Zn2+ interaction with Alzheimer amyloid beta protein calcium channels.

The Alzheimer disease 40-residue amyloid beta protein (AbetaP[1-40]) forms cation-selective channels across acidic phospholipid bilayer membranes with spontaneous transitions over a wide range of conductances ranging from 40 to 4000 pS. Zn2+ has been reported to bind to AbetaP[1-40] with high affinity, and it has been implicated in the formation of amyloid plaques. We now report the functional consequences of such Zn2+ binding for the AbetaP[1-40] channel. Provided the AbetaP[1-40] channel is expressed in the low conductance (<400 pS) mode, Zn2+ blocks the open channel in a dose- dependent manner. For AbetaP[1-40] channels in the giant conductance mode (>400 pS), Zn2+ doses in the millimolar range were required to exert substantial blockade. The Zn2+ chelator o-phenanthroline reverses the blockade. We also found that Zn2+ modulates AbetaP[1-40] channel gating and conductance only from one side of the channel. These data are consistent with predictions of our recent molecular modeling studies on AbetaP[1-40] channels indicating asymmetric Zn(2+)-AbetaP[1-40] interactions at the entrance to the pore.

Isolation of an oxygen-sensitive FNR protein of Escherichia coli: interaction at activator and repressor sites of FNR-controlled genes.

The Escherichia coli fnr gene product, FNR, is a DNA binding protein that regulates a large family of genes involved in cellular respiration and carbon metabolism during conditions of anaerobic cell growth. FNR is believed to contain a redox/O2-sensitive element for detecting the anaerobic state. To investigate this process, a fnr mutant that encodes an altered FNR protein with three amino acid substitutions in the N-terminal domain was constructed by site-directed mutagenesis. In vivo, the mutant behaved like a wild-type strain under anaerobic conditions but had a 14-fold elevated level of transcriptional activation of a reporter gene during aerobic cell growth. The altered fur gene was overexpressed in E. coli and the resultant FNR protein was purified to near homogeneity by using anaerobic chromatography procedures. An in vitro Rsa I restriction site protection assay was developed that allowed for the assessment of oxygen-dependent DNA binding of the mutant FNR protein. The FNR protein was purified as a monomer of M(r) 28,000 that contained nonheme iron at 2.05 +/- 0.34 mol of Fe per FNR monomer. In vitro DNase I protection studies were performed to establish the locations of the FNR-binding sites at the narG, narK, dmsA, and hemA promoters that are regulated by either activation or repression of their transcription. The sizes of the DNA footprints are consistent with the binding of two monomers of FNR that protect the symmetrical FNR-recognition sequence TTGAT-nnnnATCAA. Exposure of the FNR protein or protein-DNA complex to air for even short periods of time (approximately 5 min) led to the complete loss of DNA protection at a consensus FNR recognition site. A model whereby the FNR protein exists in the cell as a monomer that assembles on the DNA under anaerobic conditions to form a dimer is discussed.

Disruption of RB/E2F-1 interaction by single point mutations in E2F-1 enhances S-phase entry and apoptosis.

The retinoblastoma protein (RB) has been proposed to function as a negative regulator of cell proliferation by complexing with cellular proteins such as the transcription factor E2F. To study the biological consequences of the RB/E2F-1 interaction, point mutants of E2F-1 which fail to bind to RB were isolated by using the yeast two-hybrid system. Sequence analysis revealed that within the minimal 18-amino acid peptide of E2F-1 required for RB binding, five residues, Tyr (position 411), Glu (419), and Asp-Leu-Phe (423-425), are critical. These amino acids are conserved among the known E2F family members. While mutation of any of these five amino acids abolished binding to RB, all mutants retained their full transactivation potential. Expression of mutated E2F-1, when compared with that of wild-type, significantly accelerated entry into S phase and subsequent apoptosis. These results provide direct genetic evidence for the biological significance of the RB/E2F interaction and strongly suggest that the interplay between RB and E2F is critical for proper cell cycle progression.

Vpr protein of human immunodeficiency virus type 1 forms cation-selective channels in planar lipid bilayers.

A small (96-aa) protein, virus protein R (Vpr), of human immunodeficiency virus type 1 contains one hydrophobic segment that could form a membrane-spanning helix. Recombinant Vpr, expressed in Escherichia coli and purified by affinity chromatography, formed ion channels in planar lipid bilayers when it was added to the cis chamber and when the trans chamber was held at a negative potential. The channels were more permeable to Na+ than to Cl- ions and were inhibited when the trans potential was made positive. Similar channel activity was caused by Vpr that had a truncated C terminus, but the potential dependence of channel activity was no longer seen. Antibody raised to a peptide mimicking part of the C terminus of Vpr (AbC) inhibited channel activity when added to the trans chamber but had no effect when added to the cis chamber. Antibody to the N terminus of Vpr (AbN) increased channel activity when added to the cis chamber but had no effect when added to the trans chamber. The effects of potential and antibodies on channel activity are consistent with a model in which the positive C-terminal end of dipolar Vpr is induced to traverse the bilayer membrane when the opposite (trans) side of the membrane is at a negative potential. The C terminus of Vpr would then be available for interaction with AbC in the trans chamber, and the N terminus would be available for interaction with AbN in the cis chamber. The ability of Vpr to form ion channels in vitro suggests that channel formation by Vpr in vivo is possible and may be important in the life cycle of human immunodeficiency virus type 1 and/or may cause changes in cells that contribute to AIDS-related pathologies.

