Proton binding within a membrane protein by a protonated water cluster

Proton transfer is crucial for many enzyme reactions. Here, we show that in addition to protonatable amino acid side chains, water networks could constitute proton-binding sites in proteins. A broad IR continuum absorbance change during the proton pumping photocycle of bacteriorhodopsin (bR) indicates most likely deprotonation of a protonated water cluster at the proton release site close to the surface. We investigate the influence of several mutations on the proton release network and the continuum change, to gain information about the location and extent of the protonated water network and to reveal the participating residues necessary for its stabilization. We identify a protonated water cluster consisting in total of one proton and about five water molecules surrounded by six side chains and three backbone groups (Tyr-57, Arg-82, Tyr-83, Glu-204, Glu-194, Ser-193, Pro-77, Tyr-79, and Thr-205). The observed perturbation of proton release by many single-residue mutations is now explained by the influence of numerous side chains on the protonated H bonded network. In situ hydrogen/deuterium exchange Fourier transform IR measurements of the bR ground state, show that the proton of the release group becomes localized on Glu-204 and Asp-204 in the ground state of the mutants E194D and E204D, respectively, even though it is delocalized in the ground state of wild-type bR. Thus, the release mechanism switches between the wild-type and mutated proteins from a delocalized to a localized proton-binding site.     

Zinc alters the kinetics of IGF-II binding to cell surface receptors and binding proteins

The growth of most tissues is markedly depressed as a result of zinc deficiency by uncharacterized mechanisms that clearly involve the insulin-like growth factor (IGF) system. Herein, we describe the mechanism by which zinc (Zn²⁺) maintains IGF-II in an active form by directly regulating IGF-II binding to IGF-binding proteins (IGFBPs) and the type 1 IGF receptor (IGF-1R). The specificity of Zn²⁺ effects was confirmed by using other cations that can (Cd²⁺ and Au³⁺) or cannot (La³⁺) mimic Zn²⁺ actions. Human fibroblasts, glioblastoma cells, and murine myoblasts were used to determine the kinetics of IGF-II binding to cell surface IGFBP-3, IGFBP-5, and the IGF-1R, respectively. Zn²⁺, Cd²⁺, and Au³⁺, but not La³⁺, decreased total binding and the affinity for [¹²⁵I]IGF-II association with IGFBP-3 and IGFBP-5. These effects were a result of lowered rate of ligand association without affecting rate of dissociation. In contrast, Zn²⁺ enhanced [¹²⁵I]IGF-II binding to the IGF-1R by enhancing the rate of ligand association and decreasing the rate of dissociation. Our previous work had shown that Zn²⁺ acts at physiological levels to alter IGF binding. Together with the current work, these findings imply that Zn²⁺ acts in vivo to prevent secreted IGF-II from binding to IGFBP-3 and IGFBP-5, thus maintaining IGF-II in an “active state,” i.e., readily available for IGF-1R association.     

Recruitment of a cytoplasmic response regulator to the cell pole is linked to its cell cycle-regulated proteolysis

The response regulator CtrA, which silences the Caulobacter origin of replication and controls multiple cell cycle events, is specifically proteolyzed in cells preparing to initiate DNA replication. At the swarmer-to-stalked cell transition and in the stalked compartment of the predivisional cell, CtrA is localized to the cell pole just before its degradation. Analysis of the requirements for CtrA polar localization and CtrA proteolysis revealed that both processes require a motif within amino acids 1-56 of the CtrA receiver domain, and neither process requires CtrA phosphorylation. These results strongly suggest that CtrA polar localization is coupled to its cell cycle-regulated proteolysis. The polarly localized DivK response regulator promotes CtrA localization and proteolysis, but it does not directly recruit CtrA to the cell pole. Mutations in the divJ and pleC histidine kinases perturb the characteristic asymmetry of CtrA localization and proteolysis in the predivisional cell. We propose that polar recruitment of CtrA evolved to ensure that CtrA is degraded only in the stalked half of the predivisional cell, perhaps by localizing a proteolytic adaptor protein to the stalked pole. This is an example of controlled proteolysis of a cytoplasmic protein that is associated with its active recruitment to a specific subcellular address.     

