Structure and mechanism of the M2 proton channel of influenza A virus

The integral membrane protein M2 of influenza virus forms pH-gated proton channels in the viral lipid envelope. The low pH of an endosome activates the M2 channel before haemagglutinin-mediated fusion. Conductance of protons acidifies the viral interior and thereby facilitates dissociation of the matrix protein from the viral nucleoproteins--a required process for unpacking of the viral genome. In addition to its role in release of viral nucleoproteins, M2 in the trans-Golgi network (TGN) membrane prevents premature conformational rearrangement of newly synthesized haemagglutinin during transport to the cell surface by equilibrating the pH of the TGN with that of the host cell cytoplasm. Inhibiting the proton conductance of M2 using the anti-viral drug amantadine or rimantadine inhibits viral replication. Here we present the structure of the tetrameric M2 channel in complex with rimantadine, determined by NMR. In the closed state, four tightly packed transmembrane helices define a narrow channel, in which a 'tryptophan gate' is locked by intermolecular interactions with aspartic acid. A carboxy-terminal, amphipathic helix oriented nearly perpendicular to the transmembrane helix forms an inward-facing base. Lowering the pH destabilizes the transmembrane helical packing and unlocks the gate, admitting water to conduct protons, whereas the C-terminal base remains intact, preventing dissociation of the tetramer. Rimantadine binds at four equivalent sites near the gate on the lipid-facing side of the channel and stabilizes the closed conformation of the pore. Drug-resistance mutations are predicted to counter the effect of drug binding by either increasing the hydrophilicity of the pore or weakening helix-helix packing, thus facilitating channel opening.     

Structural alterations for proton translocation in the M state of wild-type bacteriorhodopsin

The transport of protons across membranes is an important process in cellular bioenergetics. The light-driven proton pump bacteriorhodopsin is the best-characterized protein providing this function. Photon energy is absorbed by the chromophore retinal, covalently bound to Lys 216 via a protonated Schiff base. The light-induced all-trans to 13-cis isomerization of the retinal results in deprotonation of the Schiff base followed by alterations in protonatable groups within bacteriorhodopsin. The changed force field induces changes, even in the tertiary structure, which are necessary for proton pumping. The recent report of a high-resolution X-ray crystal structure for the late M intermediate of a mutant bacteriorhopsin (with Asp 96-->Asn) displays the structure of a proton pathway highly disturbed by the mutation. To observe an unperturbed proton pathway, we determined the structure of the late M intermediate of wild-type bacteriorhodopsin (2.25 A resolution). The cytoplasmic side of our M2 structure shows a water net that allows proton transfer from the proton donor group Asp 96 towards the Schiff base. An enlarged cavity system above Asp 96 is observed, which facilitates the de- and reprotonation of this group by fluctuating water molecules in the last part of the cycle.     

Regions of variant histone His2AvD required for Drosophila development.

One way in which a distinct chromosomal domain could be established to carry out a specialized function is by the localized incorporation of specific histone variants into nucleosomes. H2AZ, one such variant of the histone protein H2A, is required for the survival of Drosophila melanogaster, Tetrahymena thermophila and mice (R. Faast et al., in preparation). To search for the unique features of Drosophila H2AZ (His2AvD, also referred to as H2AvD) that are required for its essential function, we have performed amino-acid swap experiments in which residues unique to Drosophila His2AvD were replaced with equivalently positioned Drosophila H2A.1 residues. Mutated His2AvD genes encoding modified versions of this histone were transformed into Drosophila and tested for their ability to rescue null-mutant lethality. We show that the unique feature of His2AvD does not reside in its histone fold but in its carboxy-terminal domain. This C-terminal region maps to a short alpha-helix in H2A that is buried deep inside the nucleosome core.     

Primary structure of helix C to helix G of a new retinal protein in H.sp.xz515

The gene fragment encoding the retinal protein from helix C to helix G in a new strain of halobacteria, H.sp.xz515 has been amplified by PCR method. The nucleotide sequence of this fragment has been determined. The deduced amino acid sequence has been compared with halobium br and other two br-like proteins, ar-1 and ar-2. Results show that those amino acid residues in br, essential for proton pumping and binding to retinal, are conserved. The residue M145 in br may be important for isomerization reaction of retinal.     

