THE ACTIVATION MECHANISM OF RHODOPSIN EXPLORED BY MULTISCALE METHODS

Rhodopsin is the best characterized member of the large, pharmaceutically important, family of G-protein-coupled receptors (GPCRs), and serves as a prototype for understanding GPCR activation. In this thesis, we aim at understanding the activation mechanism of rhodopsin. To this aim, we first performed an in-depth analysis of the conformational motions of rhodopsin predicted by two elastic network models, Gaussian Network Model (GNM) and Anisotropic Network Model (ANM). We compared these motions with the extensive amount of experimental data, and developed a model for rhodopsin activation. We tested the model with Meta II fluorescence decay rates measured to characterize the deactivation of rhodopsin mutants. We find that our results correctly predict 93% of the experimentally observed effects in 119 rhodopsin mutants for which the decay rates and misfolding data were measured, including a systematic analysis of Cys->Ser replacements. Next, in order to incorporate atomic details and the effects of membrane and water molecules into our model, we developed a new protocol named ANM-restrained molecular dynamics (MD). In this protocol, we used multiple ANM modes as restraints to guide MD simulations. By using this protocol, we were able to sample biologically relevant, large scale motions of the protein that are otherwise not accessible to the conventional timescales MD simulations. Furthermore, we explored the evolution of the multiple ANM global modes with realistic deformations favored by a detailed atomic force field in the presence of the explicit environment. Remarkably, with this method, we identify a highly hinge site, which does not change with several rounds of applying normal modes as restraints. This hinge site includes residues that are directly affected by the isomerization of retinal, as well as those stabilizing the resulting all-trans conformation of the chromophore. The CP ends of the helices H3, H4, H5, and H6 and the connecting loops are found to enjoy an enhanced mobility facilitated by this hinge site. Several new interactions are observed to contribute to the mechanism of signal propagation from the retinal binding pocket to the G-protein binding sites in the CP domain

Mechanism of Signal Propagation upon Retinal Isomerization: Insights from Molecular Dynamics Simulations of Rhodopsin Restrained by Normal Modes

AbstractAs one of the best studied members of the pharmaceutically relevant family of G-protein-coupled receptors, rhodopsin serves as a prototype for understanding the mechanism of G-protein-coupled receptor activation. Here, we aim at exploring functionally relevant conformational changes and signal transmission mechanisms involved in its photoactivation brought about through a cis-trans photoisomerization of retinal. For this exploration, we propose a molecular dynamics simulation protocol that utilizes normal modes derived from the anisotropic network model for proteins. Deformations along multiple low-frequency modes of motion are used to efficiently sample collective conformational changes in the presence of explicit membrane and water environment, consistent with interresidue interactions. We identify two highly stable regions in rhodopsin, one clustered near the chromophore, the other near the cytoplasmic ends of transmembrane helices H1, H2, and H7. Due to redistribution of interactions in the neighborhood of retinal upon stabilization of the trans form, local structural rearrangements in the adjoining H3–H6 residues are efficiently propagated to the cytoplasmic end of these particular helices. In the structures obtained by our simulations, all-trans retinal interacts with Cys167 on H4 and Phe203 on H5, which were not accessible in the dark state, and exhibits stronger interactions with H5, while some of the contacts made (in the cis form) with H6 are lost

Identifying Ligand Binding Conformations of the β2-Adrenergic Receptor by Using Its Agonists as Computational Probes

Recently available G-protein coupled receptor (GPCR) structures and biophysical studies suggest that the difference between the effects of various agonists and antagonists cannot be explained by single structures alone, but rather that the conformational ensembles of the proteins need to be considered. Here we use an elastic network model-guided molecular dynamics simulation protocol to generate an ensemble of conformers of a prototypical GPCR, β2-adrenergic receptor (β2AR). The resulting conformers are clustered into groups based on the conformations of the ligand binding site, and distinct conformers from each group are assessed for their binding to known agonists of β2AR. We show that the select ligands bind preferentially to different predicted conformers of β2AR, and identify a role of β2AR extracellular region as an allosteric binding site for larger drugs such as salmeterol. Thus, drugs and ligands can be used as "computational probes" to systematically identify protein conformers with likely biological significance. © 2012 Isin et al

Functional motions of influenza virus hemagglutinin: a structure-based analytical approach.

Influenza virus hemagglutinin (HA), a homotrimeric integral membrane glycoprotein essential for viral infection, is engaged in two biological functions: recognition of target cells' receptor proteins and fusion of viral and endosomal membranes, both requiring substantial conformational flexibility from the part of the glycoprotein. The different modes of collective motions underlying the functional mobility/adaptability of the protein are determined in the present study using an extension of the Gaussian network model (GNM) to treat concerted anisotropic motions. We determine the molecular mechanisms that may underlie HA function, along with the structural regions or residues whose mutations are expected to impede function. Good agreement between theoretically predicted fluctuations of individual residues and corresponding x-ray crystallographic temperature factors is found, which lends support to the GNM elucidation of the conformational dynamics of HA by focusing upon a subset of dominant modes. The lowest frequency mode indicates a global torsion of the HA trimer about its longitudinal axis, accompanied by a substantial mobility at the viral membrane connection. This mode is proposed to constitute the dominant molecular mechanism for the translocation and aggregation of HAs, and for the opening and dilation of the fusion pore. The second and third collective modes indicate a global bending, allowing for a large lateral surface exposure, which is likely to facilitate the close association of the viral and endosomal membranes before pore opening. The analysis of kinetically hot residues, in contrast, reveals a localization of energy centered around the HA2 residue Asp112, which apparently triggers the solvent exposure of the fusion peptide

RMSD profiles as a function of residue index for the ANM-restrained-MD conformations that accommodate salmeterol and epinephrine.

