Structure of the cytoplasmic beta subunit-T1 assembly of voltage-dependent K+ channels

The structure of the cytoplasmic assembly of voltage-dependent K+ channels was solved by x-ray crystallography at 2.1 angstrom resolution. The assembly includes the cytoplasmic (T1) domain of the integral membrane alpha subunit together with the oxidoreductase beta subunit in a fourfold symmetric T1(4)beta4 complex. An electrophysiological assay showed that this complex is oriented with four T1 domains facing the transmembrane pore and four beta subunits facing the cytoplasm. The transmembrane pore communicates with the cytoplasm through lateral, negatively charged openings above the T1(4)beta4 complex. The inactivation peptides of voltage-dependent K(+) channels reach their site of action by entering these openings.     

保定苜蓿锌离子跨膜转运蛋白的生物信息学分析(英文)

[目的]对保定苜蓿锌离子跨膜转运蛋白(Zinc transporter,ZnT)进行生物信息学分析。[方法]利用保定苜蓿锌离子跨膜转运蛋白氨基酸序列,应用生物信息学软件预测了该蛋白质的理化性质、分子表面亲/疏水性及二级结构,并利用不同的在线工具对其跨膜区域结构进行了对比预测。[结果]ZnT蛋白是一个整体疏水性蛋白,含有408个氨基酸,理论等电点为5.94;3种不同的跨膜预测工具预测得到相同的结果,即其存在7个可能的跨膜疏水区域,二级结构中α-helix(Hh)占48.04%、Extended strand(Ee)占9.56%、Random coi(lCc)占42.40%。这与其跨膜蛋白的性质相一致。[结论]mZnT属于锌离子跨膜转运蛋白的CDF家族,负责将细胞膜内的Zn2+转运出细胞,从而降低Zn2+的浓度与毒害。     

Structure of a site-2 protease family intramembrane metalloprotease

Regulated intramembrane proteolysis by members of the site-2 protease (S2P) family is an important signaling mechanism conserved from bacteria to humans. Here we report the crystal structure of the transmembrane core domain of an S2P metalloprotease from Methanocaldococcus jannaschii. The protease consists of six transmembrane segments, with the catalytic zinc atom coordinated by two histidine residues and one aspartate residue approximately 14 angstroms into the lipid membrane surface. The protease exhibits two distinct conformations in the crystals. In the closed conformation, the active site is surrounded by transmembrane helices and is impermeable to substrate peptide; water molecules gain access to zinc through a polar, central channel that opens to the cytosolic side. In the open conformation, transmembrane helices alpha1 and alpha6 separate from each other by 10 to 12 angstroms, exposing the active site to substrate entry. The structure reveals how zinc embedded in an integral membrane protein can catalyze peptide cleavage.     

Adenylyl cyclase amino acid sequence: possible channel- or transporter-like structure

Complementary DNA's that encode an adenylyl cyclase were isolated from a bovine brain library. Most of the deduced amino acid sequence of 1134 residues is divisible into two alternating sets of hydrophobic and hydrophilic domains. Each of the two large hydrophobic domains appears to contain six transmembrane spans. Each of the two large hydrophilic domains contains a sequence that is homologous to a single cytoplasmic domain of several guanylyl cyclases; these sequences may represent nucleotide binding sites. An unexpected topographical resemblance between adenylyl cyclase and various plasma membrane channels and transporters was observed. This structural complexity suggests possible, unappreciated functions for this important enzyme.     

Crystal structure of Cd,Zn metallothionein

The anomalous scattering data from five Cd in the native protein were used to determine the crystal structure of cadmium, zinc (Cd,Zn) metallothionein isoform II from rat liver. The structure of a 4-Cd cluster was solved by direct methods. A 2.3 A resolution electron density map was calculated by iterative single-wavelength anomalous scattering. The structure is folded into two domains. The amino terminal domain (beta) of residues 1 to 29 enfolds a three-metal cluster of one Cd and two Zn atoms coordinated by six terminal cysteine thiolate ligands and three bridging cysteine thiolates. The carboxyl terminal domain (alpha) of residues 30 to 61 enfolds a 4-Cd cluster coordinated by six terminal and five bridging cysteine thiolates. All seven metal sites have tetrahedral coordination geometry. The domains are roughly spherical, and the diameter is 15 to 20 A; there is limited contact between domains. The folding of alpha and beta is topologically similar but with opposite chirality. Redundant, short cysteine-containing sequences have similar roles in cluster formation in both alpha and beta.     

