Subscriber access provided by University of Chicago Library
B: Biophysical Chemistry and Biomolecules
Physical Origin of Thermostabilization by a Quadruple Mutation for the Adenosine A Receptor in the Active State 2a
Yuta Kajiwara, Satoshi Yasuda, Simon Hikiri, Tomohiko Hayashi, Mitsunori Ikeguchi, Takeshi Murata, and Masahiro Kinoshita J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b00443 • Publication Date (Web): 04 Apr 2018 Downloaded from http://pubs.acs.org on April 9, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
The Journal of Physical Chemistry
Physical Origin of Thermostabilization by a Quadruple Mutation for the Adenosine A2a Receptor in the Active State Yuta Kajiwara, 1 Satoshi Yasuda, 2−4 Simon Hikiri, 2,3 Tomohiko Hayashi, 4 Mitsunori Ikeguchi,5,6 Takeshi Murata,* ,2,3,7 and Masahiro Kinoshita* ,4 1
Graduate School of Energy Science, Kyoto University, Uji, Kyoto 611-0011, Japan
2
Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage, Chiba 263-8522, Japan
3
Molecular Chirality Research Center, Chiba University, 1-33 Yayoi-cho, Inage, Chiba 263-8522, Japan
4
Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011, Japan
5
Graduate School of Medical Life Science, Yokohama City University, Tsurumi, Yokohama 230-0045, Japan
6
RIKEN Medical Sciences Innovation Hub Program, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan
7
JST, PRESTO, 1-33 Yayoi-cho, Inage, Chiba 263-8522, Japan
1
ACS Paragon Plus Environment
The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ABSTRACT: The G protein-coupled receptors (GPCRs) form a large, physiologically important family of membrane proteins and are currently most attractive targets for drug discovery. We investigate the physical origin of thermostabilization of the adenosine A2a receptor (A 2a R) 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 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 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 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 A 2a R. 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.
2
ACS Paragon Plus Environment
Page 2 of 31
Page 3 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
The Journal of Physical Chemistry
INTRODUCTION The G protein-coupled receptors (GPCRs) form a large, physiologically important family of membrane proteins. 1,2 They play imperative roles in signal transduction through the cell membrane and are implicated in a number of diseases. It is no wonder that they are currently most attractive targets for drug discovery. 3 For efficient drug design, it is required that the three-dimensional (3D) structure of a target GPCR be determined with sufficiently high resolution. However, the determination is not an easy task and the 3D structures of most of GPCRs remain unsolved. The reason for this unfavorable situation is that the structural stability of a GPCR is inherently low, hindering the crystallization followed by the structure determination. An amino-acid mutation for a GPCR is known to possibly enhance its thermostability. 4−6 When the structure becomes thermally more stable, it usually exhibits higher stability against other perturbations such as the solubilization of the GPCR in detergents after removing it from the lipid bilayer. Hence, the development of a reliable theoretical method for identifying thermostabilizing mutations has been a central issue in the fields of biochemistry, structural biology, and pharmaceutical chemistry. Recent functional and biophysical studies have suggested that there exists multiple, ligand-specific conformational states for a GPCR. 7 However, the conformation of a GPCR with no ligand binding could be discussed in terms of the two-state model: There are inactive and active states which are in equilibrium with each other. 7,8 For many GPCRs, the free energy of the active state is higher than that of the inactive state. For an individual GPCR molecule, the time during which it is in the active state is significantly shorter than that during which it is in the inactive state. For an ensemble of GPCR molecules, at a certain moment, the portion of the molecules in the active state is substantially smaller than that in the inactive state. An agonist can bind to a GPCR molecule while the GPCR is in the active state. 9 Upon the binding, the active state is stabilized in the sense that its free energy is lowered. 7 Likewise, the binding of an inverse agonist to a GPCR molecule leads to the stabilization of the inactive state. If the active state is stabilized by a mutation without the agonist binding, the design of an agonist with high binding affinity will significantly be facilitated. The stabilization of the inactive state by a mutation will also be highly advantageous to efficient design of an inverse agonist with high binding affinity. In earlier works, 10−13 we developed a theoretical method for identifying thermostabilizing mutations for a membrane protein. A physics-based free-energy function (F) is incorporated in the method. F is expressed as F=Λ−TS where Λ and −TS are the energetic and entropic components, respectively, T is the absolute temperature. F is a function of the protein structure. Let ∆F (∆F=∆Λ−T∆S) be the change in F upon protein folding (i.e., the transition to the folded structure). ∆F is the criterion of the 3
ACS Paragon Plus Environment
The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