DNA-repair protein hHR23a alters its protein structure upon binding proteasomal subunit S5a

The Rad23 family of proteins, including the human homologs hHR23a and hHR23b, stimulates nucleotide excision repair and has been shown to provide a novel link between proteasome-mediated protein degradation and DNA repair. In this work, we illustrate how the proteasomal subunit S5a regulates hHR23a protein structure. By using NMR spectroscopy, we have elucidated the structure and dynamic properties of the 40-kDa hHR23a protein and show it to contain four structured domains connected by flexible linker regions. In addition, we reveal that these domains interact in an intramolecular fashion, and by using residual dipolar coupling data in combination with chemical shift perturbation analysis, we present the hHR23a structure. By itself, hHR23a adopts a closed conformation defined by the interaction of an N-terminal ubiquitin-like domain with two ubiquitin-associated domains. Interestingly, binding of the proteasomal subunit S5a disrupts the hHR23a interdomain interactions and thereby causes it to adopt an opened conformation.     

Activation of a recombinant human factor VII structural analogue alters its affinity of binding to tissue factor

A competitive enzyme-linked immunoadsorbent assay (ELISA) technique has been developed to facilitate quantitative analysis of the earliest step in the initiation of the extrinsic pathway of coagulation, i.e., complex formation of factor VII/VIIa with tissue factor. The ELISA measures the binding of biotinylated human plasma factor VII to relipidated recombinant human tissue factor. Quantitation of the relative affinity (expressed as IC₅₀) of any factor VII molecular population or structural analogue for tissue factor can be determined by competitive binding. Subnanomolar concentrations of both wild-type recombinant human factor VII (rFVII) and rFVII(R152Q), a mutation at the FVII activation site, competed effectively with biotinylated plasma-derived factor VII in binding to tissue factor. In contrast, the affinity of rFVII(R79Q), a mutation in the first epidermal growth factor-like domain, was 12-fold lower. Following activation of rFVII(R79Q), its affinity for tissue factor and enzymatic activity increased 4-fold and 6-fold, respectively. For wild-type rFVII, enzymatic activity rose significantly following activation. However, its affinity for tissue factor was unchanged. We conclude that both the activation state of factor VII and the mutation of amino-acid residues within the first epidermal growth factor-like domain may alter the affinity of factor VII for tissue factor. © 1996 Wiley-Liss, Inc.     

Identification of a herpes simplex virus 1 glycoprotein gene within a gene cluster dispensable for growth in cell culture.

The genome of herpes simplex virus 1 consists of two components, L and S, each containing unique sequences flanked by inverted repeats. Current and earlier studies have shown that 11 of the 12 open reading frames contained in the unique sequences of the S component can be deleted and are dispensable for growth in cell culture. Analyses of one recombinant virus containing a deletion in the open reading frame US7 permitted the identification of a monoclonal antibody specific for the product of this gene. The protein encoded by this gene has a predicted translated molecular weight of 41,366 and an apparent molecular weight of approximately 65,000 in denaturing polyacrylamide gels. The electrophoretic mobility of the protein synthesized by cells in the presence of inhibitory concentrations of tunicamycin is faster than that of the protein accumulating in lysates of untreated infected cells. We conclude that the product of US7 is glycoprotein subject to N-linked glycosylation, and we have designated it glycoprotein I. These studies indicate that the unique sequences of the S component encode four glycoproteins (G, D, I, and E) of which at least three (G, I, and E) are dispensable for growth in continuous lines of primate cells.     