Imaging bacteriorhodopsinlike molecules of claretmembranes from Tibet halobacteria xz515 by atomic force microscope

HalobacteriaH.sp.xz 515 was isolated from a salt lake in Tibet. Although proton release-and-uptake across claret membrane is in reverse order compared to bacteriorhodopsin in purple membrane fromHalobacterium Salinarum, and its efficiency of proton pump is much lower, AFM image shows that the molecules are still arranged in a two-dimensional hexagonal lattice of trimers. Primary structure of Cto G-helix of the archaerhodopsin shows that it has only 56% homology with bacteriorhodopsin. But the interactive amino acid residues at the interface between B and D-helixes are conserved. These amino acid residues are believed to play a significant role in the stability of protein oligomers.     

磷酸乙醇胺转移酶MCR1结构与功能的研究

本实验克隆了来源于E.coli的MCR1基因酶催化结构域,以E.coli表达系统表达蛋白.采用亲和层析、阴离子交换层析、分子筛层析等纯化方法获得纯度高、均一性好的蛋白;采用座滴和悬滴法,获得酶催化区域的蛋白质晶体;收集X射线数据后,分子置换法解析出酶催化区域的结构,其分辨率达到1.63埃.反常散射信号检测到4个锌离子信号,结构分析锌离子与周围Thr285、His465、His466、His395位氨基酸联系紧密,且Thr285被磷酸化.Thr285、His465、His466和His395点突变为丙氨酸后,宿主菌粘菌素抗性显著降低,表明该区域与酶活密切相关.本实验解析出MCR1酶活性区域的结构,鉴定出酶活性中心,为寻找抗MCR1靶向药物提供可用信息.     

cDNA cloning and expression of earthworm fibrinolytic enzyme component A

Earthworm fibrinolytic enzyme component A(EFEa),a protein with dual fibrinolytic activity ,is one of the major therapeutically important earthworm fibrinoltic enzyme components .The cDNA fragment encoded the mature protein was cloned from earthworm (Eisenia fetida )by the RT-PCR technique,The deduced amino acid sequence of the EFE component A show high homology with some members of serine proteases trypsin family,and the amino acid residues constituting the active sites are conserved in the EFEa as compared with the other proteins of the trypsin family ,The cDNA fragment was subcloned into the expression vector pQE31 and pMAL-c2X of E.coli.The resulting expression plasmids,pQE-efea and pMAL-efea ,were used to transform the E.coli strain M15.Recombinant protein bands corresponding with calcuated molecular witht were induced .The induced His6-EFEa fusion protein with pQE-efea was accumulated into inclusion body ,while the induced MBP-EFEa fusion protein with pMAL-efea was soluble and showed fibrinoloytic activities.     

Inducible expression pattern of rice Bowman-Birk inhibitor gene<Emphasis Type="Italic">OsWIP1-2</Emphasis> and its protease inhibitory activity

The WIP1-2 gene was cloned from rice. It be-longs to the Bowman-Birk inhibitor gene family. Northernblot showed that expression of this gene was induced bywounding and jasmonic acid (JA). It indicates that the OsWIPI gene plays an important role in the rice defense sys-tem. The OsWIP1-2 was cloned into pET28a and expressed inE. coli. Its expressed product was purified in the form offusion protein and tested for the inhibitory activities againsttrypsin and chymotrypsin. It was found that the fusion pro-tein could inhibit chymotrypsin, but not trypsin. It was alsofound that the His tag at its C-terminal affected its inhibitoryactivity significantly. The fusion protein with a naturalC-terminal had the inhibitory activity, while no inhibitoryactivity was detected in the fusion protein with a (His)6-tag atits C-terminal. This implies that extra amino acid residues atthe C-terminal of OsWIP1-2 may interfere with its correctfolding. The inhibitory assay indicated that the members ofrice Bowman-Birk inhibitor gene family probably differenti-ated both in their structure and function.     

Structure of the membrane domain of respiratory complex I

Complex I is the first and largest enzyme of the respiratory chain, coupling electron transfer between NADH and ubiquinone to the translocation of four protons across the membrane. It has a central role in cellular energy production and has been implicated in many human neurodegenerative diseases. The L-shaped enzyme consists of hydrophilic and membrane domains. Previously, we determined the structure of the hydrophilic domain. Here we report the crystal structure of the Esherichia coli complex I membrane domain at 3.0?? resolution. It includes six subunits, NuoL, NuoM, NuoN, NuoA, NuoJ and NuoK, with 55 transmembrane helices. The fold of the homologous antiporter-like subunits L, M and N is novel, with two inverted structural repeats of five transmembrane helices arranged, unusually, face-to-back. Each repeat includes a discontinuous transmembrane helix and forms half of a channel across the membrane. A network of conserved polar residues connects the two half-channels, completing the proton translocation pathway. Unexpectedly, lysines rather than carboxylate residues act as the main elements of the proton pump in these subunits. The fourth probable proton-translocation channel is at the interface of subunits N, K, J and A. The structure indicates that proton translocation in complex I, uniquely, involves coordinated conformational changes in six symmetrical structural elements.     

