Structure of the adenosine A2A receptor bound to an engineered G protein B. Carpenter, R. Nehmé, T. Warne, A. G. W. Leslie1 & C. G. Tate Nature, 2016, 536, 104–107 將adenosine A2A receptor 與設計過的G protein結合，並分析他們的結構 姓名 : 施曉慈 學號 : 1013489 2017/05/23

Adenosine A2A receptor (A2AR) A2AR is a class of G protein-coupled receptors (GPCRs) with adenosine as the ligand. A2AR is coupled with Gs which can stimulate the activation of adenylate cyclase . A2AR屬於結合G protein的受體，一般稱為GPCR的一員，並且A2AR以adenosine作為配體 A2AR結合的G protein在活化後可以刺激腺苷酸環化酶活化，因此稱為Gs adenosine sleeping

Ligand Agonist: a substance that fully activates the receptor. ex. adenosine, NECA, CGS21680,and UK432097 Antagonist: a substance that binds to a receptor but does not activate and can block the activity of other agonists . ex. ZM241385, XAC7, caffeine, and 1,2,4-triazines Allosteric antagonist: Na+ ion agonist adenosine antagonist caffeine

A2AR lacks the crystal structure at active conformation Structures of A2AR in the inactive state have been determined bound to the antagonists, and all the structures are very similar. Dore, A. S. et al. Structure(2011), Hino, T. et al. Nature(2012), Jaakola, V. P. et al. Science(2008). Four agonist-bound structures of A2AR are very similar and are thought to represent an active-intermediate conformation of the receptor. Lebon, G. et al, Nature 474, (2011), Xu, F. et al. Science 332, (2011). The complex structure of β2AR bound with agonist and G protein have been determined. Rasmussen, S. G. et al. Nature (2011). Inactive active- active intermediate 已經確定處於非活性狀態的A2AR的結構與拮抗劑結合，並且所有結構非常相似。 A2AR的四個激動劑結合結構非常相似，且被認為表現受體的活性中間構象。 Active β2AR-Gs

Motivation To determine the crystal structure of A2AR receptor at active state (agonist-bound). To understand the conformational influence of the G protein to A2AR receptor. 確定受體活性狀態的結構 了解G蛋白對A2AR受體的影響

Engineered G protein (mini-Gs) Structure of β2AR-Gs showed that all the contacts between the receptor and G protein were formed by the Gα subunit. Rasmussen, S. G. et al. Nature 477 (2011). Mini-Gs comprises a truncated form of the GTPase domain of Gαs, and eight point mutation. The truncations removed the switch III region, 25 amino acids from the N-ter and the α -helical domain. 他人的研究表示:β2AR-Gs的結構顯示受體與G蛋白之間的所有接觸均由Gα亞基形成 較小的G protein可促進GPCR-Gs複合物的結晶 Mini-Gs包含截短GTP酶結構域的Gα、8個點突變、3個截短位，分別切除了switch III region(深淺藍交界)、N-ter的25個氨基酸(極淺藍)、α-螺旋結構域(深藍)。 8個點突變以在沒有Gβγ的情況下和在洗滌劑存在下穩定蛋白質 Gs mini-Gs

Mini-Gs increases the affinity of ligands Mini-Gs increases the binding affinity of agonist NECA and allosteric antagonist Na+ to A2AR. 作者想要知道mini-Gs對於A2AR結合ligand的親和性的影響 因此，作法… 3H-ZM241385 (同位素標定的) The nanobody(Nb35) facilitates crystallization, Nb35 binds to the Gs-Gβ interface Mini-Gs增加激動劑NECA和變構拮抗劑Na +對A2AR的結合親和力。 inverse agonist ZM241385 agonist NECA

Mini-Gs increases the thermostability The complex was considerably more thermostable, particularly in short-chain detergents. 熱穩定性測試 該複合物更加耐高溫，特別是在短鏈detergent中。

A2AR-Mini-Gs complex is in active state The conformation of mini-Gs is virtually identical to active β2AR–Gs structure. Active G protein can couple to A2AR and induce high-affinity agonist binding. 比較Mini-Gs與活化的β2AR-Gs結構。 Mini-Gs與活化的β2AR-Gs中觀察到的結構幾乎相同。 G protein呈活性狀態，與其(前人所說)和A2AR結合並誘導對激動劑高親和力結合的能力一致 GDP在微型Gs結構中的存在反映了工程化G蛋白的性質，其在復合物形成後對GTPγ介導的解離不敏感 RMSD = 1.7 Å A2AR -mini-Gs with NECA and GDP Active β2AR-Gs

Interactions between A2AR and mini-Gs The interactions between A2AR and G protein are majorly located at the intracellular end of helices and α5 helix, respectively. A2AR mini-Gs 以topology觀看A2AR-min-Gs結構中A2AR和min-Gs的topology 灰色陰影的殘基無序;二硫鍵為粉色線 A2AR和G protein之間的相互作用主要位於helices的細胞內末端和mini-Gs的α5螺旋。 Blue: positively charged; red: negatively charged; green: hydrophobic; yellow: hydrophilic