Determination of ligand cluster and binding site within VP40 of Ebola virus:clue for drug development

正Dear Editor. Ebola virus infection is the present public health threct that posed the trend of wordwide pandemic[1].At present.there is still no effective drug and vaccine for trearment of Ebola     

Increased binding of concanavalin a at the cell surface following exposure to thyroliberin

Thyroliberin (THR) binds specifically to SD1 rat prolactin cells and increases prolactin release. THR-induced modifications of surface membrane of intact SD1 cells were looked for, using concanavalin A (Con A) as a probe. At the electron microscope level the binding was restricted to the cell surface.Preexposure of the cells to TRH (27 nM) for 30 min at 37°C increased the binding of Con A by 28–120%. Such an increase was not observed with low doses of TRH (13.5 and 2.7 nM) nor after only a 10-min exposure to 27 nM TRH. This effect is specific for TRH; it was not observed with other peptides. Simultaneous exposure to Con A and [³H]TRH did not alter [³H]TRH binding, but preexposure to Con A reduced the [³H]TRH binding by 10%, which may be due to steric hindrance. It is concluded that TRH induces an increased exposure of surface membrane glycoproteins in intact SD1 cells.     

Cloning of mouse immunoglobulin epsilon gene and its location within the heavy chain gene cluster.

Mouse immunoglobulin epsilon chain gene was cloned from DNA of a hybridoma producing anti-dinitrophenyl IgE, which was constructed by fusing a spleen cell of a BALB/c mouse with a variant clone of MOPC21 myeloma (IgG1 producer). Because a given active heavy chain constant region (CH) gene is linked to a heavy chain joining segment (JH) gene at its 5' side, the expressed C epsilon gene of the hybridoma was cloned from a phage library containing partial Sau3A digests of IgE hybridoma DNA by using a J gene fragment as a probe. Among 6 X 10(5) phages screened, five positive clones were obtained and three of them were identified as C epsilon gene clones by restriction mapping, Southern blot hybridization, R-loop formation, and partial nucleotide sequence determination. The determined nucleotide sequence predicted the amino acid sequence which resembles a part of the CH3 domain of human epsilon chain. The deletion profile of the C epsilon gene in various myelomas expressing different CH genes indicates that the C epsilon gene is located between the C gamma 2a and C alpha genes. The linkage (5'-epsilon-alpha-3') was directly confirmed by molecular cloning of the overlapping chromosomal segments from newborn mouse DNA.     

Identification of a recent recombination event within the human beta-globin gene cluster.

In a detailed study of inheritance of DNA sequence polymorphism in a large reference pedigree, an individual was identified with an apparent genetic recombination event within the human beta-globin gene cluster. Analysis of the haplotypes of relevant individuals within this pedigree suggested that the meiotic crossing-over event is likely to have occurred within a 19.8-kilobase-pair region of the beta-globin gene cluster. Analysis of other DNA markers closely linked to the beta-globin gene cluster--segment 12 of chromosome 11 (D11S12) and loci for insulin, the cellular oncogene c-Ha-ras, and preproparathyroid hormone--confirmed that a crossover event must have occurred within the region of chromosome 11 between D11S12 and the beta-globin gene cluster. It is suggested that the event observed has occurred within a DNA region compatible with recombinational "hot spots" suggested by population studies.     

Interaction of single-chain urokinase with its receptor induces the appearance and disappearance of binding epitopes within the resultant complex for other cell surface proteins

