HOW MUCH BOVINE RHODOPSIN CRYSTAL STRUCTURE IS USEFUL FOR MODELING HUMAN GPCRS? - β2-Adrenergic Receptor as a Test Case (original) (raw)
Related papers
2009
Availability of realistic models for human G-Protein Coupled Receptors (hGPCRs) will aid structure-based drug design (SBDD), thus shortening the time period needed for drug development and minimizing crossreactivity of drugs with other hGPCRs. Many researchers have constructed models for hGPCRs with homology modeling techniques based on the X-ray structure of bovine rhodopsin and recently to β2adrenergic receptor which are the only two GPCRs that have high resolution crystal structures. In this study, we evaluate the usefulness of the bovine rhodopsin crystal structures for modeling hGPCRs by analysis of large database of human G-protein coupled receptors that are members of family A (rhodopsin family). The recently released structure of β2-adrenergic receptor was used as a test case for validation purposes of our findings. From pair-wise sequence alignment of each of the receptors in the database to bovine rhodopsin, we come to the conclusion that only for few hGPCRs, X-ray structure of rhodopsin could be used as a template for modeling the trans-membrane domains (TMDs).The detailed analysis of the whole database shows that in general, similarity to bovine rhodopsin is found more in the middle/endoplasmic part than in the exoplasmic part. The shift in the cytoplasmic end of TMD-6 that has been seen in the recently released crystal structure of β2-adrenergic receptor could be understood well from our bioinformatics study. On the basis of our results from this research, we propose to regard specific parts from the endoplasmic domain of the rhodopsin helices as appropriate template for constructing models of other GPCRs, while most of the exoplasmic parts of GPCRs in this family require other techniques for their modeling, due to the low sequence similarity between the receptors and rhodopsin in that region.
Rhodopsin crystal: new template yielding realistic models of G-protein-coupled receptors?
Trends in Pharmacological Sciences, 2003
Molecular modelling is of major help to understand structure -function data on G-protein-coupled receptors (GPCRs). Since the first determination of the structure of rhodopsin, at high resolution, the view has emerged that it will be now easy to automatically obtain realistic models for any GPCR by homology modeling. Our experience on cholecystokinin CCK 1 receptor modelling together with available data on other GPCRs leads us to rule out this opinion. We believe that construction of realistic models of certain GPCRs still remains timeconsuming and requires many refinements of the models in close association with experiments. This conclusion has important consequences for modelling orphan GPCRs.
Crystal structure of the human β2 adrenergic G-protein-coupled receptor
Nature, 2007
Structural analysis of G-protein-coupled receptors (GPCRs) for hormones and neurotransmitters has been hindered by their low natural abundance, inherent structural flexibility, and instability in detergent solutions. Here we report a structure of the human b 2 adrenoceptor (b 2 AR), which was crystallized in a lipid environment when bound to an inverse agonist and in complex with a Fab that binds to the third intracellular loop. Diffraction data were obtained by high-brilliance microcrystallography and the structure determined at 3.4 Å / 3.7 Å resolution. The cytoplasmic ends of the b 2 AR transmembrane segments and the connecting loops are well resolved, whereas the extracellular regions of the b 2 AR are not seen. The b 2 AR structure differs from rhodopsin in having weaker interactions between the cytoplasmic ends of transmembrane (T M)3 and T M 6, involving the conserved E / DRY sequences. These differences may be responsible for the relatively high basal activity and structural instability of the b 2 AR, and contribute to the challenges in obtaining diffraction-quality crystals of non-rhodopsin GPCRs.
Journal of Computer-Aided Molecular Design, 2006
It is well known that G protein-coupled receptors are prime targets for drug discovery. At the present time there is only one protein from this class that has an X-ray crystal structure, bovine rhodopsin. Crystal structures of rhodopsin have become invaluable templates for the modeling of class-A G proteincoupled receptors as they likely represent the overall topology of this family of proteins. However, because of low sequence homology within the class and the inherent mobility of integral membrane proteins, it is unlikely that this single structural template reflects the ensemble of conformations accessible for any given receptor. We have devised a procedure based upon comparative modeling that uses induced fit modeling coupled with binding site expansion. The modeling protocol enables an ensemble approach to binding mode prediction. The utility of models for b-2 adrenergic receptor will be discussed.