Interactions between A2AR and mini-Gs The interface between A2AR and mini-Gs is formed between 20 amino acid residues from the receptor and 17 residues in mini-Gs. 作者觀察結構及序列後得知:A2AR和mini-Gs之間的界面在受體的20個氨基酸殘基和mini-Gs之間形成17個殘基 在mini-Gs中，接觸主要由α5螺旋形成，包括14個殘基，其對A2AR的H3、CL2、H5、H6、H7、H8的殘基進行包裝 另外的相互作用包括S1，S3，S2-S3loop、α5中的殘基形成疏水口袋，其中A2AR的CL2中的Leu110側鏈被螯合 A2AR和mini-Gs中的氨基酸殘基形成互補表面，其主要（〜90％）的接觸通過范德瓦相互作用以及六個極性相互作用 線厚度代表氨基酸殘基之間相互作用的相對數。 藍: 正電 ; 紅:負電 ; 綠:疏水 ; 黃:親水。 Blue: positively charged; red: negatively charged; green: hydrophobic; yellow: hydrophilic

Mini-Gs is different from the full-length Gs Two receptors have very similar architectures, but mini-Gs does not superimpose exactly on the Gα subunit of the G protein. There is a difference in orientation of ~ 15° between mini-Gs and Gα subunit . The most substantial difference is an 8° tilt between the respective α5 helices. 為了清楚起見，Gαs、Gβγ、Νb35的α -helical domain全部被省略。 兩個受體具有非常相似的結構，包含向外拉的H6細胞膜內端，使得螺旋α5可突出到A2AR細胞質面內的裂縫，但mini-Gs不完全疊加在與β2AR結合的Gα亞基上 G protein 間的差異較大，Mini-Gs和Gα亞基之間的方向差異為〜15°。 主要原因為a5 helix間有8°的偏移 作者認為是A2AR與B2ar的組成的氨基酸不同導致與G protein結合的位置不同造成 這可能是A2AR與β2AR具有不同氨基酸殘基，導致G蛋白與受體的包裝略有不同，或是晶格接觸的影響。 A2AR-mini-Gs和β2AR-Gs複合物的比較:A2AR為彩色 ; β2AR為灰色 ; mini-Gs為紫色 ; Gs為深灰色 黃: C ; 藍: N ; 紅: O; 橙: P A2AR: rainbow coloration β2AR: grey Gαs: dark grey mini-Gs: purple carbon: yellow nitrogen: blue oxygen: red phosphorous: orange RMSD(total) = 1.7 Å RMSD(G protein) = 0.92 Å

Ballesteros–Weinstein numbering and common Gα numbering The Ballesteros–Weinstein numbering (B-W) and common Gα numbering (CGN) allow relating every amino acid residue position to equivalent positions in homologous proteins. The most conserved residues are used as anchor point. Ballesteros–Weinstein numbering (GPCR)  common Gα numbering (G protein) TM1 Asn55 (1.50)   TM2 Asp83 (2.50)   TM3 Arg135(3.50)   TM4 Trp161(4.50)   TM5 Pro215(5.50) TM6 Pro267(6.50) TM7 Pro303(7.50) Ballesteros-Weinstein編號（B-W）和普通Gα編號（CGN）將每個氨基酸殘基位置與同源蛋白中的等同位置相聯繫 最保守的殘基用作錨點

Hydrogen bonds between A2AR receptor and G protein The 8° tilt between the respective α 5 helices results in a 3.7 Å displacement of the Cα of Tyr391H5.23 in mini-Gs. The interactions between A2AR and G protein are highly conserved in β2AR-Gs complex. 詳細比較各G蛋白和受體之間的氫鍵（紅色虛線） 之前提到，a5 helix間有8°的偏移 A2AR中，α5螺旋的頂點Tyr391偏移3.7 A，離開G protein的中央 下圖中，明顯可看到a5 helix的頂端越來越遠離H5 EH5.24

Conserved hydrogen bonds Although the exact orientation and atomic contacts may differ, many of the contacts between residues in the α5 helix of the G protein and the receptors are conserved. 換個角度可清楚地看到胺基酸間的interaction A2AR的mini-Gs主要與H3、H5形成氫鍵，頂端Tyr與H7形成氫鍵 β2AR的Gs主要與H3、H5形成氫鍵，頂端胺基酸與H6形成氫鍵 儘管精確的方向和原子的接觸可能都不同，但G protein的α5螺旋中的residue與受體之間的許多接觸是保守(conserved)的

Conserved hydrophobic interactions There is a highly conserved interaction around CL2, Leu110 in A2AR and Phe139 in β 2AR, and residues His41S1.2, Val217S3.1 and Asp215s2s3.1 in Gαs. 在其他地方也有高度保守的相互作用，存在於A2AR中的Leu110和β2AR中的Phe139和Gαs中殘基His41S1.2，Val217S3.1和Asp215s2s3.1之間。 Green: A2AR Blue: β2AR Purple: mini-Gs Grey: Gαs

