Structure of the dengue virus envelope protein after membrane fusion

Dengue virus enters a host cell when the viral envelope glycoprotein, E, binds to a receptor and responds by conformational rearrangement to the reduced pH of an endosome. The conformational change induces fusion of viral and host-cell membranes. A three-dimensional structure of the soluble E ectodomain (sE) in its trimeric, postfusion state reveals striking differences from the dimeric, prefusion form. The elongated trimer bears three 'fusion loops' at one end, to insert into the host-cell membrane. Their structure allows us to model directly how these fusion loops interact with a lipid bilayer. The protein folds back on itself, directing its carboxy terminus towards the fusion loops. We propose a fusion mechanism driven by essentially irreversible conformational changes in E and facilitated by fusion-loop insertion into the outer bilayer leaflet. Specific features of the folded-back structure suggest strategies for inhibiting flavivirus entry.     

Epstein-Barr virus latent membrane protein inhibits human epithelial cell differentiation

Epstein-Barr virus (EBV), a human herpesvirus, is strongly linked with two relatively rare forms of B-cell lymphoma and with a much more prevalent epithelial malignancy, undifferentiated nasopharyngeal carcinoma (NPC). The availability of suitable culture systems has allowed detailed analysis of EBV-induced growth transformation in B lymphocytes, but little is known about the virus--epithelial cell interaction or about the possible effector role of viral proteins in the pathogenesis of NPC. Here we describe an experimental system to monitor the effects of introduced viral or cellular genes upon human epithelial cell growth and differentiation. We transfected a human epithelial cell line, which retains several features of normal keratinocyte behaviour in vitro, with the EBV gene encoding latent membrane protein (LMP), one of only two viral proteins known to be expressed in NPC cells in vivo. LMP expression was accompanied by changes in the epithelial cell surface phenotype, mimicking surface changes observed in NPC cells, and by severe impairment of the cellular response to differentiation signals. The ability of LMP to inhibit terminal differentiation indicates a mechanism whereby EBV infection of squamous epithelium could contribute to the multi-step pathogenesis of NPC.     

Structural differences between a ras oncogene protein and the normal protein

One of the most commonly found transforming ras oncogenes in human tumours has a valine codon replacing the glycine codon at position 12 of the normal c-Ha-ras gene. To understand the structural reasons behind cell transformation arising from this single amino acid substitution, we have determined the crystal structure of the GDP-bound form of the mutant protein, p21(Val-12), encoded by this oncogene. We report here the overall structure of p21(Val-12) at 2.2 A resolution and compare it with the structure of the normal c-Ha-ras protein. One of the major differences is that the loop of the transforming ras protein that binds the beta-phosphate of the guanine nucleotide is enlarged. Such a change in the 'catalytic site' conformation could explain the reduced GTPase activity of the mutant, which keeps the protein in the GTP bound 'signal on' state for a prolonged period time, ultimately causing cell transformation.     

Two configurations of a channel-forming membrane protein

The protein oligomer forming the gap junction channel has been analysed in two Ca2+-sensitive states by electron microscopy of membranes in frozen aqueous solutions. Switching between states occurs by a small cooperative rearrangement involving tilting of the subunits, which may be responsible for the effect of Ca2+ on channel permeability in vivo.     

Solving the membrane protein folding problem

One of the great challenges for molecular biologists is to learn how a protein sequence defines its three-dimensional structure. For many years, the problem was even more difficult for membrane proteins because so little was known about what they looked like. The situation has improved markedly in recent years, and we now know over 90 unique structures. Our enhanced view of the structure universe, combined with an increasingly quantitative understanding of fold determination, engenders optimism that a solution to the folding problem for membrane proteins can be achieved.     

Structural basis of West Nile virus neutralization by a therapeutic antibody

West Nile virus is a mosquito-borne flavivirus closely related to the human epidemic-causing dengue, yellow fever and Japanese encephalitis viruses. In establishing infection these icosahedral viruses undergo endosomal membrane fusion catalysed by envelope glycoprotein rearrangement of the putative receptor-binding domain III (DIII) and exposure of the hydrophobic fusion loop. Humoral immunity has an essential protective function early in the course of West Nile virus infection. Here, we investigate the mechanism of neutralization by the E16 monoclonal antibody that specifically binds DIII. Structurally, the E16 antibody Fab fragment engages 16 residues positioned on four loops of DIII, a consensus neutralizing epitope sequence conserved in West Nile virus and distinct in other flaviviruses. The E16 epitope protrudes from the surface of mature virions in three distinct environments, and docking studies predict Fab binding will leave five-fold clustered epitopes exposed. We also show that E16 inhibits infection primarily at a step after viral attachment, potentially by blocking envelope glycoprotein conformational changes. Collectively, our results suggest that a vaccine strategy targeting the dominant DIII epitope may elicit safe and effective immune responses against flaviviral diseases.     