耐热碱性磷酸酯酶基因的DNA序列分析

从栖热菌中克隆到产耐热碱性磷酸酯酶（FD－TAP）基因并进行了DNA序列分析,结果表明此2．0kb的片段含有一个1056bp的开放阅读框,编码501个氨基酸的蛋白质,其N端有一26个氨基酸的信号肽．在起始密码子的上游5bp处有一个5＇－GGAGGT－3＇的SD序列．基因编码区的（G＋C）％为68．7％,第3位密码子（G＋C）％为92．7％．FD－TAP的氨基酸序列与大肠杆菌等生物的碱性磷酸酯酶氨基酸序列比较,相同性为27％,相似性为38％．中央β－折叠区及与活性中心相关的氨基酸残基高度保守．表明FD－TAP具有与大肠杆菌碱性磷酸酯酶相似的结构和作用机制．在相当于大肠杆菌碱性磷酸酯酶的His370至His412两个金属离子结合部位之间,FD－TAP有一72个氨基酸的插入片段,提示该插入片段与FD－TAP的高耐热性相关．     

Crystal structure of the plasma membrane proton pump

A prerequisite for life is the ability to maintain electrochemical imbalances across biomembranes. In all eukaryotes the plasma membrane potential and secondary transport systems are energized by the activity of P-type ATPase membrane proteins: H+-ATPase (the proton pump) in plants and fungi, and Na+,K+-ATPase (the sodium-potassium pump) in animals. The name P-type derives from the fact that these proteins exploit a phosphorylated reaction cycle intermediate of ATP hydrolysis. The plasma membrane proton pumps belong to the type III P-type ATPase subfamily, whereas Na+,K+-ATPase and Ca2+-ATPase are type II. Electron microscopy has revealed the overall shape of proton pumps, however, an atomic structure has been lacking. Here we present the first structure of a P-type proton pump determined by X-ray crystallography. Ten transmembrane helices and three cytoplasmic domains define the functional unit of ATP-coupled proton transport across the plasma membrane, and the structure is locked in a functional state not previously observed in P-type ATPases. The transmembrane domain reveals a large cavity, which is likely to be filled with water, located near the middle of the membrane plane where it is lined by conserved hydrophilic and charged residues. Proton transport against a high membrane potential is readily explained by this structural arrangement.     

磺化聚醚醚酮(sPEEK)/SiO2杂化质子交换膜的制备与表征

为提高磺化聚醚醚酮(sPEEK)质子交换膜的耐甲醇渗透性能,用正硅酸乙酯为前驱体制得硅溶胶,在sPEEK中原位生成SiO2,制备了直接甲醇燃料电池用sPEEK/SiO2杂化质子交换膜材料,用核磁共振(1H-NMR)和Fourier红外光谱(FT-IR)表征了膜的化学结构,用扫描电子显微镜(SEM)观察了sPEEK与SiO2的复合形态,用交流阻抗仪和气相色谱仪分别测定了膜的质子传导率和甲醇渗透系数。实验结果表明,在质子传导率没有严重降低的同时,杂化膜的阻醇性得到了较好的改善。     

Structure of Sindbis virus core protein reveals a chymotrypsin-like serine proteinase and the organization of the virion

Sindbis virus consists of a nucleocapsid core surrounded by a lipid membrane through which penetrate 80 glycoprotein trimers. The structure of the core protein comprising the coat surrounding the genomic RNA has been determined. The polypeptide fold from residue 114 to residue 264 is homologous to that of chymotrypsin-like serine proteinases with catalytic residues His 141, Asp 163 and Ser 215 of the core protein positioned as in other serine proteinases. The C-terminal tryptophan remains in the P1 substrate site subsequent to the autocatalytic cis cleavage of the capsid protein, thus rendering the proteinase inactive. Model building of the Sindbis core protein dimer shows that the nucleocapsid is likely to have T = 4 quasisymmetry.     