Enterococcus faecalis pheromone binding protein, PrgZ, recruits a chromosomal oligopeptide permease system to import sex pheromone cCF10 for induction of conjugation.

Conjugative transfer of the plasmid pCF10 by Enterococcus faecalis donor cells occurs in response to a peptide sex pheromone, cCF10, secreted by recipients. The plasmid-encoded cCF10 binding protein, PrgZ, is similar in sequence to binding proteins (OppAs) encoded by oligopeptide permease (opp) operons. Mutation of prgZ decreased the sensitivity of donor cells to pheromone, whereas inactivation of the chromosomal E. faecalis opp operon abolished response at physiological concentrations of pheromone. Affinity chromatography experiments demonstrated the interaction of the pheromone with several putative intracellular regulatory molecules, including an RNA molecule required for positive regulation of conjugation functions. These data suggest that processing of the pheromone signal involves recruitment of a chromosomal Opp system by PrgZ and that signaling occurs by direct interaction of internalized pheromone with intracellular effectors.

Interaction between the phage HK022 Nun protein and the nut RNA of phage lambda.

The nun gene product of prophage HK022 excludes phage lambda infection by blocking the expression of genes downstream from the lambda nut sequence. The Nun protein functions both by competing with lambda N transcription-antitermination protein and by actively inducing transcription termination on the lambda chromosome. We demonstrate that Nun binds directly to a stem-loop structure within nut RNA, boxB, which is also the target for the N antiterminator. The two proteins show comparable affinities for boxB and they compete with each other. Their interactions with boxB are similar, as shown by RNase protection experiments, NMR spectroscopy, and analysis of boxB mutants. Each protein binds the 5' strand of the boxB stem and the adjacent loop. The stem does not melt upon the binding of Nun or N, as the 3' strand remains sensitive to a double-strand-specific RNase. The binding of RNA partially protects Nun from proteolysis and changes its NMR spectra. Evidently, although Nun and N bind to the same surface of boxB RNA, their respective complexes interact differently with RNA polymerase, inducing transcription termination or antitermination, respectively.

Production of avian leukosis virus particles in mammalian cells can be mediated by the interaction of the human immunodeficiency virus protein Rev and the Rev-responsive element.

In human immunodeficiency virus type 1-infected cells, the efficient expression of viral proteins from unspliced and singly spliced RNAs is dependent on two factors: the presence in the cell of the viral protein Rev and the presence in the viral RNA of the Rev-responsive element (RRE). We show here that the HIV-1 Rev/RRE system can increase the expression of avian leukosis virus (ALV) structural proteins in mammalian cells (D-17 canine osteosarcoma) and promote the release of mature ALV virions from these cells. In this system, the Rev/RRE interaction appears to facilitate the export of full-length unspliced ALV RNA from the nucleus to the cytoplasm, allowing increased production of the ALV structural proteins. Gag protein is produced in the cytoplasm of the ALV-transfected cells even in the absence of a Rev/RRE interaction. However, a functional Rev/RRE interaction increases the amount of Gag present intracellularly and, more strikingly, results in the release of mature ALV particles into the supernatant. RCAS virus containing an RRE is replication-competent in chicken embryo fibroblasts; however, we have been unable to determine whether the particles produced in D-17 cells are as infectious as the particles produced in chicken embryo fibroblasts.

Prion protein (PrP) synthetic peptides induce cellular PrP to acquire properties of the scrapie isoform.

Conversion of the cellular isoform of prion protein (PrPC) into the scrapie isoform (PrPSc) involves an increase in the beta-sheet content, diminished solubility, and resistance to proteolytic digestion. Transgenetic studies argue that PrPC and PrPSc form a complex during PrPSc formation; thus, synthetic PrP peptides, which mimic the conformational pluralism of PrP, were mixed with PrPC to determine whether its properties were altered. Peptides encompassing two alpha-helical domains of PrP when mixed with PrPC produced a complex that displayed many properties of PrPSc. The PrPC-peptide complex formed fibrous aggregates and up to 65% of complexed PrPC sedimented at 100,000 x g for 1 h, whereas PrPC alone did not. These complexes were resistant to proteolytic digestion and displayed a high beta-sheet content. Unexpectedly, the peptide in a beta-sheet conformation did not form the complex, whereas the random coil did. Addition of 2% Sarkosyl disrupted the complex and rendered PrPC sensitive to protease digestion. While the pathogenic A117V mutation increased the efficacy of complex formation, anti-PrP monoclonal antibody prevented interaction between PrPC and peptides. Our findings in concert with transgenetic investigations argue that PrPC interacts with PrPSc through a domain that contains the first two putative alpha-helices. Whether PrPC-peptide complexes possess prion infectivity as determined by bioassays remains to be established.