Crystal structure of a human membrane protein involved in cysteinyl leukotriene biosynthesis

The cysteinyl leukotrienes, namely leukotriene (LT)C4 and its metabolites LTD4 and LTE4, the components of slow-reacting substance of anaphylaxis, are lipid mediators of smooth muscle constriction and inflammation, particularly implicated in bronchial asthma. LTC4 synthase (LTC4S), the pivotal enzyme for the biosynthesis of LTC4 (ref. 10), is an 18-kDa integral nuclear membrane protein that belongs to a superfamily of membrane-associated proteins in eicosanoid and glutathione metabolism that includes 5-lipoxygenase-activating protein, microsomal glutathione S-transferases (MGSTs), and microsomal prostaglandin E synthase 1 (ref. 13). LTC4S conjugates glutathione to LTA4, the endogenous substrate derived from arachidonic acid through the 5-lipoxygenase pathway. In contrast with MGST2 and MGST3 (refs 15, 16), LTC4S does not conjugate glutathione to xenobiotics. Here we show the atomic structure of human LTC4S in a complex with glutathione at 3.3 A resolution by X-ray crystallography and provide insights into the high substrate specificity for glutathione and LTA4 that distinguishes LTC4S from other MGSTs. The LTC4S monomer has four transmembrane alpha-helices and forms a threefold symmetric trimer as a unit with functional domains across each interface. Glutathione resides in a U-shaped conformation within an interface between adjacent monomers, and this binding is stabilized by a loop structure at the top of the interface. LTA4 would fit into the interface so that Arg 104 of one monomer activates glutathione to provide the thiolate anion that attacks C6 of LTA4 to form a thioether bond, and Arg 31 in the neighbouring monomer donates a proton to form a hydroxyl group at C5, resulting in 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTC4). These findings provide a structural basis for the development of LTC4S inhibitors for a proinflammatory pathway mediated by three cysteinyl leukotriene ligands whose stability and potency are different and by multiple cysteinyl leukotriene receptors whose functions may be non-redundant.     

Human nectin-like 1, a novel membrane protein interacting with protein 4.1 R

Human nectin-like 1 (NECL1) full-length cDNA was cloned by bioinformatics method when searching for candidate membrane proteins interacting with members of protein 4.1 family. The cytoplasmic and extracellular regions of NECL1 were expressed in and purified from E. coli, and the polyclonal antibody was produced. Interaction between the cytoplasmic region of NECL1 and the 30 kD membrane binding domain of protein 4.1 on red blood cell (4. 1R) was demonstrated by IAsys-biosensor system and GST pull-down experiment. Results of biotin-labeled peptide ELISA further demonstrated the key amino acids for the binding. The interaction research of NECL1's cytoplasmic domain provides basis for further study of the functions of NECL1 in nervous system.     

YidC mediates membrane protein insertion in bacteria

The basic machinery for the translocation of proteins into or across membranes is remarkably conserved from Escherichia coli to humans. In eukaryotes, proteins are inserted into the endoplasmic reticulum using the signal recognition particle (SRP) and the SRP receptor, as well as the integral membrane Sec61 trimeric complex (composed of alpha, beta and gamma subunits). In bacteria, most proteins are inserted by a related pathway that includes the SRP homologue Ffh, the SRP receptor FtsY, and the SecYEG trimeric complex, where Y and E are related to the Sec61 alpha and gamma subunits, respectively. Proteins in bacteria that exhibit no dependence on the Sec translocase were previously thought to insert into the membrane directly without the aid of a protein machinery. Here we show that membrane insertion of two Sec-independent proteins requires YidC. YidC is essential for E. coli viability and homologues are present in mitochondria and chloroplasts. Depletion of YidC also interferes with insertion of Sec-dependent membrane proteins, but it has only a minor effect on the export of secretory proteins. These results provide evidence for an additional component of the translocation machinery that is specialized for the integration of membrane proteins.     

Regulation of the association of membrane skeletal protein 4.1 with glycophorin by a polyphosphoinositide

Many of the physical properties of the erythrocyte membrane appear to depend on the membrane skeleton, which is attached to the membrane through associations with transmembrane proteins. A membrane skeletal protein, protein 4.1, is pivotal in the assembly of the membrane skeleton because of its ability to promote associations between spectrin and actin. Protein 4.1 also binds to the membrane through at least two sites: a high-affinity site on the glycophorins and a site of lower affinity associated with band 3 (ref. 11). The glycophorin-protein 4.1 association has been proposed to be involved in maintenance of cell shape. Here we show that the association between glycophorin and protein 4.1 is regulated by a polyphosphoinositide cofactor. This observation suggests a mechanism which may explain the recently reported dependence of red cell shape on the level of polyphosphoinositides in the membrane.     

Synchronised transmembrane insertion and glycosylation of a nascent membrane protein.

Studies of the synthesis and incorporation of the vesicular stomatitis virus glycoprotein into membranes in a synchronised cell-free system demonstrate a tight coupling between polypeptide synthesis and membrane insertion, as a result of which the nascent chain crosses the membrane. The studies reveal a surprisingly precise sequence by which the nascent chain of this membrane glycoprotein is glycosylated in two steps. These findings have important implications for the mechanisms of membrane assembly.     

大型水电站垫层蜗壳座环结构特性分析

为研究蜗壳和外围混凝土联合受力情况下座环的结构特性,结合一实际工程垫层蜗壳结构,采用三维有限元方法,从座环应力、位移和变形以及抗剪性能几个方面分析座环结构的受力特性.结果表明:相比于座环结构强度,其位移和变形更值得重视,垫层变形模量和摩擦系数的大小应该成为座环结构分析的重要考虑因素;座环承受较大的剪力,垫层蜗壳结构设计中不能忽视此问题.