Ligand binding to human prostaglandin E receptor EP4 at the lipid-bilayer interface.

Prostaglandin E receptor EP4, a G-protein-coupled receptor, is involved in disorders such as cancer and autoimmune disease. Here, we report the crystal structure of human EP4 in complex with its antagonist ONO-AE3-208 and an inhibitory antibody at 3.2 Å resolution. The structure reveals that the extracellular surface is occluded by the extracellular loops and that the antagonist lies at the interface with the lipid bilayer, proximal to the highly conserved Arg316 residue in the seventh transmembrane domain. Functional and docking studies demonstrate that the natural agonist PGE2 binds in a similar manner. This structural information also provides insight into the ligand entry pathway from the membrane bilayer to the EP4 binding pocket. Furthermore, the structure reveals that the antibody allosterically affects the ligand binding of EP4. These results should facilitate the design of new therapeutic drugs targeting both orthosteric and allosteric sites in this receptor family.

Analyses based on statistical thermodynamics for large difference between thermophilic rhodopsin and xanthorhodopsin in terms of thermostability.

Although the two membrane proteins, thermophilic rhodopsin (TR) and xanthorhodopsin (XR), share a high similarity in amino-acid sequence and an almost indistinguishable three-dimensional structure, TR is much more thermostable than XR. This is counterintuitive also because TR possesses only a smaller number of intramolecular hydrogen bonds (HBs) than XR. Here we investigate physical origins of the remarkable difference between XR and TR in the stability. Our free-energy function (FEF) is improved so that not only the portion within the transmembrane (TM) region but also the extracellular and intracellular portions within the water-immersed (WI) regions can be considered in assessing the stability. The assessment is performed on the basis of the FEF change upon protein folding, which is calculated for the crystal structure of XR or TR. Since the energetics within the TM region is substantially different from that within the WI regions, we determine the TM and WI portions of XR or TR by analyzing the distribution of water molecules using all-atom molecular dynamics simulations. The energetic component of the FEF change consists of a decrease in energy arising from the formation of intramolecular HBs and an increase in energy caused by the break of protein-water HBs referred to as "energetic dehydration penalty." The entropic component is a gain of the translational, configurational entropies of hydrocarbon groups within the lipid bilayer and of water molecules. The entropic component is calculated using the integral equation theory combined with our morphometric approach. The energetic one is estimated by a simple but physically reasonable method. We show that TR is much more stable than XR for the following reasons: The decrease in energy within the TM region is larger, and the energetic dehydration penalty within the WI regions is smaller, leading to higher energetic stabilization, and tighter packing of side chains accompanying the association of seven helices confers higher entropic stabilization on TR.

Identification of thermostabilizing mutations for a membrane protein whose three-dimensional structure is unknown.

We recently developed a physics-based method for identifying thermostabilizing mutations of a membrane protein. The method uses a free-energy function F where the importance of translational entropy of hydrocarbon groups within the lipid bilayer is emphasized. All of the possible mutations can rapidly be examined. The method was illustrated for the adenosine A2a receptor (A2a R) whose three-dimensional (3D) structure experimentally determined was utilized as the wild-type structure. Nine single mutations and a double mutation predicted to be stabilizing or destabilizing were checked by referring to the experimental results: The success rate was remarkably high. In this work, we postulate that the 3D structure of A2a R is unknown. We construct candidate models for the 3D structure using the homology modeling and select the model giving the lowest value to the change in F on protein folding. The performance achieved is only slightly lower than that in the recent work. © 2016 Wiley Periodicals, Inc.

Physical Origin of Thermostabilization by a Quadruple Mutation for the Adenosine A2a Receptor in the Active State.

