Structural basis of multiple drug-binding capacity of the AcrB multidrug efflux pump

Multidrug efflux pumps cause serious problems in cancer chemotherapy and treatment of bacterial infections. Yet high-resolution structures of ligand transporter complexes have previously been unavailable. We obtained x-ray crystallographic structures of the trimeric AcrB pump from Escherichia coli with four structurally diverse ligands. The structures show that three molecules of ligands bind simultaneously to the extremely large central cavity of 5000 cubic angstroms, primarily by hydrophobic, aromatic stacking and van der Waals interactions. Each ligand uses a slightly different subset of AcrB residues for binding. The bound ligand molecules often interact with each other, stabilizing the binding.     

Associative conditioning of single sensory neurons suggests a cellular mechanism for learning

A cellular analog of associative learning has been demonstrated in individual sensory neurons of the tail withdrawal reflex of Aplysia. Sensory cells activated by intracellular current injection shortly before a sensitizing shock to the animal's tail display significantly more facilitation of their monosynaptic connections to a tail motor neuron than cells trained either with intracellular stimulation unpaired to tail shock or with tail shock alone. This associative effect is acquired rapidly and is expressed as a temporally specific amplification of heterosynaptic facilitation. The results suggest that activity-dependent neuromodulation may be a mechanism underlying associative information storage and point to aspects of subcellular processes that might be involved in the formation of neural associations.     

Tryptophan 7-halogenase (PrnA) structure suggests a mechanism for regioselective chlorination

Chlorinated natural products include vancomycin and cryptophycin A. Their biosynthesis involves regioselective chlorination by flavin-dependent halogenases. We report the structural characterization of tryptophan 7-halogenase (PrnA), which regioselectively chlorinates tryptophan. Tryptophan and flavin adenine dinucleotide (FAD) are separated by a 10 angstrom-long tunnel and bound by distinct enzyme modules. The FAD module is conserved in halogenases and is related to flavin-dependent monooxygenases. On the basis of biochemical studies, crystal structures, and by analogy with monooxygenases, we predict that FADH2 reacts with O2 to make peroxyflavin, which is decomposed by Cl-. The resulting HOCl is guided through the tunnel to tryptophan, where it is activated to participate in electrophilic aromatic substitution.     

Structural evidence for a two-metal-ion mechanism of group I intron splicing

We report the 3.4 angstrom crystal structure of a catalytically active group I intron splicing intermediate containing the complete intron, both exons, the scissile phosphate, and all of the functional groups implicated in catalytic metal ion coordination, including the 2'-OH of the terminal guanosine. This structure suggests that, like protein phosphoryltransferases, an RNA phosphoryltransferase can use a two-metal-ion mechanism. Two Mg2+ ions are positioned 3.9 angstroms apart and are directly coordinated by all six of the biochemically predicted ligands. The evolutionary convergence of RNA and protein active sites on the same inorganic architecture highlights the intrinsic chemical capacity of the two-metal-ion catalytic mechanism for phosphoryl transfer.     

Structural mechanism for statin inhibition of HMG-CoA reductase

HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase (HMGR) catalyzes the committed step in cholesterol biosynthesis. Statins are HMGR inhibitors with inhibition constant values in the nanomolar range that effectively lower serum cholesterol levels and are widely prescribed in the treatment of hypercholesterolemia. We have determined structures of the catalytic portion of human HMGR complexed with six different statins. The statins occupy a portion of the binding site of HMG-CoA, thus blocking access of this substrate to the active site. Near the carboxyl terminus of HMGR, several catalytically relevant residues are disordered in the enzyme-statin complexes. If these residues were not flexible, they would sterically hinder statin binding.     

Structural mechanism of endosome docking by the FYVE domain

The recruitment of trafficking and signaling proteins to membranes containing phosphatidylinositol 3-phosphate [PtdIns(3)P] is mediated by FYVE domains. Here, the solution structure of the FYVE domain of the early endosome antigen 1 protein (EEA1) in the free state was compared with the structures of the domain complexed with PtdIns(3)P and mixed micelles. The multistep binding mechanism involved nonspecific insertion of a hydrophobic loop into the lipid bilayer, positioning and activating the binding pocket. Ligation of PtdIns(3)P then induced a global structural change, drawing the protein termini over the bound phosphoinositide by extension of a hinge. Specific recognition of the 3-phosphate was determined indirectly and directly by two clusters of conserved arginines.     