<p>Red and blue curves represent RMSD per residue between the starting structure and the conformations that accommodate salmeterol and epinephrine, respectively. In both complexes, not only cytoplasmic region that binds to G-protein but also the extracellular region exhibits higher motilities. Although the overall RMSD profile of the extracellular region tends to maintain similar features in both conformations, 8M has larger structural rearrangements at EC2 and at the extracellular end of the helices that are connected by this loop. These structural arrangements lead to the accommodation of salmeterol at the extracellular region. The sequence ranges of the helices (H1–H8) are indicated by the labels on the upper abscissa and distinguished by gray bands. The color code of the helices that are used in the ribbon diagrams of β₂AR are also displayed as colored bands at the bottom of the graph.</p

Binding of salmeterol to the active BI-167107 bound form of β₂AR.

<p>Salmeterol occupies the same location in the crystal structure of the active β₂AR as we identified by ANM-restrained-MD for the conformation-salmeterol complex. The residues that have atoms in 3.5 Å are displayed and those that are not interacting with salmeterol in ANM-restrained-MD conformation are labeled in red. These are Trp286 at H6, Ile 309 at H7, and Phe194 at EC2. Carbon atoms of salmeterol are colored in pink. The rest of the atoms are colored the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050186#pone-0050186-g003" target="_blank">Figure 3</a>.</p

The complexes of epinephrine with an ANM-restrained-MD conformation and the active crystal structure.

<p><b>A.</b> ANM-restrained-MD conformation-Epinephrine Complex. Epinephrine is located at the orthosteric binding site. The meta- and para- hydroxyl groups of the catechol ring are interacting with Ser203 and Ser204 at H5, respectively. Both β-hydroxyl and amine groups of epinephrine are forming hydrogen bonds with Asp113. The carbon atoms of epinephrine are colored green. <b>B.</b> BI-167107 bound active structure-Epinephrine Complex. The first pose of epinephrine in the BI-167107 bound active structure is located at the orthosteric binding pocket near the experimentally verified ligand binding residues active crystal structure. However, it is not forming any hydrogen bonds with any of these residues such as serines at H5 and Asn113 at H3.</p

Protocol for ANM-restrained-MD simulations and β₂AR conformations.

<p><b>A.</b> Structure of β₂AR. Transmembrane helices 1–7 are labeled by numbers and colored in red, orange, yellow, green, blue, purple, and pink, respectively. Cytoplasmic helix 8 and the short extracellular helix below the binding cavity in extracellular loop 2 are colored in cyan. Ligand- and G-protein binding sites are shown by arrows. The palmitolyl group that is anchored to the membrane from the H8 is also shown in cyan. <b>B.</b> The protocol for generating the ensemble of conformations by ANM-restrained-MD algorithm. <b>C.</b> Ribbon diagrams of β₂AR conformations. Front view (top) and back view (bottom) of β2AR conformers generated by ANM-restrained-MD are shown.</p

Microdomains of β₂AR stabilized by new interactions and water molecules.

<p><b>A</b>. Water molecules stabilizing the conserved NPXXY motif (left) and the Asn-Asp pair (right) within the transmembrane region, and (<b>B</b>) the critical catecholamine binding Ser203 and Ser207 residues at H5 in conformations where they both point to the ligand binding pocket and connected through a water molecule. <b>C</b>. The motion of extracellular loop two (EC2) and the residues that form the salt bridge at the EC site. The motion found by ANM-restrained-MD to break the salt bridge at the extracellular site and the opening of the extracellular site is shown. ANM-restrained-MD conformation and the carazolol-bound structures are in solid and transparent colors, respectively. The side chains of Asp192 at EC2 and Lys305 at H7 that form the salt bridge at the inactive state of β₂AR are displayed on both structures. The motion of the EC2 including the short helix is depicted by red arrows.</p

Binding of salmeterol to β₂AR.

<p><b>A.</b> Interactions of salmeterol at the binding site of an ANM-restrained-MD conformation; <b>B.</b> The location of salmeterol tail forming a ‘lid’ with the extracellular loop 2 (EC2) of β₂AR from the extracellular site view. The flexible tail of salmeterol folds parallel to the EC2 and stabilizes the rest of the ligand forming a lid at the extracellular site. The green arrow shows the direction that the salmeterol tail runs similar to a beta-sheet structure. <b>C.</b> The residues lining the salmeterol binding pocket; <b>D.</b> The stabilization of the aromatic ring of salmeterol by Val114, Thr118 at H3, Ser203 and Ser207 at H5, and Phe290 atH6. Carbon, oxygen, nitrogen and hydrogen atoms of salmeterol is colored green, red, blue and white; respectively. ANM-restrained-MD conformation that is able to accommodate inside the ligand binding pocket is shown in ribbon diagram with transparent helices. The residues with the majority of atoms within the 3.5 Å making specific interactions with salmeterol are displayed. The EC2 that closely interacts with salmeterol is also show in green ribbon diagram.</p