Binding of urokinase-type plasminogen activator (uPA) to its glycosylphosphatidylinositol-anchored receptor (uPAR) initiates signal transduction, adhesion, and migration in certain cell types. To determine whether some of these activities may be mediated by associations between the uPA/uPAR complex and other cell surface proteins, we studied the binding of complexes composed of recombinant, soluble uPA receptor (suPAR) and single chain uPA (scuPA) to a cell line (LM-TK- fibroblasts) that does not express glycosylphosphatidylinositol (GPI)-anchored proteins to eliminate potential competition by endogenous uPA receptors. scuPA induced the binding of suPAR to LM-TK- cells. Binding of labeled suPAR/scuPA was inhibited by unlabeled complex, but not by scuPA or suPAR added separately, indicating cellular binding sites had been formed that are not present in either component. Binding of the complex was inhibited by low molecular weight uPA (LMW-uPA) indicating exposure of an epitope found normally in the isolated B chain of two chain uPA (tcuPA), but hidden in soluble scuPA. Binding of LMW-uPA was independent of its catalytic site and was associated with retention of its enzymatic activity. Additional cell binding epitopes were generated within suPAR itself by the aminoterminal fragment of scuPA, which itself does not bind to LM-TK- cells. When scuPA bound to suPAR, a binding site for alpha 2-macroglobulin receptor/LDL receptor-related protein (alpha 2 MR/LRP) was lost, while binding sites for cell-associated vitronectin and thrombospondin were induced. In accord with this, the internalization and degradation of cell-associated tcuPA and tcuPA-PAI- 1 complexes proceeded less efficiently in the presence of suPAR. Further, little degradation of suPAR was detected, suggesting that cell- bound complex dissociated during the initial stages of endocytosis. Thus, the interaction of scuPA with its receptor causes multiple functional changes within the complex including the dis-appearance of an epitope in scuPA involved in its clearance from the cell surface and the generation of novel epitopes that promote its binding to proteins involved in cell adhesion and signal transduction.     

分化抗原簇63在非小细胞肺癌中的表达及临床意义

目的 检测分化抗原簇63(cluster of differentiation 63,CD63)在非小细胞肺癌(non-small-cell lung cancer,NSCLC)组织中的表达水平与临床特征和生存的相关性.方法 收集2013年6月至2015年1月在安阳市第二人民医院接受手术治疗的NSCLC患者126例,收...     

Targeting a binding pocket within the trimer-of-hairpins: small-molecule inhibition of viral fusion

Trimeric class I virus fusion proteins undergo a series of conformational rearrangements that leads to the association of C- and N-terminal heptad repeat domains in a "trimer-of-hairpins" structure, facilitating the apposition of viral and cellular membranes during fusion. This final fusion hairpin structure is sustained by protein-protein interactions, associations thought initially to be refractory to small-molecule inhibition because of the large surface area involved. By using a photoaffinity analog of a potent respiratory syncytial virus fusion inhibitor, we directly probed the interaction of the inhibitor with its fusion protein target. Studies have shown that these inhibitors bind within a hydrophobic cavity formed on the surface of the N-terminal heptad-repeat trimer. In the fusogenic state, this pocket is occupied by key amino acid residues from the C-terminal heptad repeat that stabilize the trimer-of-hairpins structure. The results indicate that a low-molecular-weight fusion inhibitor can interfere with the formation or consolidation of key structures within the hairpin moiety that are essential for membrane fusion. Because analogous cavities are present in many class I viruses, including HIV, these results demonstrate the feasibility of this approach as a strategy for drug discovery.     

A cluster of mutations within a short triplet repeat in the C1 inhibitor gene.

Mutations in the C1 inhibitor gene that result in low functional levels of C1 inhibitor protein cause hereditary angioneurotic edema. This disease is characterized by episodic edema leading to considerable morbidity and death. Among 60 unreported kindred with the disease, four patients were discovered to have mutations clustered within a 12-bp segment of exon 5 from nucleotide 8449 to nucleotide 8460. This short segment of DNA contains three direct repeats of the triplet CAA and is immediately preceded by a similar adenosine-rich sequence (CAAGAACAC). These triplet repeats make this region susceptible to mutation by a slipped mispairing mechanism. There are two other short triplet repeat elements in the coding region for this gene, but they have not become mutated in any kindred examined. This suggests that the apparent enhanced mutation rate in this region of exon 5 may be influenced by DNA structural characteristics.     