Structure of human T-cell receptors specific for an immunodominant myelin basic protein peptide: positioning of T-cell receptors on HLA-DR2/peptide complexes.

T-cell receptors (TCRs) recognize peptide bound within the relatively conserved structural framework of major histocompatibility complex (MHC) class I or class II molecules but can discriminate between closely related MHC molecules. The structural basis for the specificity of ternary complex formation by the TCR and MHC/peptide complexes was examined for myelin basic protein (MBP)-specific T-cell clones restricted by different DR2 subtypes. Conserved features of this system allowed a model for positioning of the TCR on DR2/peptide complexes to be developed: (i) The DR2 subtypes that presented the immunodominant MBP peptide differed only at a few polymorphic positions of the DR beta chain. (ii) TCR recognition of a polymorphic residue on the helical portion of the DR beta chain (position DR beta 67) was important in determining the MHC restriction. (iii) The TCR variable region (V) alpha 3.1 gene segment was used by all of the T-cell clones. TCR V beta usage was more diverse but correlated with the MHC restriction--i.e., with the polymorphic DR beta chains. (iv) Two clones with conserved TCR alpha chains but different TCR beta chains had a different MHC restriction but a similar peptide specificity. The difference in MHC restriction between these T-cell clones appeared due to recognition of a cluster of polymorphic DR beta-chain residues (DR beta 67-71). MBP-(85-99)-specific TCRs therefore appeared to be positioned on the DR2/peptide complex such that the TCR beta chain contacted the polymorphic DR beta-chain helix while the conserved TCR alpha chain contacted the nonpolymorphic DR alpha chain.

Solution structure of the Shc SH2 domain complexed with a tyrosine-phosphorylated peptide from the T-cell receptor.

She is a widely expressed adapter protein that plays an important role in signaling via a variety of cell surface receptors and has been implicated in coupling the stimulation of growth factor, cytokine, and antigen receptors to the Ras signaling pathway. She interacts with several tyrosine-phosphorylated receptors through its C-terminal SH2 domain, and one of the mechanisms of T-cell receptor-mediated Ras activation involves the interaction of the Shc SH2 domain with the tyrosine-phosphorylated zeta chain of the T-cell receptor. Here we describe a high-resolution NMR structure of the Shc SH2 domain complexed to a phosphopeptide (GHDGLpYQGLSTATK) corresponding to a portion of the zeta chain of the T-cell receptor. Although the overall architecture of the protein is similar to other SH2 domains, distinct structural differences were observed in the smaller beta-sheet, BG loop, (pY + 3) phosphopeptide-binding site, and relative position of the bound phosphopeptide.

Interaction of herpes simplex virus 1 origin-binding protein with DNA polymerase alpha.

The herpes simplex virus 1 (HSV-1) genome encodes seven polypeptides that are required for its replication. These include a heterodimeric DNA polymerase, a single-strand-DNA-binding protein, a heterotrimeric helicase/primase, and a protein (UL9 protein) that binds specifically to an HSV-1 origin of replication (oris). We demonstrate here that UL9 protein interacts specifically with the 180-kDa catalytic subunit of the cellular DNA polymerase alpha-primase. This interaction can be detected by immunoprecipitation with antibodies directed against either of these proteins, by gel mobility shift of an oris-UL9 protein complex, and by stimulation of DNA polymerase activity by the UL9 protein. These findings suggest that enzymes required for cellular DNA replication also participate in HSV-1 DNA replication.

Determinants of the phagocytic signal mediated by the type IIIA Fc gamma receptor, Fc gamma RIIIA: sequence requirements and interaction with protein-tyrosine kinases.