Chimeric NGF-EGF receptors define domains responsible for neuronal differentiation

To determine the domains of the low-affinity nerve growth factor (NGF) receptor required for appropriate signal transduction, a series of hybrid receptors were constructed that consisted of the extracellular ligand-binding domain of the human epidermal growth factor (EGF) receptor (EGFR) fused to the transmembrane and cytoplasmic domains of the human low-affinity NGF receptor (NGFR). Transfection of these chimeric receptors into rat pheochromocytoma PC12 cells resulted in appropriate cell surface expression. Biological activity mediated by the EGF-NGF chimeric receptor was assayed by the induction of neurite outgrowth in response to EGF in stably transfected cells. Furthermore, the chimeric receptor mediated nuclear signaling, as evidenced by the specific induction of transin messenger RNA, an NGF-responsive gene. Neurite outgrowth was not observed with chimeric receptors that contained the transmembrane domain from the EGFR, suggesting that the membrane-spanning region and cytoplasmic domain of the low-affinity NGFR are necessary for signal transduction.     

Reconstitution of Ca2+-regulated membrane fusion by synaptotagmin and SNAREs

We investigated the effect of synaptotagmin I on membrane fusion mediated by neuronal SNARE proteins, SNAP-25, syntaxin, and synaptobrevin, which were reconstituted into vesicles. In the presence of Ca2+, the cytoplasmic domain of synaptotagmin I (syt) strongly stimulated membrane fusion when synaptobrevin densities were similar to those found in native synaptic vesicles. The Ca2+ dependence of syt-stimulated fusion was modulated by changes in lipid composition of the vesicles and by a truncation that mimics cleavage of SNAP-25 by botulinum neurotoxin A. Stimulation of fusion was abolished by disrupting the Ca2+-binding activity, or by severing the tandem C2 domains, of syt. Thus, syt and SNAREs are likely to represent the minimal protein complement for Ca2+-triggered exocytosis.     

Crystal structure of Escherichia coli MscS,a voltage-modulated and mechanosensitive channel

The mechanosensitive channel of small conductance (MscS) responds both to stretching of the cell membrane and to membrane depolarization. The crystal structure at 3.9 angstroms resolution demonstrates that Escherichia coli MscS folds as a membrane-spanning heptamer with a large cytoplasmic region. Each subunit contains three transmembrane helices (TM1, -2, and -3), with the TM3 helices lining the pore, while TM1 and TM2, with membrane-embedded arginines, are likely candidates for the tension and voltage sensors. The transmembrane pore, apparently captured in an open state, connects to a large chamber, formed within the cytoplasmic region, that connects to the cytoplasm through openings that may function as molecular filters. Although MscS is likely to be structurally distinct from other ion channels, similarities in gating mechanisms suggest common structural elements.     

A zinc clasp structure tethers Lck to T cell coreceptors CD4 and CD8

The T cell coreceptors CD4 and CD8 both associate via their cytoplasmic tails with the N-terminus of the Src-family tyrosine kinase Lck. These interactions require zinc and are critical for T cell development and activation. We examined the folding and solution structures of ternary CD4-Lck-Zn2+ and CD8alpha-Lck-Zn2+ complexes. The coreceptor tails and the Lck N-terminus are unstructured in isolation but assemble in the presence of zinc to form compactly folded heterodimeric domains. The cofolded complexes have similar "zinc clasp" cores that are augmented by distinct structural elements. A dileucine motif required for clathrin-mediated endocytosis of CD4 is masked by Lck.     