Crystal structure of the human beta2 adrenergic G-protein-coupled receptor
Nature, 2007
Structural analysis of G-protein-coupled receptors (GPCRs) for hormones and neurotransmitters has been hindered by their low natural abundance, inherent structural flexibility, and instability in detergent solutions. Here we report a structure of the human b 2 adrenoceptor (b 2 AR), which was crystallized in a lipid environment when bound to an inverse agonist and in complex with a Fab that binds to the third intracellular loop. Diffraction data were obtained by high-brilliance microcrystallography and the structure determined at 3.4 Å / 3.7 Å resolution. The cytoplasmic ends of the b 2 AR transmembrane segments and the connecting loops are well resolved, whereas the extracellular regions of the b 2 AR are not seen. The b 2 AR structure differs from rhodopsin in having weaker interactions between the cytoplasmic ends of transmembrane (T M)3 and T M 6, involving the conserved E / DRY sequences. These differences may be responsible for the relatively high basal activity and structural instability of the b 2 AR, and contribute to the challenges in obtaining diffraction-quality crystals of non-rhodopsin GPCRs.
High-Resolution Crystal Structure of an Engineered Human beta2Adrenergic G Protein-Coupled Receptor
Science, 2007
G protein-coupled receptors comprise the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human β 2 -adrenergic receptor-T4 lysozyme fusion protein bound to the partial inverse agonist carazolol at 2.4 Å resolution. The structure provides a high-resolution view of a human G protein-coupled receptor bound to a diffusible ligand. Ligand-binding site accessibility is enabled by the second extracellular loop which is held out of the binding cavity by a pair of closely spaced disulfide bridges and a short helical segment within the loop. Cholesterol, a necessary component for crystallization, mediates an intriguing parallel association of receptor molecules in the crystal lattice. Although the location of carazolol in the β 2 -adrenergic receptor is very similar to that of retinal in rhodopsin, structural differences in the ligand binding site and other regions highlight the challenges in using rhodopsin as a template model for this large receptor family. $ These authors contributed equally Author Contributions: RCS and BKK independently pushed the GPCR structural biology projects for more than 15 years. BKK managed the protein design, production and purification. RCS managed novel crystallization and data collection methods development and experiments. VC developed novel methods for, and performed LCP crystallization, LCP crystal mounting, LCP data collection, model refinement, analyzed the results, and was involved in manuscript preparation. DMR supplied protein materials for all crystallization trials, grew and collected data from the bicelle crystals, collected, processed and refined the 3.5 Å LCP structure, refined the 2.4 Å structure, analyzed the results, and was involved in manuscript preparation. MAH designed the blind crystal screening protocol and collected the 2.4 Å data set, processed the 2.4 Å data, solved the structure by MR at 3.5 Å and 2.4 Å resolution, wrote the initial draft of the manuscript and created all figures. SGFR assisted with the final stages of β 2 AR-T4L purification. FST expressed β 2 AR-T4L in insect cells and, together with TSK, performed the initial stage of β 2 AR purification. HJC assisted with the refinement. PK assisted in developing novel methods to screen the transparent crystals, data collection, refinement, and was involved in manuscript preparation. WIW assisted with low resolution data collection and processing, solved the β 2 AR-T4L molecular replacement problem at 3.5 Å, participated in the 2.4 Å refinement process, and participated in structure analysis and manuscript preparation. BKK additionally assisted with β 2 AR-T4L purification, β 2 AR-T4L 3.5 Å synchrotron data collection, structure analysis and manuscript preparation. BKK and DMR designed the β 2 AR-T4L fusion protein strategy. RCS additionally assisted with β 2 AR-T4L crystallization, 2.4 Å data collection, structure solution, refinement, structure analysis and manuscript preparation.