Contact atoms of A2AR The Tyr391H5.23 makes van der Waals interactions with Arg1023.50 that forms the whole upper surface of the cleft. The total buried surface area is 1,048 Å2 on the A2AR receptor. Green: carbon Blue: nitrogen Red: oxygen Pink: interact with Arg3.50 將與G protein接觸的原子著色，綠:C ; 藍:N ; 紅:O ; 粉紅: α5螺旋的頂點Tyr391^H5.23與受體的Arg102^3.50進行廣泛的范德瓦相互作用，形成裂縫的整個上表面 受體上的總掩埋面積為1,048Å2。

Additional difference affects receptor-G protein contact The main difference between the A2AR–mini-Gs interface compared to the β 2AR–Gs interface occurs at the H5, H7 and H8. The majority of amino acid residues at the interface between the receptor and G protein are identical, but there are different interaction in specific regions. 比較A2AR-mini-Gs複合物和β2AR-Gs複合物中與G protein有interaction的residue 主要的差異為A2AR的H7、H8額外與mini-Gs接觸，而b2AR的H5比較長，與Gs形成較多的交互作用 儘管在受體和G蛋白之間的界面處的大多數氨基酸殘基是相同的，但在特定的區域有所差異 接觸的受體的氨基酸: 紅:帶負電荷 ; 藍:帶正電 ; 綠:疏水 ; 黃:親水，白色代表沒有與G protein接觸，每受體所佔比例相似 B–W number表示跨膜α -helices中residues的B-W數，虛線連接H8或loop的residues 氨基酸殘基5.71-5.77在A2AR-mini-Gs結構中無序(loop)。 Red: negatively charged Blue: positively charged Green: hydrophobic Yellow: hydrophilic White: no contact

Mini-Gs does not affect ligand-bound conformation of A2AR Compare the structure of NECA -bound A2AR with the structure of A2AR bound to mini-Gs. There are no different at the ligand binding site of receptor. 比較結合NECA的A2AR與A2AR-Mini-Gs的結構。 受體配體結合位點沒有差異 A2AR-mini-Gs: rainbow coloration A2AR: grey

Mini-Gs activate the A2AR Comparison the active-intermediate state of agonist-bound A2AR and A2AR -mini-Gs. Major rearrangements in the cytoplasmic half of the receptor, and transition from the active-intermediate state to the activated G-protein-bound conformation. 比較UK432097綁定的A2AR和A2AR-mini-Gs的活躍中間狀態。(由細胞質端觀看) 受體核心的細胞質半部分的主要重排以適應G蛋白結合，並從活性中間狀態轉變為活化的G蛋白結合構象。 有以下幾點改變: 通過H6向外彎曲，H6的細胞質末端離開受體芯離開14Å H5、H7對H6的細胞質末端的包裝有顯著變化。 A2AR-mini-Gs: rainbow coloration A2AR: grey

Mini-Gs activate the A2AR H6運動的程度取決於A2AR的Lys227^6.29、Ala231^6.33、Leu235^6.37與mini-Gs的Leu393^H5.25、C-ter之間的范德華力相互作用。 高度保守的Tyr197和Tyr288採用旋轉異構體來填充Leu235、Ile238的空間，Arg102^3.50改變，與Tyr391^H5.23作用

Conclusion The transition of the receptor from agonist-bound active-intermediate state to active state has the following characteristics: 14 Å shift of the cytoplasmic end H6 away from the receptor core slight changes in the cytoplasmic ends of H5 and H7 rotamer changes of the amino acid side chains Arg3.50, Tyr5.58 and Tyr7.53 受體從激動劑結合的活性中間狀態轉變為活性狀態具有以下特徵：  跨膜螺旋6（H6）的細胞質末端離受體核心的轉移 H5和H7的細胞質末端略有變化 氨基酸側鏈Arg3.50，Tyr5.58和Tyr7.53的旋轉異構體變化 在活性中間狀態中描述的配體結合口袋的配置描述了結合A2AR的NECA的高親和力狀態。

Alignment of β2AR and A2AR

Alignment of mini-Gs with Gs 這可能是A2AR與β2AR具有不同氨基酸殘基，導致G蛋白與受體的相互作用區域略有不同

Prepare recombinant baculoviruses to expressing A2AR purification Mini-Gs A2AR Insert mini-Gs gene into plasmid and transform to BL21(DE3)RIL Prepare recombinant baculoviruses to expressing A2AR Incubate cell and induct with 50 μ M IPTG for 20 h at 25 °C. Incubate insect cell and infect with A2AR baculovirus for 72 h Collect cells and lyse by sonication in lysis buffer Collect cells and prepare membrane by two ultracentrifugation steps purify by Ni2+ Sepharose FF column purify by Ni-NTA column Add TEV protease and purify by Ni2+- NTA resin Add TEV protease and purify by Ni2+- NTA resin