Structure of the δ-opioid receptor bound to naltrindole

The opioid receptor family comprises three members, the μ-, δ- and κ-opioid receptors, which respond to classical opioid alkaloids such as morphine and heroin as well as to endogenous peptide ligands like endorphins. They belong to the G-protein-coupled receptor (GPCR) superfamily, and are excellent therapeutic targets for pain control. The δ-opioid receptor (δ-OR) has a role in analgesia, as well as in other neurological functions that remain poorly understood. The structures of the μ-OR and κ-OR have recently been solved. Here we report the crystal structure of the mouse δ-OR, bound to the subtype-selective antagonist naltrindole. Together with the structures of the μ-OR and κ-OR, the δ-OR structure provides insights into conserved elements of opioid ligand recognition while also revealing structural features associated with ligand-subtype selectivity. The binding pocket of opioid receptors can be divided into two distinct regions. Whereas the lower part of this pocket is highly conserved among opioid receptors, the upper part contains divergent residues that confer subtype selectivity. This provides a structural explanation and validation for the 'message-address' model of opioid receptor pharmacology, in which distinct 'message' (efficacy) and 'address' (selectivity) determinants are contained within a single ligand. Comparison of the address region of the δ-OR with other GPCRs reveals that this structural organization may be a more general phenomenon, extending to other GPCR families as well.     

Structure of a beta1-adrenergic G-protein-coupled receptor

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 A resolution crystal structure of a beta(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 alpha-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 beta(1)-adrenergic receptor and binding of carazolol to the beta(2)-adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the beta(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.     

Methylated lysine 79 of histone H3 targets 53BP1 to DNA double-strand breaks

The mechanisms by which eukaryotic cells sense DNA double-strand breaks (DSBs) in order to initiate checkpoint responses are poorly understood. 53BP1 is a conserved checkpoint protein with properties of a DNA DSB sensor. Here, we solved the structure of the domain of 53BP1 that recruits it to sites of DSBs. This domain consists of two tandem tudor folds with a deep pocket at their interface formed by residues conserved in the budding yeast Rad9 and fission yeast Rhp9/Crb2 orthologues. In vitro, the 53BP1 tandem tudor domain bound histone H3 methylated on Lys 79 using residues that form the walls of the pocket; these residues were also required for recruitment of 53BP1 to DSBs. Suppression of DOT1L, the enzyme that methylates Lys 79 of histone H3, also inhibited recruitment of 53BP1 to DSBs. Because methylation of histone H3 Lys 79 was unaltered in response to DNA damage, we propose that 53BP1 senses DSBs indirectly through changes in higher-order chromatin structure that expose the 53BP1 binding site.     

Structure of the human κ-opioid receptor in complex with JDTic

Opioid receptors mediate the actions of endogenous and exogenous opioids on many physiological processes, including the regulation of pain, respiratory drive, mood, and--in the case of κ-opioid receptor (κ-OR)--dysphoria and psychotomimesis. Here we report the crystal structure of the human κ-OR in complex with the selective antagonist JDTic, arranged in parallel dimers, at 2.9?? resolution. The structure reveals important features of the ligand-binding pocket that contribute to the high affinity and subtype selectivity of JDTic for the human κ-OR. Modelling of other important κ-OR-selective ligands, including the morphinan-derived antagonists norbinaltorphimine and 5'-guanidinonaltrindole, and the diterpene agonist salvinorin A analogue RB-64, reveals both common and distinct features for binding these diverse chemotypes. Analysis of site-directed mutagenesis and ligand structure-activity relationships confirms the interactions observed in the crystal structure, thereby providing a molecular explanation for κ-OR subtype selectivity, and essential insights for the design of compounds with new pharmacological properties targeting the human κ-OR.     

嗜酸氧化亚铁硫杆菌的核糖-5-磷酸异构酶的同源建模研究

采用同源建模技术和分子动力学模拟方法,构建了嗜酸氧化亚铁硫杆菌Acidithiobacillus ferrooxidans(A.f)的核糖-5-磷酸异构酶(rpiA)基因编码的蛋白质三维分子结构模型。将结构模型进行绑定位点搜索并与底物核糖-5-磷酸(R5P)进行柔性分子对接,结果显示,R5P被招募到A.ferrooxidans的RpiA的活性位点并随后被激活;残基Asp81,Thr31,Lys121,Ser30,Glu103,Asp84,Lys94,Asp118,Lys7,Gly97,Gly29,Gly95,Thr28和H2O对底物绑定或催化起重要作用,其中,Gly97,Gly29,Gly95和Thr28是新识别的残基,它们在其他生物体的RpiA中相当保守但未被发现。     