Structural basis of gating by the outer membrane transporter FecA

Siderophore-mediated acquisition systems facilitate iron uptake. We present the crystallographic structure of the integral outer membrane receptor FecA from Escherichia coli with and without ferric citrate at 2.5 and 2.0 angstrom resolution. FecA is composed of three distinct domains: the barrel, plug, and NH2-terminal extension. Binding of ferric citrate triggers a conformational change of the extracellular loops that close the external pocket of FecA. Ligand-induced allosteric transitions are propagated through the outer membrane by the plug domain, signaling the occupancy of the receptor in the periplasm. These data establish the structural basis of gating for receptors dependent on the cytoplasmic membrane protein TonB. By compiling available data for this family of receptors, we propose a mechanism for the energy-dependent transport of siderophores.     

Three-dimensional structure of a recombinant gap junction membrane channel

Gap junction membrane channels mediate electrical and metabolic coupling between adjacent cells. The structure of a recombinant cardiac gap junction channel was determined by electron crystallography at resolutions of 7.5 angstroms in the membrane plane and 21 angstroms in the vertical direction. The dodecameric channel was formed by the end-to-end docking of two hexamers, each of which displayed 24 rods of density in the membrane interior, which is consistent with an alpha-helical conformation for the four transmembrane domains of each connexin subunit. The transmembrane alpha-helical rods contrasted with the double-layered appearance of the extracellular domains. Although not indicative for a particular type of secondary structure, the protein density that formed the extracellular vestibule provided a tight seal to exclude the exchange of substances with the extracellular milieu.     

耐铅锌离子微生物的筛选及其吸附特性

为获得去除铅、锌污染的高效菌株,采用浓度梯度筛选法,从受铅锌冶炼污染土壤中分离筛选耐铅锌离子微生物,并研究其对Pb2+和Zn2+离子的吸附特性。结果表明:筛选的菌株(编号0a)在试验条件范围内吸附基本符合Langmuir单分子层吸附行为,重金属离子浓度为300mg/L时0a菌对Zn2+、Pb2+离子最大吸附量分别达23.6mg/g和31.7mg/g;pH为3～6时对Zn2+、Pb2+的去除率较高,pH值为7.5时分别对Zn2+、Pb2+的吸附量最高,为30.96mg/g和23.5mg/g;吸附率与吸附时间符合吸附+细胞膜传输模型。初步鉴定该菌株为绿色木霉(Trichoderma viride)。     

超声波处理种子和基肥施锌对香稻 干物质、锌积累特性的影响

为了研究超声波处理种子和基肥施锌对香稻干物质、锌积累特性的影响,以常规香稻品种农香18 和美 香占为材料进行大田试验,设置锌肥处理和超声波种子处理,测定两香稻品种各生育时期茎、叶、穗的干物质积累量 和锌含量等指标。结果表明,在超声波处理种子和基肥施锌处理下,农香18 和美香占各器官的干物质积累量、锌含 量、锌积累量均显著高于对照。随着生育期的推移,叶锌的含量呈先降低后升高的趋势,在Zn0 处理下茎锌的含量 先降低后升高,在Zn2 和Zn4 处理下则呈先升高后降低再升高的趋势。Zn0 处理下叶和茎锌含量在分蘖期最高,施 锌处理（Zn2、Zn4）下则成熟期最高。结论院（1）超声波处理种子和合适的基施锌肥浓度可以提高香稻的干物质积累 量、锌含量和锌积累量;（2）高浓度的锌肥（Zn4）可以提高香稻生育后期吸收锌的能力。     

Structure and mechanism of the lactose permease of Escherichia coli

Membrane transport proteins that transduce free energy stored in electrochemical ion gradients into a concentration gradient are a major class of membrane proteins. We report the crystal structure at 3.5 angstroms of the Escherichia coli lactose permease, an intensively studied member of the major facilitator superfamily of transporters. The molecule is composed of N- and C-terminal domains, each with six transmembrane helices, symmetrically positioned within the permease. A large internal hydrophilic cavity open to the cytoplasmic side represents the inward-facing conformation of the transporter. The structure with a bound lactose homolog, beta-D-galactopyranosyl-1-thio-beta-D-galactopyranoside, reveals the sugar-binding site in the cavity, and residues that play major roles in substrate recognition and proton translocation are identified. We propose a possible mechanism for lactose/proton symport (co-transport) consistent with both the structure and a large body of experimental data.     

