Identification of motions in membrane proteins by elastic network models and their experimental validation.

Identifying the functional motions of membrane proteins is difficult because they range from large-scale collective dynamics to local small atomic fluctuations at different timescales that are difficult to measure experimentally due to the hydrophobic nature of these proteins. Elastic Network Models, and in particular their most widely used implementation, the Anisotropic Network Model (ANM), have proven to be useful computational methods in many recent applications to predict membrane protein dynamics. These models are based on the premise that biomolecules possess intrinsic mechanical characteristics uniquely defined by their particular architectures. In the ANM, interactions between residues in close proximity are represented by harmonic potentials with a uniform spring constant. The slow mode shapes generated by the ANM provide valuable information on the global dynamics of biomolecules that are relevant to their function. In its recent extension in the form of ANM-guided molecular dynamics (MD), this coarse-grained approach is augmented with atomic detail. The results from ANM and its extensions can be used to guide experiments and thus speedup the process of quantifying motions in membrane proteins. Testing the predictions can be accomplished through (a) direct observation of motions through studies of structure and biophysical probes, (b) perturbation of the motions by, e.g., cross-linking or site-directed mutagenesis, and (c) by studying the effects of such perturbations on protein function, typically through ligand binding and activity assays. To illustrate the applicability of the combined computational ANM-experimental testing framework to membrane proteins, we describe-alongside the general protocols-here the application of ANM to rhodopsin, a prototypical member of the pharmacologically relevant G-protein coupled receptor family.

Identifying ligand binding conformations of the ß2-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, ß(2)-adrenergic receptor (ß(2)AR). 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 ß(2)AR. We show that the select ligands bind preferentially to different predicted conformers of ß(2)AR, and identify a role of ß(2)AR 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.

Mechanism of signal propagation upon retinal isomerization: insights from molecular dynamics simulations of rhodopsin restrained by normal modes.

As 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 Cys(167) on H4 and Phe(203) 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.

Predisposition of the dark state of rhodopsin to functional changes in structure.

As the only member of the family of G-protein-coupled receptors for which atomic coordinates are available, rhodopsin is widely studied for insight into the molecular mechanism of G-protein-coupled receptor activation. The currently available structures refer to the inactive, dark state, of rhodopsin, rather than the light-activated metarhodopsin II (Meta II) state. A model for the Meta II state is proposed here by analyzing elastic network normal modes in conjunction with experimental data. Key mechanical features and interactions broken/formed in the proposed model are found to be consistent with the experimental data. The model is further tested by using a set of Meta II fluorescence decay rates measured to empirically characterize the deactivation of rhodopsin mutants. The model is found to correctly predict 93% of the experimentally observed effects in 119 rhodopsin mutants for which the decay rates and misfolding data have been measured, including a systematic analysis of Cys-->Ser replacements reported here. Based on the detailed comparison between model and experiments, a cooperative activation mechanism is deduced that couples retinal isomerization to concerted changes in conformation, facilitated by the intrinsic dynamics of rhodopsin. A global hinge site is identified near the retinal-binding pocket that ensures the efficient propagation of signals from the central transmembrane region to both cytoplasmic and extracellular ends. The predicted activation mechanism opens the transmembrane helices at the critical G-protein binding cytoplasmic domain. This model provides a detailed, mechanistic description of the activation process, extending experimental observations and yielding new insights for further tests.

Identification of core amino acids stabilizing rhodopsin.

Rhodopsin is the only G protein-coupled receptor (GPCR) whose 3D structure is known; therefore, it serves as a prototype for studies of the GPCR family of proteins. Rhodopsin dysfunction has been linked to misfolding, caused by chemical modifications that affect the naturally occurring disulfide bond between C110 and C187. Here, we identify the structural elements that stabilize rhodopsin by computational analysis of the rhodopsin structure and comparison with data from previous in vitro mutational studies. We simulate the thermal unfolding of rhodopsin by breaking the native-state hydrogen bonds sequentially in the order of their relative strength, using the recently developed Floppy Inclusion and Rigid Substructure Topography (FIRST) method [Jacobs, D. J., Rader, A. J., Kuhn, L. A. & Thorpe, M. F. (2001) Proteins 44, 150-165]. Residues most stable under thermal denaturation are part of a core, which is assumed to be important for the formation and stability of folded rhodopsin. This core includes the C110-C187 disulfide bond at the center of residues forming the interface between the transmembrane and the extracellular domains near the retinal binding pocket. Fast mode analysis of rhodopsin using the Gaussian network model also identifies the disulfide bond and the retinal ligand binding pocket to be the most rigid region in rhodopsin. Experiments confirm that 90% of the amino acids predicted by the FIRST method to be part of the core cause misfolding upon mutation. The observed high degree of conservation (78.9%) of this disulfide bond across all GPCR classes suggests that it is critical for the stability and function of GPCRs.

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.