The G protein-coupled receptors (GPCRs) form a large, physiologically important family of membrane proteins and are currently the most attractive targets for drug discovery. We investigate the physical origin of thermostabilization of the adenosine A2a receptor (A2aR) in the active state, which was experimentally achieved by another research group using the four point mutations: L48A, A54L, T65A, and Q89A. The investigation is performed on the basis of our recently developed physics-based free-energy function (FEF), which has been quite successful for the thermodynamics of GPCRs in the inactive state. The experimental condition for solving the wild-type and mutant crystal structures was substantially different from that for comparing their thermostabilities. Therefore, all-atom molecular dynamics simulations are necessitated, which also allows us to account for the structural fluctuations of the membrane protein. We show that the quadruple mutation leads to the enlargement of the solvent-entropy gain upon protein folding. The solvent is formed by hydrocarbon groups constituting nonpolar chains within the lipid bilayer, and the entropy is relevant to the thermal motion of the hydrocarbon groups. From an energetic point of view (e.g., in terms of protein intramolecular hydrogen bonds), the mutation confers no improvement upon the structural stability of A2aR. The reliability of our FEF and the crucial importance of the solvent-entropy effect have thus been demonstrated for a GPCR in the active state. We are now ready to identify thermostabilizing mutations of GPCRs not only in the inactive state but also in the active one.

Hot-Spot Residues to be Mutated Common in G Protein-Coupled Receptors of Class A: Identification of Thermostabilizing Mutations Followed by Determination of Three-Dimensional Structures for Two Example Receptors.

G protein-coupled receptors (GPCRs), which are indispensable to life and also implicated in a number of diseases, construct important drug targets. For the efficient structure-guided drug design, however, their structural stabilities must be enhanced. An amino-acid mutation is known to possibly lead to the enhancement, but currently available experimental and theoretical methods for identifying stabilizing mutations suffer such drawbacks as the incapability of exploring the whole mutational space with minor effort and the unambiguous physical origin of the enhanced or lowered stability. In general, after the identification is successfully made for a GPCR, the whole procedure must be followed all over again for the identification for another GPCR. Here we report a theoretical strategy by which many different GPCRs can be considered at the same time. The strategy is illustrated for three GPCRs of Class A in the inactive state. We argue that a mutation of the residue at a position of NBW = 3.39 (NBW is the Ballesteros-Weinstein number), a hot-spot residue, leads to substantially higher stability for significantly many GPCRs of Class A in the inactive state. The most stabilizing mutations of the residues with NBW = 3.39 are then identified for two of the three GPCRs, using the improved version of our free-energy function. These identifications are experimentally corroborated, which is followed by the determination of new three-dimensional (3D) structures for the two GPCRs. We expect that on the basis of the strategy, the 3D structures of many GPCRs of Class A can be solved for the first time in succession.

Identification of Thermostabilizing Mutations for Membrane Proteins: Rapid Method Based on Statistical Thermodynamics.

Membrane proteins are responsible for the communication between cells and their environments. They are indispensable to the expression of life phenomena and also implicated in a number of diseases. Nevertheless, the studies on membrane proteins are far behind those on water-soluble proteins, primarily due to their low structural stability. Introduction of mutations can enhance their thermostability and stability in detergents, but the stabilizing mutations are currently identified by experiments. The recently reported computational methods suffer such drawbacks as the exploration of only limited mutational space and the empiricism whose results are difficult to physically interpret. Here we develop a rapid method that allows us to treat all of the possible mutations. It employs a free-energy function (FEF) that takes into account the translational entropy of hydrocarbon groups within the lipid bilayer as well as the protein intramolecular hydrogen bonding. The method is illustrated for the adenosine A2a receptor whose wild-type structure is known and utilized. We propose a reliable strategy of finding key residues to be mutated and selecting their mutations, which will lead to considerably higher stability. Representative single mutants predicted to be stabilizing or destabilizing were experimentally examined and the success rate was found to be remarkably high. The melting temperature Tm for two of them was substantially higher than that of the wild type. A double mutant with even higher Tm was also obtained. Our FEF captures the essential physics of the stability changes upon mutations.