Light Activation of Rhodopsin: Insights from Molecular Dynamics Simulations Guided by Solid-State NMR Distance Restraints
ABSTRACT Structural restraints provided by solid-state NMR measurements of the metarhodopsin II intermediate are combined with molecular dynamics simulations to help visualize structural changes in the light activation of rhodopsin. Since the timescale for the formation of the metarhodopsin II intermediate (> 1 ms) is beyond that readily accessible by molecular dynamics, we use NMR distance restraints derived from 13C dipolar recoupling measurements to guide the simulations. The simulations yield a working model for how photoisomerization of the 11-cis retinylidene chromophore bound within the interior of rhodopsin is coupled to transmembrane helix motion and receptor activation. The mechanism of activation that emerges is that multiple switches on the extracellular (or intradiscal) side of rhodopsin trigger structural changes that converge to disrupt the ionic lock between helices H3 and H6 on the intracellular side of the receptor.
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ABSTRACT: G protein-coupled receptors (GPCRs) are one of the most important target classes in the central nervous system (CNS) drug discovery, however the fact they are integral membrane proteins and are unstable when purified out of the cell precludes them from a wide range of structural and biophysical techniques that are used for soluble proteins. In this study we demonstrate how protein engineering methods can be used to identify mutations which can both increase the thermostability of receptors, when purified in detergent, as well as biasing the receptor towards a specific physiologically relevant conformational state. We demonstrate this method for the adenosine A(2A) receptor and muscarinic M(1) receptor. The resultant stabilised receptors (known as StaRs) have a pharmacological profile consistent with the inverse agonist conformation. The stabilised receptors can be purified in large quantities, whilst retaining correct folding, thus generating reagents suitable for a broad range of structural and biophysical studies. In the case of the A(2A)-StaR we demonstrate that surface plasmon resonance can be used to profile the association and dissociation rates of a range of antagonists, a technique that can be used to improve the in vivo efficacy of receptor antagonists.Neuropharmacology 01/2011; 60(1):36-44. DOI:10.1016/j.neuropharm.2010.07.001 · 4.82 Impact Factor
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ABSTRACT: Membrane proteins are a large, diverse group of proteins, representing about 20-30% of the proteomes of most organisms, serving a multitude of cellular functions and more than 40% of drug targets. Knowledge of a membrane protein structure enables us insight into its function and dynamics, and can be used for further rational drug design. Owing to the intrinsic hydrophobicity, flexibility, and instability of membrane proteins, solid-state NMR may offer an unique opportunity to study membrane protein structure, ligand binding, and activation at atomic resolution in the native membrane environment on a wide ranging time scale. Over the past several years, solid-state NMR has made tremendous progress, showing its capability of determining membrane protein structure, ligand binding, and protein dynamic conformation on a variety of time scales at atomic resolution. In this chapter we will mainly discuss some recent achievements on membrane protein structure determination, ligand conformation and binding, structure changes upon activation, and structure of insoluble fibrous proteins investigated by using magic-angle spinning solid-state NMR from the structural biology point of view. Protein dynamics, sensitivity enhancement, and the possibility of chemical shift-based structure determination in solid-state NMR are also briefly touched upon.Topics in current chemistry 12/2011; 326:187-213. DOI:10.1007/128_2011_287 · 4.61 Impact Factor
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ABSTRACT: Extracellular adenosine mediates most of its physiological effects via an interaction with four G protein-coupled receptors (GPCRs), the adenosine receptors (ARs). These ARs are important pharmacological targets in the treatment of a wide variety of diseases from central nervous system disorders to ischemic injury. As for other GPCRs, drug development for the ARs has been hampered by the lack of structural data for this class of membrane proteins. However, in the past 3 years, this situation has changed with the elucidation of structures for the turkey β(1)-adrenoceptor, the human β(2)-adrenoceptor, squid rhodopsin, the activated form of bovine (rhod)opsin, the human adenosine A(2A) receptor, and most recently the CXCR4 chemokine receptor. In this review, the structural features of the human adenosine A(2A) receptor will be discussed with a particular focus on the ligand binding site. Further, the implications of this structural information for AR ligand selectivity, drug screening, homology modeling, and virtual ligand screening will be discussed.Advances in pharmacology (San Diego, Calif.) 01/2011; 61:1-40. DOI:10.1016/B978-0-12-385526-8.00001-1