Structure of the nociceptin/orphanin FQ receptor in complex with a peptide mimetic

Members of the opioid receptor family of G-protein-coupled receptors (GPCRs) are found throughout the peripheral and central nervous system, where they have key roles in nociception and analgesia. Unlike the 'classical' opioid receptors, δ, κ and μ (δ-OR, κ-OR and μ-OR), which were delineated by pharmacological criteria in the 1970s and 1980s, the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP, also known as ORL-1) was discovered relatively recently by molecular cloning and characterization of an orphan GPCR. Although it shares high sequence similarity with classical opioid GPCR subtypes (～60%), NOP has a markedly distinct pharmacology, featuring activation by the endogenous peptide N/OFQ, and unique selectivity for exogenous ligands. Here we report the crystal structure of human NOP, solved in complex with the peptide mimetic antagonist compound-24 (C-24) (ref. 4), revealing atomic details of ligand-receptor recognition and selectivity. Compound-24 mimics the first four amino-terminal residues of the NOP-selective peptide antagonist UFP-101, a close derivative of N/OFQ, and provides important clues to the binding of these peptides. The X-ray structure also shows substantial conformational differences in the pocket regions between NOP and the classical opioid receptors κ (ref. 5) and μ (ref. 6), and these are probably due to a small number of residues that vary between these receptors. The NOP-compound-24 structure explains the divergent selectivity profile of NOP and provides a new structural template for the design of NOP ligands.     

Structural and functional conservation of key domains in InsP3 and ryanodine receptors

Inositol-1,4,5-trisphosphate receptors (InsP(3)Rs) and ryanodine receptors (RyRs) are tetrameric intracellular Ca(2+) channels. In each of these receptor families, the pore, which is formed by carboxy-terminal transmembrane domains, is regulated by signals that are detected by large cytosolic structures. InsP(3)R gating is initiated by InsP(3) binding to the InsP(3)-binding core (IBC, residues 224-604 of InsP(3)R1) and it requires the suppressor domain (SD, residues 1-223 of InsP(3)R1). Here we present structures of the amino-terminal region (NT, residues 1-604) of rat InsP(3)R1 with (3.6??) and without (3.0??) InsP(3) bound. The arrangement of the three NT domains, SD, IBC-β and IBC-α, identifies two discrete interfaces (α and β) between the IBC and SD. Similar interfaces occur between equivalent domains (A, B and C) in RyR1 (ref. 9). The orientations of the three domains when docked into a tetrameric structure of InsP(3)R and of the ABC domains docked into RyR are remarkably similar. The importance of the α-interface for activation of InsP(3)R and RyR is confirmed by mutagenesis and, for RyR, by disease-causing mutations. Binding of InsP(3) causes partial closure of the clam-like IBC, disrupting the β-interface and pulling the SD towards the IBC. This reorients an exposed SD loop ('hotspot' (HS) loop) that is essential for InsP(3)R activation. The loop is conserved in RyR and includes mutations that are associated with malignant hyperthermia and central core disease. The HS loop interacts with an adjacent NT, suggesting that activation re-arranges inter-subunit interactions. The A domain of RyR functionally replaced the SD in full-length InsP(3)R, and an InsP(3)R in which its C-terminal transmembrane region was replaced by that from RyR1 was gated by InsP(3) and blocked by ryanodine. Activation mechanisms are conserved between InsP(3)R and RyR. Allosteric modulation of two similar domain interfaces within an N-terminal subunit reorients the first domain (SD or A domain), allowing it, through interactions of the second domain of an adjacent subunit (IBC-β or B domain), to gate the pore.     

Structural basis for specific cleavage of Lys 63-linked polyubiquitin chains

Deubiquitinating enzymes (DUBs) remove ubiquitin from conjugated substrates to regulate various cellular processes. The Zn(2+)-dependent DUBs AMSH and AMSH-LP regulate receptor trafficking by specifically cleaving Lys 63-linked polyubiquitin chains from internalized receptors. Here we report the crystal structures of the human AMSH-LP DUB domain alone and in complex with a Lys 63-linked di-ubiquitin at 1.2 A and 1.6 A resolutions, respectively. The AMSH-LP DUB domain consists of a Zn(2+)-coordinating catalytic core and two characteristic insertions, Ins-1 and Ins-2. The distal ubiquitin interacts with Ins-1 and the core, whereas the proximal ubiquitin interacts with Ins-2 and the core. The core and Ins-1 form a catalytic groove that accommodates the Lys 63 side chain of the proximal ubiquitin and the isopeptide-linked carboxy-terminal tail of the distal ubiquitin. This is the first reported structure of a DUB in complex with an isopeptide-linked ubiquitin chain, which reveals the mechanism for Lys 63-linkage-specific deubiquitination by AMSH family members.     