锌与尿素结合对锌有效性及尿素转化的影响

【目的】通过研究锌与尿素以不同方式结合施用对土壤中锌有效性及尿素在土壤中转化的影响,探究氮锌相互作用机制,为锌与尿素科学配伍及养分高效利用提供科学依据。【方法】将七水硫酸锌按0.5%和5%的重量份与尿素分别进行物理掺混（U+Zn）和熔融混合（UZn）,制备含锌尿素试验产品：U+Zn0.5、U+Zn5、UZn0.5和UZn5。采用土壤培养试验,研究锌与尿素以不同方式结合施用对土壤有效锌含量、土壤酰胺态氮含量、土壤NO₃^–-N和NH₄⁺-N含量及土壤脲酶活性的影响,并结合X射线光电子能谱和核磁共振波谱分析锌与尿素不同结合方式对锌有效性和尿素转化的影响机制。试验设置8个处理：①CK（对照）,不施任何肥料;②U,施用普通尿素;③Zn0.5,单施ZnSO₄·7H₂O;④Zn5,单施ZnSO₄·7H₂O;⑤U+Zn0.5,施用含锌尿素U+Zn0.5;⑥U+Zn5,施用含锌尿素U+Zn5;⑦UZn0.5,施用含锌尿素UZn0.5;⑧UZn5,施用含锌尿素UZn5。其中,处理②、⑤、⑥、⑦和⑧的氮用量相同,处理③、⑤和⑦的锌用量相同,处理④同⑥和⑧的锌用量。【结果】（1）与单施锌肥相比,锌与尿素以物理掺混和熔融混合方式结合后施用均可提高土壤有效锌含量,且熔融混合方式对锌有效性的提高效果强于物理掺混。在0.5%水平下,锌与尿素混合施用较锌肥单施土壤有效锌含量平均提高17.3%,而熔融混合较物理掺混平均提高了10.9%;在5%水平下,锌与尿素混合施用较锌肥单施土壤有效锌含量平均提高13.1%,熔融混合较物理掺混则平均提高了12.7%;在熔融混合方式下,0.5%用量（UZn0.5）的锌固定率较5%用量（UZn5）的降低了23.93个百分点。（2）与普通尿素（U）相比,4种含锌尿素均可减缓尿素水解,其中锌与尿素熔融结合较物理掺混结合更有利于延缓尿素水解,且以0.5%的用量时二者差异达到显著水平（P<0.05）。（3）锌与尿素结合可在培养后期提高土壤NH₄⁺-N含量,以UZn0.5提高幅度最明显。与普通尿素（U）相比,U+Zn5、UZn0.5和UZn5处理在培养后期可显著提高土壤NO₃^--N含量,且UZn0.5处理提高幅度显著高于UZn5处理。（4）锌与尿素熔融混合在培养后期可提高土壤矿质态氮含量,与U处理相比,UZn0.5和UZn5处理土壤矿质态氮含量分别提高了7.6%和1.9%,且UZn0.5较UZn5处理土壤矿质态氮含量仍高出5.6%,差异达显著水平（P<0.05）。（5）锌与尿素结合在培养前期可抑制土壤脲酶活性,熔融混合较物理掺混抑制效果更强;锌与尿素熔融混合可在培养后期提高土壤脲酶活性,UZn0.5处理提高土壤脲酶活性的程度高于UZn5处理。【结论】锌与尿素结合（物理掺混、熔融混合）均可减少土壤对锌的固定,提高土壤有效锌含量,以氮锌熔融混合效果更好。锌与尿素结合能够延缓尿素水解,在培养后期提高土壤NH₄⁺-N、NO₃^–-N和矿质态氮含量,以熔融混合方式和锌添加量以0.5%效果较好。0.5%添加量的七水硫酸锌与尿素熔融混合制成含锌尿素产品,在生产中具有推广前景。     