High-Resolution Crystal Structure of an Engineered Human b2Adrenergic G Protein–Coupled Receptor
Science
G protein-coupled receptors comprise the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human β 2 -adrenergic receptor-T4 lysozyme fusion protein bound to the partial inverse agonist carazolol at 2.4 Å resolution. The structure provides a high-resolution view of a human G protein-coupled receptor bound to a diffusible ligand. Ligand-binding site accessibility is enabled by the second extracellular loop which is held out of the binding cavity by a pair of closely spaced disulfide bridges and a short helical segment within the loop. Cholesterol, a necessary component for crystallization, mediates an intriguing parallel association of receptor molecules in the crystal lattice. Although the location of carazolol in the β 2 -adrenergic receptor is very similar to that of retinal in rhodopsin, structural differences in the ligand binding site and other regions highlight the challenges in using rhodopsin as a template model for this large receptor family. $ These authors contributed equally Author Contributions: RCS and BKK independently pushed the GPCR structural biology projects for more than 15 years. BKK managed the protein design, production and purification. RCS managed novel crystallization and data collection methods development and experiments. VC developed novel methods for, and performed LCP crystallization, LCP crystal mounting, LCP data collection, model refinement, analyzed the results, and was involved in manuscript preparation. DMR supplied protein materials for all crystallization trials, grew and collected data from the bicelle crystals, collected, processed and refined the 3.5 Å LCP structure, refined the 2.4 Å structure, analyzed the results, and was involved in manuscript preparation. MAH designed the blind crystal screening protocol and collected the 2.4 Å data set, processed the 2.4 Å data, solved the structure by MR at 3.5 Å and 2.4 Å resolution, wrote the initial draft of the manuscript and created all figures. SGFR assisted with the final stages of β 2 AR-T4L purification. FST expressed β 2 AR-T4L in insect cells and, together with TSK, performed the initial stage of β 2 AR purification. HJC assisted with the refinement. PK assisted in developing novel methods to screen the transparent crystals, data collection, refinement, and was involved in manuscript preparation. WIW assisted with low resolution data collection and processing, solved the β 2 AR-T4L molecular replacement problem at 3.5 Å, participated in the 2.4 Å refinement process, and participated in structure analysis and manuscript preparation. BKK additionally assisted with β 2 AR-T4L purification, β 2 AR-T4L 3.5 Å synchrotron data collection, structure analysis and manuscript preparation. BKK and DMR designed the β 2 AR-T4L fusion protein strategy. RCS additionally assisted with β 2 AR-T4L crystallization, 2.4 Å data collection, structure solution, refinement, structure analysis and manuscript preparation.
Structure of a β1-adrenergic G-protein-coupled receptor
Nature, 2008
G-protein-coupled receptors have a major role in transmembrane signalling in most eukaryotes and many are important drug targets. Here we report the 2.7 Å resolution crystal structure of a b 1 -adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey (Meleagris gallopavo) receptor was selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane a-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilized by two disulphide bonds and a sodium ion. Binding of cyanopindolol to the b 1 -adrenergic receptor and binding of carazolol to the b 2 -adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the b 2 -adrenergic receptor, directly interacts by means of a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation.
2009
G protein-coupled receptors (GPCRs) constitute a very large family of heptahelical, integral membrane proteins that mediate a wide variety of physiological processes, ranging from the transmission of the light and odorant signals to the mediation of neurotransmission and hormonal actions. GPCRs are dysfunctional or deregulated in several human diseases and are estimated to be the target of more than 40% of drugs used in clinical medicine today. The crystal structures of rhodopsin and the recent published crystal structures of beta-adrenergic receptors and human A2A Adrenergic Receptor provide the information of the three-dimensional structure of GPCRs, which supports homology modeling studies and structure-based drug-design approaches. Rhodopsin-based homology modeling has represented for many years a widely used approach to built GPCR three-dimensional models. Structural models can be used to describe the interatomic interactions between ligand and receptor and how the binding info...