Inheritance of a cardiac arterial asymmetry in mice

A pair of coronary arteries supplies the interventricular septum of the heart of Mus musculus. The members of this pair tend to be of unequal size, which permits distiniction between specimens displaying left asymmetry and those showing righit asymmetry. Three inbred strains (C57BL/1O, DBA/1, and Bulb/c) differ with regard to this asymmetry. However, variation exists within strains, which suggests that nongenetic factors also influence the development of the asymmetry. Right arterial asymmetry is dominant over left asymmetry.     

Structural mechanism for STI-571 inhibition of abelson tyrosine kinase

The inadvertent activation of the Abelson tyrosine kinase (Abl) causes chronic myelogenous leukemia (CML). A small-molecule inhibitor of Abl (STI-571) is effective in the treatment of CML. We report the crystal structure of the catalytic domain of Abl, complexed to a variant of STI-571. Critical to the binding of STI-571 is the adoption by the kinase of an inactive conformation, in which a centrally located "activation loop" is not phosphorylated. The conformation of this loop is distinct from that in active protein kinases, as well as in the inactive form of the closely related Src kinases. These results suggest that compounds that exploit the distinctive inactivation mechanisms of individual protein kinases can achieve both high affinity and high specificity.     

偏置分流叶片离心泵内流动机理及仿真分析

偏置分流叶片离心泵内流动机理是水力机械研究的热点问题之一.通过对偏置分流叶片离心泵内流场的流动机理进行分析,并采用FLUENT软件,对IS65-40-250型偏置分流叶片离心泵进行流场的数值模拟计算,揭示了流体在离心泵内的速度和压力分布规律.研究表明,采用分流叶片偏置方法,有利于提高离心泵内流场速度和压力分布的均匀性,...     

啤酒花自动采摘机的结构设计及机理分析

介绍了1种啤酒花自动采摘机的主要结构设计及工作机理.通过对啤酒花品种‘青岛大花’的力学特征分析,采用了捋花采摘的设计思想,机器摘花时作用于花瓣上的力较小,采摘动作柔和,使啤酒花采摘时的破碎率降低,损失率减小,并通过调整摘花轮之间的差速度提高啤酒花的摘净率.对于不同品种啤酒花,因其力学特性与‘青岛大花’不同,可通过调整摘花轮之间的差速系统达到对不同品种啤酒花的采摘要求.     

螺杆泵式水田化肥深施机的排送肥机构研究

在水田化肥深施方案（即将化肥与适量水混合后，用强制性排送肥机构将其排入土中）的基础上，对排送肥机构的结构进行了研究。研制了一种对各排肥管轮换供肥的分配器，并对相关参数进行了优化，实验表明，这种排送肥机构能满足农业生产的要求。     

Structural insight into the ion-exchange mechanism of the sodium/calcium exchanger

Sodium/calcium (Na(+)/Ca(2+)) exchangers (NCX) are membrane transporters that play an essential role in maintaining the homeostasis of cytosolic Ca(2+) for cell signaling. We demonstrated the Na(+)/Ca(2+)-exchange function of an NCX from Methanococcus jannaschii (NCX_Mj) and report its 1.9 angstrom crystal structure in an outward-facing conformation. Containing 10 transmembrane helices, the two halves of NCX_Mj share a similar structure with opposite orientation. Four ion-binding sites cluster at the center of the protein: one specific for Ca(2+) and three that likely bind Na(+). Two passageways allow for Na(+) and Ca(2+) access to the central ion-binding sites from the extracellular side. Based on the symmetry of NCX_Mj and its ability to catalyze bidirectional ion-exchange reactions, we propose a structure model for the inward-facing NCX_Mj.     