The Fc gamma receptor-associated gamma and zeta subunits contain a conserved cytoplasmic motif, termed the immunoglobulin gene tyrosine activation motif, which contains a pair of YXXL sequences. The tyrosine residues within these YXXL sequences have been shown to be required for transduction of a phagocytic signal. We have previously reported that the gamma subunit of the type IIIA Fc gamma receptor (Fc gamma RIIIA) is approximately 6 times more efficient in mediating phagocytosis than the zeta subunit of Fc gamma RIIIA. By exchanging regions of the cytoplasmic domains of the homologous gamma and zeta chains, we observed that the cytoplasmic area of the gamma chain bearing a pair of the conserved YXXL sequences is important in phagocytic signaling. Further specificity of phagocytic signaling is largely determined by the two internal XX amino acids in the YXXL sequences. In contrast, the flanking amino acids of the YXXL sequences including the seven intervening amino acids between the two YXXL sequences do not significantly affect the phagocytic signal. Furthermore, the protein-tyrosine kinase Syk, but not the related kinase ZAP-70, stimulated Fc gamma RIIIA-mediated phagocytosis. ZAP-70, however, increased phagocytosis when coexpressed with the Src family kinase Fyn. These data demonstrate the importance of the two specific amino acids within the gamma subunit YXXL cytoplasmic sequences in phagocytic signaling and explain the difference in phagocytic efficiency of the gamma and zeta chains. These results indicate the importance of Syk in Fc gamma RIIIA-mediated phagocytosis and demonstrate that ZAP-70 and syk differ in their requirement for a Src-related kinase in signal transduction.

Polyomavirus VP1 phosphorylation: coexpression with the VP2 capsid protein modulates VP1 phosphorylation in Sf9 insect cells.

The polyomavirus virion has an outer capsid comprised of 72 pentamers of the VP1 protein associated with the minor virion proteins, VP2 and VP3, and the viral minichromosome. To investigate the interaction between VP1 and VP2/VP3, we mapped VP1 phosphorylation sites and assayed VP1 recognition by anti-peptide antibodies after coexpression of VP1 with VP2 or VP3 by using recombinant baculovirus vectors. VP1, expressed either alone or with VP3, was phosphorylated on serine residues, which are not modified during polyomavirus infection of mouse cells. When VP1 was coexpressed with VP2, the nonphysiologic serine phosphorylation of VP1 was decreased, and a tryptic peptide containing Thr-63, a site modified during virus infection of mouse cells, was phosphorylated. An anti-peptide antibody directed against the VP1 BC loop domain containing Thr-63 recognized VP1 expressed alone but not VP1 coexpressed with VP2 or VP3. The change in phosphorylation resulting from coexpression of two structural proteins identifies the potential of the baculovirus system for studying protein-protein interactions and defines a functional role for the VP1-VP2 interaction.

The bend in RNA created by the trans-activation response element bulge of human immunodeficiency virus is straightened by arginine and by Tat-derived peptide.

The trans-activation response element (TAR) found near the 5' end of the viral RNA of the human immunodeficiency virus contains a 3-nt bulge that is recognized by the virally encoded trans-activator protein (Tat), an important mediator of transcriptional activation. Insertion of the TAR bulge into double-stranded RNA is known to result in reduced electrophoretic mobility, suggestive of a bulge-induced bend. Furthermore, NMR studies indicate that Arg causes a change in the structure of the TAR bulge, possibly reducing the bulge angle. However, neither of these effects has been quantified, nor have they been compared with the effects of the TAR-Tat interaction. Recently, an approach for the quantification of bulge-induced bends has been described in which hydrodynamic measurements, employing the method of transient electric birefringence, have yielded precise estimates for the angles of a series of RNA bulges, with the angles ranging from 7 degrees to 93 degrees. In the current study, transient electric birefringence measurements indicate that the TAR bulge introduces a bend of 50 degrees +/- 5 degrees in the absence of Mg2+. Addition of Arg leads to essentially complete straightening of the helix (to < 10 degrees) with a transition midpoint in the 1 mM range. This transition demonstrates specificity for the TAR bulge: no comparable transition was observed for U3 or A3 (control) bulges with differing flanking sequences. An essentially identical structural transition is observed for the Tat-derived peptide, although the transition midpoint for the latter is near 1 microM. Finally, low concentrations of Mg2+ alone reduce the bend angle by approximately 50%, consistent with the effects of Mg2+ on other pyrimidine bulges. This last observation is important in view of the fact that most previous structural/binding studies were performed in the absence of Mg2+.

The crystal structure of an N-terminal two-domain fragment of vascular cell adhesion molecule 1 (VCAM-1): a cyclic peptide based on the domain 1 C-D loop can inhibit VCAM-1-alpha 4 integrin interaction.

Vascular cell adhesion molecule 1 (VCAM-1) represents a structurally and functionally distinct class of immunoglobulin superfamily molecules that bind leukocyte integrins and are involved in inflammatory and immune functions. X-ray crystallography defines the three-dimensional structure of the N-terminal two-domain fragment that participates in ligand binding. Residues in domain 1 important for ligand binding reside in the C-D loop, which projects markedly from one face of the molecule near the contact between domains 1 and 2. A cyclic peptide that mimics this loop inhibits binding of alpha 4 beta 1 integrin-bearing cells to VCAM-1. These data demonstrate how crystallographic structural information can be used to design a small molecule inhibitor of biological function.