Crystal structures of a multidrug transporter reveal a functionally rotating mechanism

AcrB is a principal multidrug efflux transporter in Escherichia coli that cooperates with an outer-membrane channel, TolC, and a membrane-fusion protein, AcrA. Here we describe crystal structures of AcrB with and without substrates. The AcrB-drug complex consists of three protomers, each of which has a different conformation corresponding to one of the three functional states of the transport cycle. Bound substrate was found in the periplasmic domain of one of the three protomers. The voluminous binding pocket is aromatic and allows multi-site binding. The structures indicate that drugs are exported by a three-step functionally rotating mechanism in which substrates undergo ordered binding change.     

Crystal structure of bacterial multidrug efflux transporter AcrB

AcrB is a major multidrug exporter in Escherichia coli. It cooperates with a membrane fusion protein, AcrA, and an outer membrane channel, TolC. We have determined the crystal structure of AcrB at 3.5 A resolution. Three AcrB protomers are organized as a homotrimer in the shape of a jellyfish. Each protomer is composed of a transmembrane region 50 A thick and a 70 A protruding headpiece. The top of the headpiece opens like a funnel, where TolC might directly dock into AcrB. A pore formed by three alpha-helices connects the funnel with a central cavity located at the bottom of the headpiece. The cavity has three vestibules at the side of the headpiece which lead into the periplasm. In the transmembrane region, each protomer has twelve transmembrane alpha-helices. The structure implies that substrates translocated from the cell interior through the transmembrane region and from the periplasm through the vestibules are collected in the central cavity and then actively transported through the pore into the TolC tunnel.     

Structural basis of anticodon loop recognition by glutaminyl-tRNA synthetase

The refined crystal structure of Escherichia coli glutaminyl transfer RNA synthetase complexed with transfer RNA(Gln) and ATP reveals that the structure of the anticodon loop of the enzyme-bound tRNA(Gln) differs extensively from that of the known crystal structures of uncomplexed tRNA molecules. The anticodon stem is extended by two non-Watson-Crick base pairs, leaving the three anti-codon bases unpaired and splayed out to bind snugly into three separate complementary pockets in the protein. These interactions suggest that the entire anticodon loop provides essential sites for glutaminyl tRNA synthetase discrimination among tRNA molecules.     

东濮凹陷轴向重力流水道沉积研究

利用地质、测井和地震等多种资料进行的综合研究表明,东濮凹陷桥口和新霍地区沙三段中发育轴向重力流水道沉积,它们主要由灰黑色块状泥岩与平行层理砂岩、块状层理砂岩、递变层理砂岩及合卵石砂岩构成,在垂向上组合成鲍玛序列的AE、BE、ACE、BCE和CE等。依据研究区轴向重力流水道沉积特征,将其划分成近源和远源水道、近源和远源水道侧源等4个微相,并描述了这4个微相的沉积特征,建立了沉积模式。油气勘探实践表明,重力流水道砂体是我国东部陆相断陷湖盆中重要的油气勘探新目标,它具有良好的储集物性,并含有丰富的油气。     

液体包装机灌装记忆的实现

在医药用液体包装机的灌装过程中,药液的漏灌和无瓶灌装会造成很大的经济损失,在生产过程中必须禁止,然而,由于流水线上药瓶的不连续传送以及停电、紧急停车等故障的发生,都会产生漏灌和无瓶空灌的现象发生。本文主要对液体包装机灌装控制中出现的问题加以分析,并提出了一套行之有效的控制方法。     

Crystal structure of the µ-opioid receptor bound to a morphinan antagonist

Opium is one of the world's oldest drugs, and its derivatives morphine and codeine are among the most used clinical drugs to relieve severe pain. These prototypical opioids produce analgesia as well as many undesirable side effects (sedation, apnoea and dependence) by binding to and activating the G-protein-coupled μ-opioid receptor (μ-OR) in the central nervous system. Here we describe the 2.8?? crystal structure of the mouse μ-OR in complex with an irreversible morphinan antagonist. Compared to the buried binding pocket observed in most G-protein-coupled receptors published so far, the morphinan ligand binds deeply within a large solvent-exposed pocket. Of particular interest, the μ-OR crystallizes as a two-fold symmetrical dimer through a four-helix bundle motif formed by transmembrane segments 5 and 6. These high-resolution insights into opioid receptor structure will enable the application of structure-based approaches to develop better drugs for the management of pain and addiction.     

Structural basis for transcription elongation by bacterial RNA polymerase

The RNA polymerase elongation complex (EC) is both highly stable and processive, rapidly extending RNA chains for thousands of nucleotides. Understanding the mechanisms of elongation and its regulation requires detailed information about the structural organization of the EC. Here we report the 2.5-A resolution structure of the Thermus thermophilus EC; the structure reveals the post-translocated intermediate with the DNA template in the active site available for pairing with the substrate. DNA strand separation occurs one position downstream of the active site, implying that only one substrate at a time can specifically bind to the EC. The upstream edge of the RNA/DNA hybrid stacks on the beta'-subunit 'lid' loop, whereas the first displaced RNA base is trapped within a protein pocket, suggesting a mechanism for RNA displacement. The RNA is threaded through the RNA exit channel, where it adopts a conformation mimicking that of a single strand within a double helix, providing insight into a mechanism for hairpin-dependent pausing and termination.     

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.     