An amino-acid taste receptor

The sense of taste provides animals with valuable information about the nature and quality of food. Mammals can recognize and respond to a diverse repertoire of chemical entities, including sugars, salts, acids and a wide range of toxic substances. Several amino acids taste sweet or delicious (umami) to humans, and are attractive to rodents and other animals. This is noteworthy because L-amino acids function as the building blocks of proteins, as biosynthetic precursors of many biologically relevant small molecules, and as metabolic fuel. Thus, having a taste pathway dedicated to their detection probably had significant evolutionary implications. Here we identify and characterize a mammalian amino-acid taste receptor. This receptor, T1R1+3, is a heteromer of the taste-specific T1R1 and T1R3 G-protein-coupled receptors. We demonstrate that T1R1 and T1R3 combine to function as a broadly tuned L-amino-acid sensor responding to most of the 20 standard amino acids, but not to their D-enantiomers or other compounds. We also show that sequence differences in T1R receptors within and between species (human and mouse) can significantly influence the selectivity and specificity of taste responses.     

The structural basis of agonist-induced activation in constitutively active rhodopsin

G-protein-coupled receptors (GPCRs) comprise the largest family of membrane proteins in the human genome and mediate cellular responses to an extensive array of hormones, neurotransmitters and sensory stimuli. Although some crystal structures have been determined for GPCRs, most are for modified forms, showing little basal activity, and are bound to inverse agonists or antagonists. Consequently, these structures correspond to receptors in their inactive states. The visual pigment rhodopsin is the only GPCR for which structures exist that are thought to be in the active state. However, these structures are for the apoprotein, or opsin, form that does not contain the agonist all-trans retinal. Here we present a crystal structure at a resolution of 3 ? for the constitutively active rhodopsin mutant Glu 113 Gln in complex with a peptide derived from the carboxy terminus of the α-subunit of the G protein transducin. The protein is in an active conformation that retains retinal in the binding pocket after photoactivation. Comparison with the structure of ground-state rhodopsin suggests how translocation of the retinal β-ionone ring leads to a rotation of transmembrane helix 6, which is the critical conformational change on activation. A key feature of this conformational change is a reorganization of water-mediated hydrogen-bond networks between the retinal-binding pocket and three of the most conserved GPCR sequence motifs. We thus show how an agonist ligand can activate its GPCR.     

Structure of the HP1 chromodomain bound to histone H3 methylated at lysine 9

Specific modifications to histones are essential epigenetic markers---heritable changes in gene expression that do not affect the DNA sequence. Methylation of lysine 9 in histone H3 is recognized by heterochromatin protein 1 (HP1), which directs the binding of other proteins to control chromatin structure and gene expression. Here we show that HP1 uses an induced-fit mechanism for recognition of this modification, as revealed by the structure of its chromodomain bound to a histone H3 peptide dimethylated at Nzeta of lysine 9. The binding pocket for the N-methyl groups is provided by three aromatic side chains, Tyr21, Trp42 and Phe45, which reside in two regions that become ordered on binding of the peptide. The side chain of Lys9 is almost fully extended and surrounded by residues that are conserved in many other chromodomains. The QTAR peptide sequence preceding Lys9 makes most of the additional interactions with the chromodomain, with HP1 residues Val23, Leu40, Trp42, Leu58 and Cys60 appearing to be a major determinant of specificity by binding the key buried Ala7. These findings predict which other chromodomains will bind methylated proteins and suggest a motif that they recognize.     

Structure and dynamics of the M3 muscarinic acetylcholine receptor

Acetylcholine, the first neurotransmitter to be identified, exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences. We describe here the structure of the G(q/11)-coupled M3 mAChR ('M3 receptor', from rat) bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the G(i/o)-coupled M2 receptor, offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.     

一种非肽CCK拮抗剂的设计和合成

缩胆囊素是在激素，在消化道和神经传导方向起重要的作用。许多非肽类物质与缩胆囊一也有很强的亲合力，成为人们感兴越的研究课题。该文用四氢嘧啶酮环系替代吡咯环，设计合了非肽缩胆囊拮抗剂（＋）ＲＰ６６８０３的类似物。合成反应以天冬酰胺叔丁酯为起始原料，先与苯甲醛形成亚胺，再与甘氨酸酰氯发生环化缩化，生成中间体３－苯基－４－氨甲基羰基－５－叔丁氧羰基四氢嘧啶酮，最后与间甲苯异异氰酸酯反应，即得目标产物。以天