支持热拔插的高效率电荷泵设计

提出了一种高效率的电荷泵电路结构,在相同输入电压条件下,其输出电压比传统电荷泵高出1个阈值电压.此外,针对传统电荷泵电路在上电过程中存在浪涌电流的缺点,设计了一个两级限流电路,实现电荷泵电路的软启动,确保芯片在热拔插过程中,不会影响输入电源线上的其他芯片的正常工作.芯片测试结果表明在电源电压为2.7 V、负载电容为4.5μF、负载电阻为600Ω的条件下,提供4.8 V的输出电压,上升时间约为36 ms,实现可靠性工作,效率最高达到83%.     

Friction anisotropy and asymmetry of a compliant monolayer induced by a small molecular tilt

Lateral force microscopy in the wearless regime was used to study the friction behavior of a lipid monolayer on mica. In the monolayer, condensed domains with long-range orientational order of the lipid molecules were present. The domains revealed unexpectedly strong friction anisotropies and non-negligible friction asymmetries. The angular dependency of these effects correlated well with the tilt direction of the alkyl chains of the monolayer, as determined by electron diffraction and Brewster angle microscopy. The molecular tilt causing these frictional effects was less than 15 degrees, demonstrating that even small molecular tilts can make a major contribution to friction.     

Structural analysis of the mechanism of adenovirus binding to its human cellular receptor, CAR

Binding of virus particles to specific host cell surface receptors is known to be an obligatory step in infection even though the molecular basis for these interactions is not well characterized. The crystal structure of the adenovirus fiber knob domain in complex with domain I of its human cellular receptor, coxsackie and adenovirus receptor (CAR), is presented here. Surface-exposed loops on knob contact one face of CAR, forming a high-affinity complex. Topology mismatches between interacting surfaces create interfacial solvent-filled cavities and channels that may be targets for antiviral drug therapy. The structure identifies key determinants of binding specificity, which may suggest ways to modify the tropism of adenovirus-based gene therapy vectors.     

N-Cadherin, a cell adhesion molecule involved in establishment of embryonic left-right asymmetry

Within the bilaterally symmetric vertebrate body plan, many organs develop asymmetrically. Here, it is demonstrated that a cell adhesion molecule, N-cadherin, is one of the earliest proteins to be asymmetrically expressed in the chicken embryo and that its activity is required during gastrulation for proper establishment of the left-right axis. Blocking N-cadherin function randomizes heart looping and alters the expression of Snail and Pitx2, later components of the molecular cascade that regulate left-right asymmetry. However, the expression of other components of this cascade (Nodal and Lefty) was unchanged after blocking N-cadherin function, suggesting the existence of parallel pathways in the establishment of left-right morphogenesis. Here, the results suggest that N-cadherin-mediated cell adhesion events are required for establishment of left-right asymmetry.     

Isolation of an olfactory cDNA: similarity to retinol-binding protein suggests a role in olfaction

Molecular cloning techniques were used to isolate and characterize a protein possibly involved in the signal transducing system in olfactory tissue of the frog Rana pipiens. A complementary DNA library was constructed with messenger RNA obtained from frog olfactory neuroepithelium. A 700-base pair complementary DNA clone encoding a protein with a molecular weight of 20,300 was identified by differential hybridization analysis with polyadenylated RNA from olfactory epithelium and nonsensory respiratory epithelium. The messenger RNA corresponding to this clone was abundant in the cells of Bowman's glands in olfactory tissue but not in respiratory epithelium nor in several other tissues. The predicted sequence of this protein is homologous to members of a family of proteins that bind and transport small molecules in serum, suggesting that this protein may also bind and transport odorants in the mucus secreted by Bowman's glands.     

Structural basis of a phototropin light switch

Phototropins are light-activated kinases important for plant responses to blue light. Light initiates signaling in these proteins by generating a covalent protein-flavin mononucleotide (FMN) adduct within sensory Per-ARNT-Sim (PAS) domains. We characterized the light-dependent changes of a phototropin PAS domain by solution nuclear magnetic resonance spectroscopy and found that an alpha helix located outside the canonical domain plays a key role in this activation process. Although this helix associates with the PAS core in the dark, photoinduced changes in the domain structure disrupt this interaction. We propose that this mechanism couples light-dependent bond formation to kinase activation and identifies a signaling pathway conserved among PAS domains.