Structure of acid-sensing ion channel 1 at 1.9 A resolution and low pH

Acid-sensing ion channels (ASICs) are voltage-independent, proton-activated receptors that belong to the epithelial sodium channel/degenerin family of ion channels and are implicated in perception of pain, ischaemic stroke, mechanosensation, learning and memory. Here we report the low-pH crystal structure of a chicken ASIC1 deletion mutant at 1.9 A resolution. Each subunit of the chalice-shaped homotrimer is composed of short amino and carboxy termini, two transmembrane helices, a bound chloride ion and a disulphide-rich, multidomain extracellular region enriched in acidic residues and carboxyl-carboxylate pairs within 3 A, suggesting that at least one carboxyl group bears a proton. Electrophysiological studies on aspartate-to-asparagine mutants confirm that these carboxyl-carboxylate pairs participate in proton sensing. Between the acidic residues and the transmembrane pore lies a disulphide-rich 'thumb' domain poised to couple the binding of protons to the opening of the ion channel, thus demonstrating that proton activation involves long-range conformational changes.     

Structure and function of a membrane component SecDF that enhances protein export

Protein translocation across the bacterial membrane, mediated by the secretory translocon SecYEG and the SecA ATPase, is enhanced by proton motive force and membrane-integrated SecDF, which associates with SecYEG. The role of SecDF has remained unclear, although it is proposed to function in later stages of translocation as well as in membrane protein biogenesis. Here, we determined the crystal structure of Thermus thermophilus SecDF at 3.3?? resolution, revealing a pseudo-symmetrical, 12-helix transmembrane domain belonging to the RND superfamily and two major periplasmic domains, P1 and P4. Higher-resolution analysis of the periplasmic domains suggested that P1, which binds an unfolded protein, undergoes functionally important conformational changes. In vitro analyses identified an ATP-independent step of protein translocation that requires both SecDF and proton motive force. Electrophysiological analyses revealed that SecDF conducts protons in a manner dependent on pH and the presence of an unfolded protein, with conserved Asp and Arg residues at the transmembrane interface between SecD and SecF playing essential roles in the movements of protons and preproteins. Therefore, we propose that SecDF functions as a membrane-integrated chaperone, powered by proton motive force, to achieve ATP-independent protein translocation.     

Crystal structure of a membrane-embedded H+-translocating pyrophosphatase

H(+)-translocating pyrophosphatases (H(+)-PPases) are active proton transporters that establish a proton gradient across the endomembrane by means of pyrophosphate (PP(i)) hydrolysis. H(+)-PPases are found primarily as homodimers in the vacuolar membrane of plants and the plasma membrane of several protozoa and prokaryotes. The three-dimensional structure and detailed mechanisms underlying the enzymatic and proton translocation reactions of H(+)-PPases are unclear. Here we report the crystal structure of a Vigna radiata H(+)-PPase (VrH(+)-PPase) in complex with a non-hydrolysable substrate analogue, imidodiphosphate (IDP), at 2.35?? resolution. Each VrH(+)-PPase subunit consists of an integral membrane domain formed by 16 transmembrane helices. IDP is bound in the cytosolic region of each subunit and trapped by numerous charged residues and five Mg(2+) ions. A previously undescribed proton translocation pathway is formed by six core transmembrane helices. Proton pumping can be initialized by PP(i) hydrolysis, and H(+) is then transported into the vacuolar lumen through a pathway consisting of Arg?242, Asp?294, Lys?742 and Glu?301. We propose a working model of the mechanism for the coupling between proton pumping and PP(i) hydrolysis by H(+)-PPases.     

Binding Sites of La~(3 ) on Camodulin

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Structural basis for signal transduction by the Toll/interleukin-1 receptor domains

Toll-like receptors (TLRs) and the interleukin-1 receptor superfamily (IL-1Rs) are integral to both innate and adaptive immunity for host defence. These receptors share a conserved cytoplasmic domain, known as the TIR domain. A single-point mutation in the TIR domain of murine TLR4 (Pro712His, the Lps(d) mutation) abolishes the host immune response to lipopolysaccharide (LPS), and mutation of the equivalent residue in TLR2, Pro681His, disrupts signal transduction in response to stimulation by yeast and gram-positive bacteria. Here we report the crystal structures of the TIR domains of human TLR1 and TLR2 and of the Pro681His mutant of TLR2. The structures have a large conserved surface patch that also contains the site of the Lps(d) mutation. Mutagenesis and functional studies confirm that residues in this surface patch are crucial for receptor signalling. The Lps(d) mutation does not disturb the structure of the TIR domain itself. Instead, structural and functional studies indicate that the conserved surface patch may mediate interactions with the down-stream MyD88 adapter molecule, and that the Lps(d) mutation may abolish receptor signalling by disrupting this recruitment.