stability of the folded structure: Lower ∆F implies higher stability. As the most important feature of ∆F, the entropic effect originating from the thermal motion of hydrocarbon groups constituting nonpolar chains within the lipid bilayer10−14 is taken into account in ∆S using an integral equation theory (IET)15 combined with our morphometric approach (MA). 16,17 The hydrocarbon groups act as the solvent for a membrane protein, and ∆S denotes the solvent-entropy gain upon protein folding. The IET is a statistical-mechanical theory of solvation, and the MA is capable of relating ∆S to the polyatomic characteristics of the folded structure with quantitative accuracy. In the energetic component, we account for protein intramolecular electrostatic energy by regarding intramolecular hydrogen bonding as a pivotal factor. 13 In contrast with the alanine (Ala) scanning mutagenesis, 18−20 the method can explore the whole mutational space with no heavy computational burden. Its high performance has already been demonstrated for GPCRs in the inactive state. 10−13 The most important success was that we discovered the existence of key and hot-spot residues. 12,13 Many of the mutations of a key residue are stabilizing, and a GPCR possesses multiple key residues. A hot-spot residue is the key residue common in significantly many different GPCRs. We showed that the residue at a position of N BW =3.39 (N BW is the Ballesteros-Weinstein number) is a hot-spot residue for GPCRs of Class A in the inactive state and its mutation to arginine (Arg) or lysine (Lys) can be highly stabilizing. 12,13 On the basis of this concept, we were successful in stabilizing the structures of several GPCRs including the adenosine A 2a receptor (A 2a R), muscarinic acetylcholine receptor 2 (M2R), and prostaglandin E receptor 4 (EP4). 13 We wish to extend our theoretical method to GPCRs in the active state. To this end, we examine the applicability of our free-energy function to a GPCR in the active state as an important first step. It has recently been reported that a quadruple mutation for A2a R enhances its structural stability in the active state to a significant extent. 21 However, the physical origin of this enhancement is still unknown, though the structures of both the wild type and the quadruple mutant have been determined by experiments. 22,23 In this study, we apply ∆F to these two structures and compare their stabilities, with the aid of all-atom molecular dynamics simulations necessitated for the following reason: The experimental condition for solving the wild-type and mutant crystal structures is substantially different from that for comparing their thermostabilities; and the structures matching the condition adopted for the comparison need to be constructed by modifying the crystal structures. The result can be summarized as follows: ∆F of the mutant is considerably lower than that of the wild type and the mutant is significantly more stable; the two structures share almost the same value of ∆Λ (∆Λ of the mutant is slightly higher than that of the wild type); and the enhanced stability is attributed to considerably more negative −T∆S. This result is 4
ACS Paragon Plus Environment
Page 4 of 31
Page 5 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
The Journal of Physical Chemistry
significant in the following two respects: Our free-energy function is applicable not only to the inactive state but also to the active one; and the entropic effect originating from the thermal motion of hydrocarbon groups constituting nonpolar chains within the lipid bilayer is crucially important. THEORETICAL METHOD Our basic stance was to develop the simplest possible method still capturing the essential physics so that all the possible mutations could be examined in quite a short computation time. Detailed descriptions of our free-energy function (FEF) were given in our earlier publications 10−13 and need not be repeated. Here, we briefly summarize the principal features of the FEF and the techniques introduced to efficiently calculate the FEF. (The model and the theoretical method are recapitulated in Supporting Information (SI) by comparing thermodynamics for a membrane protein with that for a water-soluble one.) Free-energy Function (FEF). The stability of a GPCR should be governed by that of its transmembrane (TM) region. 10−13 Therefore, only the TM region is considered. GPCR folding is accompanied by a gain of solvent entropy, loss of protein conformational entropy, and lowering of energy. The solvent is formed by hydrocarbon groups constituting nonpolar chains within the lipid bilayer, and its entropy is presumed to be governed by the element originating from the translational displacement of these hydrocarbon groups. 10−14 The solvent-entropy gain arising from the close packing of side chains in the side-to-side association of the seven helices is considered as the dominant contributor to the net gain. Assuming that the wild type and its mutant share the same loss of conformational entropy, we omit this loss. Protein intramolecular van der Waals (vdW) energy decreases upon the folding, but this decrease is assumed to be cancelled out by the increase in protein-solvent vdW energy. Since such cancellation does not occur for electrostatic energy, we account for the decrease in protein intramolecular electrostatic energy which is attributable primarily to the formation of intramolecular hydrogen bonds (IHBs). 13 Denoting the gain of solvent entropy and the lowering of energy thus defined by ∆S and ∆Λ, respectively, we express the change of the FEF upon GPCR folding (∆F) as ∆F/(k B T 0 )=∆Λ/(k B T 0 )−T∆S/(k B T 0 )=∆Λ/(k B T 0 )−∆S/k B
(1)
where T is the absolute temperature and set at T=T 0 , T0 =298 K, and k B is Boltzmann’s constant. We found that ∆Λ and ∆S are both strongly dependent on the GPCR structure (∆Λ0). ∆Λ/(k B T0 ) and −∆S/k B are referred to as “energetic and entropic components” of ∆F/(k B T0 ), respectively. A lower value of ∆F implies higher structural 5
ACS Paragon Plus Environment
The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
stability. A mutation which lowers ∆F is identified as a thermostabilizing one. In our interpretation, the statement, “the active state is stabilized”, implies that ∆F of the active state (i.e., the free-energy change occurring when a protein folds into the active state) is lowered. Method of Calculating the FEF. There are backbone-backbone, backbone-side chain, and side chain-side chain IHBs. We examine donors and acceptors of the IHBs within the TM region of a folded structure given. When an IHB is formed, an energy decrease of D is considered. D is made dependent on the distance between centers of H (the hydrogen atom covalently bound to the donor) and the acceptor. ∆Λ calculated can be expressed as xN IHB E* where N IHB denotes the number of IHBs and 0