Structural basis for gene regulation by a thiamine pyrophosphate-sensing riboswitch

Riboswitches are metabolite-sensing RNAs, typically located in the non-coding portions of messenger RNAs, that control the synthesis of metabolite-related proteins. Here we describe a 2.05 angstroms crystal structure of a riboswitch domain from the Escherichia coli thiM mRNA that responds to the coenzyme thiamine pyrophosphate (TPP). TPP is an active form of vitamin B1, an essential participant in many protein-catalysed reactions. Organisms from all three domains of life, including bacteria, plants and fungi, use TPP-sensing riboswitches to control genes responsible for importing or synthesizing thiamine and its phosphorylated derivatives, making this riboswitch class the most widely distributed member of the metabolite-sensing RNA regulatory system. The structure reveals a complex folded RNA in which one subdomain forms an intercalation pocket for the 4-amino-5-hydroxymethyl-2-methylpyrimidine moiety of TPP, whereas another subdomain forms a wider pocket that uses bivalent metal ions and water molecules to make bridging contacts to the pyrophosphate moiety of the ligand. The two pockets are positioned to function as a molecular measuring device that recognizes TPP in an extended conformation. The central thiazole moiety is not recognized by the RNA, which explains why the antimicrobial compound pyrithiamine pyrophosphate targets this riboswitch and downregulates the expression of thiamine metabolic genes. Both the natural ligand and its drug-like analogue stabilize secondary and tertiary structure elements that are harnessed by the riboswitch to modulate the synthesis of the proteins coded by the mRNA. In addition, this structure provides insight into how folded RNAs can form precision binding pockets that rival those formed by protein genetic factors.     

基于二维DCT的OFDMA系统信道估计方法

在OFDMA(orthogonal frequency division multiple access)系统中,每个用户通过占用若干时频资源块以实现多址传输,此时传统的基于DFT(discrete Fourier transform)的高效信道估计算法将会产生难以消除的平底效应;另一方面,当终端高速移动时,基于DFT的时域插值亦将面临较为严重的性能损失.为了解决上述问题,提出一种基于二维DCT(discrete cosine transform)的信道估计算法.仿真结果表明,所提出的信道估计算法能以较低的实现复杂度获取逼近最优信道估计器的性能,且在低信噪比条件下亦可获取高精度的信道估计,有助于实现"绿色通信".     

Molecular simulation study of the binding mechanism of [α-PTi<Subscript>2</Subscript>W<Subscript>10</Subscript>O<Subscript>40</Subscript>]<Superscript>7−</Superscript> for its promising broad-spectrum inhibitory activity to FluV-A neuraminidase

Polyoxometalate (POM) has promising antiviral activities. It shows broad-spectrum inhibiting ability, high efficiency, and low tox-icity. Experimental assays show that titanium containing polyoxotungstates have anti-influenza-virus activity. In this paper, the bind-ing mechanisms of five isomers of di-Ti-substituted polyoxotungstate, [α-1,2-PTi₂W₁₀O₄₀]^7– (α-1,2), [α-1,6-PTi₂W₁₀O₄₀]^7– (α-1,6), [α-1,5-PTi₂W₁₀O₄₀]^7– (α-1,5), [α-1,4-PTi₂W₁₀O₄₀]^7– (α-1,4) and [α-1,11-PTi₂W₁₀O₄₀]^7– (α-1,11), to five subtypes of influenza virus A neuraminidase (FluV-A NA) were investigated in the context of aqueous solution by using molecular docking and molecular dynamics studies. The results show that the isomer α-1,2 is superior to other isomers as a potential inhibitor to neuraminidase. The positively charged arginine residues around the active site of NA could be induced by negatively charged POM to adapt themselves and could form salt bridge interactions and hydrogen bond interactions with POM. The binding free energies of POM/NA complexes range from –5.36 to –8.31 kcal mol^–1. The electrostatic interactions are found to be the driving force during the binding process of POM to NA. The conformational analysis shows that POM tends to bind primarily with N1 and N8 at the edge of the active pocket, which causes the conformational change of the pincers structure comprising residue 347 and loop 150. Whereas, the active pockets of N2, N9 and N4 are found to be more spacious, which allows POM to enter into the active pockets directly and anchor there firmly. This study shows that negatively charged ligand as POM could induce the reorganization of the active site of NA and highlights POM as a promising inhibitor to NA despite the ever increasing mutants of NA.     

The role of the distal histidine in myoglobin and haemoglobin

The distal E7 histidine in vertebrate myoglobins and haemoglobins has been strongly conserved during evolution and is thought to be important in fine-tuning the ligand affinities of these proteins. A hydrogen bond between the N epsilon proton of the distal histidine and the second oxygen atom may stabilize O2 bound to the haem iron. The proximity of the imidazole side chain to the sixth coordination position, which is required for efficient hydrogen bonding, has been postulated to inhibit sterically the binding of CO and alkyl isocyanides. To test these ideas, engineered mutants of sperm whale myoglobin and the alpha- and beta-subunits of human haemoglobin were prepared in which E7 histidine was replaced by glycine. Removal of the distal imidazole in myoglobin and the alpha-subunits of intact, R-state haemoglobin caused significant changes in the affinity for oxygen, carbon monoxide and methyl isocyanide; in contrast, the His-E7 to Gly substitution produced little or no effect on the rates and extents of O2, CO and methyl isocyanide binding to beta-chains within R-state haemoglobin. In the beta-subunit the distal histidine seems to be less significant in regulating the binding of ligands to the haem iron in the high affinity quaternary conformation. Structural differences in the oxygen binding pockets shown by X-ray crystallographic studies account for the functional differences of these proteins.     

Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid

The three-dimensional structures of influenza virus haemagglutinins complexed with cell receptor analogues show sialic acids bound to a pocket of conserved amino acids surrounded by antibody-binding sites. Sialic acid fills the conserved pocket, demonstrating that it is the influenza virus receptor. The proximity of the antibody-binding sites suggests that antibodies neutralize virus infectivity by preventing virus-to-cell binding. The structures suggest approaches to the design of anti-viral drugs that could block attachment of viruses to cells.