NifS-directed assembly of a transient [2Fe-2S] cluster within the NifU protein

The NifS and NifU proteins from Azotobacter vinelandii are required for the full activation of nitrogenase. NifS is a homodimeric cysteine desulfurase that supplies the inorganic sulfide necessary for formation of the Fe-S clusters contained within the nitrogenase component proteins. NifU has been suggested to complement NifS either by mobilizing the Fe necessary for nitrogenase Fe-S cluster formation or by providing an intermediate Fe-S cluster assembly site. As isolated, the homodimeric NifU protein contains one [2Fe-2S](2+, +) cluster per subunit, which is referred to as the permanent cluster. In this report, we show that NifU is able to interact with NifS and that a second, transient [2Fe-2S] cluster can be assembled within NifU in vitro when incubated in the presence of ferric ion, L-cysteine, and catalytic amounts of NifS. Approximately one transient [2Fe-2S] cluster is assembled per homodimer. The transient [2Fe-2S] cluster species is labile and rapidly released on reduction. We propose that transient [2Fe-2S] cluster units are formed on NifU and then released to supply the inorganic iron and sulfur necessary for maturation of the nitrogenase component proteins. The role of the permanent [2Fe-2S] clusters contained within NifU is not yet known, but they could have a redox function involving either the formation or release of transient [2Fe-2S] cluster units assembled on NifU. Because homologs to both NifU and NifS, respectively designated IscU and IscS, are found in non-nitrogen fixing organisms, it is possible that the function of NifU proposed here could represent a general mechanism for the maturation of Fe-S cluster-containing proteins.     

Transformation by polyoma virus alters expression of a cell mutation affecting cycle traverse.

A temperature-sensitive mutant of hamster BHK 21/13 cells, tsAF8, which at 39 degrees becomes arrested in the G1 (G0) phase of the cell cycle, is phenotypically altered with respect to temperature sensitivity after transformation with polyoma virus. Polyoma transformation does not produce reversion to a non-temperature-sensitive phenotype but causes increased entry into S and increased rate of cell death at the nonpermissive temperature, compared to untransformed tsAF8 cells. The increased frequency of cells synthesizing DNA is not accompanied by an increased frequency of mitosis, since most of the polyoma-transformed tsAF8 cells that synthesize DNA at the nonpermissive temperature do not divide. At the permissive temperature, polyoma-transformed tsAF8 cells, unlike tsAF8, also lose viability when exposed to other methods of arresting cells in G1. The most likely explanation for this phenomenon is that polyoma virus transformation interferes with the cellular response to this mutation as well as to other conditions that cause cell cycle arrest in G1.     

Reversible redistribution of phytochrome within the cell upon conversion to its physiologically active form.

The intracellular localization of phytochrome was seen in dark-grown oat (Avena sativa L., cv. Garry) and rice (Oryza sativa L., cv. unknown) shoots after various light treatments using an indirect peroxidase-antiperoxidase antibody labeling method. Phytochrome is generally distributed throughout the cytoplasm in cells of tissue that had not been exposed to light prior to fixation. Within, at most, 8 min after the onset of saturating red irradiation, phytochrome, now present in the far-red-absorbing form, becomes associated with discrete regions of the cell. These regions do not appear to be nuclei, plastids, or mitochondria. After phototransformation back to the red-absorbing form originally present, phytochrome slowly resumes its general distribution. It is possible that this discrete localization of the far-red-absorbing form of phytochrome represents a physiologically significant binding with a receptor site in the cell.     

Insulin binding to rat mammary gland at various stages of cell isolation and purification

Epithelial cell-rich fractions of rat mammary gland were prepared using percoll gradients after collagenase dispersion. Their insulin-binding characteristics were similar to those of crude acini but superior to unfractionated isolated cells. Optimal binding was obtained after 60 min at 20°C or 16 h at 4°C at pH 7.8. Binding at 37°C was lower due probably to an enhanced rate of insulin degradation.48 h after ovariectomy of 18-day pregnant rats insulin binding to acini doubled due to an increase in the number of insulin receptors. Progesterone but not bromocriptine (which prevented the rise in serum prolactin which occurred after ovariectomy) prevented this increase in insulin binding. These results illustrate that the change in serum progesterone rather than prolactin increases insulin binding to the mammary cell at parturition whilst insulin binding decreases in adipose tissue at the same time (Flint et al., Mol. Cell EndocrinoL, 20, 101–111, 1981) enabling coordinated changes in the metabolism of these 2 tissues to take place during the perinatal period.