甘蓝纤维素合成酶基因BoCesA的克隆与序列分析

为研究甘蓝纤维素合成酶BoCesA基因在干旱胁迫下的表达模式,通过同源克隆得到甘蓝纤维素合成酶基因的cDNA全长,利用生物信息学软件分析该基因的特征;构建含BoCesA和GFP的融合表达载体,通过基因枪转化洋葱表皮细胞试验对该基因进行亚细胞定位;利用荧光定量PCR分析基因在不同组织中的表达情况及干旱胁迫下的表达模式。甘蓝纤维素合成酶BoCesA基因的cDNA全长为2 721bp,编码906个氨基酸,在N端含有锌指结构域和2个跨膜区,C端含有6个跨膜区,除包含植物纤维素合成酶基因共有的保守区外,还包含2个高突变区。通过氨基酸序列比对分析,发现甘蓝的该基因与拟南芥的AtCesA3基因同源性较高。亚细胞定位表明BoCesA基因初步定位于细胞质膜上。荧光定量PCR分析表明该基因在叶中的表达量高于茎和根中的表达量,在NaCl、PEG处理下BoCesA表达量呈现先升高后下降的趋势。通过对甘蓝纤维素合成酶BoCesA基因在NaCl、PEG胁迫下的表达分析,预测该基因对干旱胁迫有响应,它的表达受干旱胁迫的影响,这为进一步研究基因功能奠定了基础。     

Zinc mediation of the binding of human growth hormone to the human prolactin receptor

Human growth hormone (hGH) elicits a diverse set of biological activities including lactation that derives from binding to the prolactin (PRL) receptor. The binding affinity of hGH for the extracellular binding domain of the hPRL receptor (hPRLbp) was increased about 8000-fold by addition of 50 micromolar ZnCl2. Zinc was not required for binding of hGH to the hGH binding protein (hGHbp) or for binding of hPRL to the hPRLbp. Other divalent metal ions (Ca2+, Mg2+, Cu2+, Mn2+, and Co2+) at physiological concentrations did not support such strong binding. Scatchard analysis indicated a stoichiometry of one Zn2+ per hGH.hPRLbp complex. Mutational analysis showed that a cluster of three residues (His18, His21, and Glu174) in hGH and His188 from the hPRLbp (conserved in all PRL receptors but not GH receptors) are probable Zn2+ ligands. This polypeptide hormone.receptor "zinc sandwich" provides a molecular mechanism to explain why nonprimate GHs are not lactogenic and offers a molecular link between zinc deficiency and its association with altered functions of hGH.     

Mechanism of membrane anchoring affects polarized expression of two proteins in MDCK cells

The signals that direct membrane proteins to the apical or basolateral plasma membrane domains of polarized epithelial cells are not known. Several of the class of proteins anchored in the membrane by glycosyl-phosphatidylinositol (GPI) are expressed on the apical surface of such cells. However, it is not known whether the mechanism of membrane anchorage or the polypeptide sequence provides the sorting information. The conversion of the normally basolateral vesicular stomatitis virus glycoprotein (VSV G) to a GPI-anchored protein led to its apical expression. Conversely, replacement of the GPI anchor of placental alkaline phosphatase with the transmembrane and cytoplasmic domains of VSV G shifted its expression from the apical to the basolateral surface. Thus, the mechanism of membrane anchorage can determine the sorting of proteins to the apical or basolateral surface, and the GPI anchor itself may provide an apical transport signal.     

扶桑绵粉蚧钙调蛋白基因的克隆与生物信息学分析

钙调蛋白(calmodulin,CaM)对生物体内多种Ca2+依赖的细胞功能和酶体系都有重要的调节作用。为研究扶桑绵粉蚧的信号转导受体蛋白,首次克隆了扶桑绵粉蚧钙调蛋白基因PsCaM的cDNA全长序列,其开放阅读框(ORF)包含447bp的片段,编码148个氨基酸。PsCaM基因由3个内含子和4个外显子组成。3个内含子的长度分别为73、81、72bp,分隔的4个外显子的长度分别为33、133、183、98bp。功能域分析结果显示:该蛋白具有2个EF-hand结构域,有13个Ca2+结合位点;该蛋白的理论等电点是6.21,属于稳定蛋白,且没有跨膜区域;通过同源建模获得了其蛋白的三维结构。多序列比较显示,PsCaM基因相对较保守。