs from the MD simulation onto the protein 3-D structure. The different colors in the network mapping of the LSFs represent regions of correlated fluctuations. (c) The spectrum of the fluctuations from the MD simulation of Meta II showing the rotamer torsion (blue), torsional oscillation of the retinal (orange), vibrational fluctuation of the Asn302 network comprising the NPxxY motif (magenta), and the spectral signature of the localized solvent relaxation (LSR) mode (red) that couples with the conserved network of H-bonds. (d) A 2-D mapping of the LSFs from (b) where solid links correspond to strong interactions between residues and dotted links are associated with weaker connections. The numbers correspond to the stages of activation outlined at the bottom of the figure.

Figure 6

(a) MSA of the rhodopsin-like family of proteins broken into communities with each community containing different colors. In communities, nodes within the same community are highly connected and communicate with one another efficiently in a localized manner, whereas nodes in different communities have fewer connections and require intermodular edges (or links) for forming long-distance communication. The nodes represent amino acid residues, the links are the connections between the residues, and the size of the nodes denotes the number of connections (links) to other nodes. The reference structure for the MSA is bovine rhodopsin (opsd_bovin) with pdb ID 1u19. (b) Cα− atom representation of rhodopsin showing the global hinge-like elongation torsion of Meta II from the dominant PCA mode (PCA1) from the MD simulation. The global torsion has been divided into structural domains where each domain is colored separately. The line shown depicts the bisector region of the separate domains.

Figure 7

(a) Cartoon representation of active-state rhodopsin bound with arrestin showing the mapping of the LSFs from the MD simulation onto the rhodopsin 3-D structure. The different colors in the network mapping of the LSFs represent regions of correlated fluctuations. (b) A 2-D mapping of the LSFs from (a). (c) The spectrum of the fluctuations from the MD simulation of the arrestin-bound rhodopsin showing the rotamer torsion (blue), torsional oscillation of the retinal (orange), vibrational fluctuation of the Asn302 network comprising the NPxxY motif (magenta). The inset in (c) shows the spectral signature of the solvent H-bonding fluctuations (red) that couple with the conserved network of water-mediated H-bonds in rhodopsin. (d) Illustration of the dominant PCA mode (PCA1) of rhodopsin from the MD simulation of rhodopsin bound with arrestin where the areas in blue show regions with less mobility and the areas in red show regions with more mobility.