Changes in high K+-evoked serotonin release and serotonin 2A/2C receptor binding in the frontal cortex of rats with thioacetamide-induced hepatic encephalopathy

Protein import into the nucleus is generally considered to involve specific nuclear localization signals (NLS) though it is becoming increasingly clear that efficient and well controlled import of proteins which lack a canonical NLS also occurs in cells. Human immunodeficiency virus type 1 (HIV-1) Vpr is one such protein which does not have an identifiable canonical NLS and yet efficiently localizes to the nuclear compartment. Here, we use confocal microscopy to demonstrate that mutations in the putative central hydrophobic helix of Vpr result in the retention of the protein in highly localized ring-like structures around the nuclear periphery with striking impairment in their ability to enter the nuclear interior. By characterizing other biological activities associated with this protein, such as its ability to incorporate into budding virions and its ability to arrest cells in G2, we show that this helical domain is specific for the nuclear translocation of the protein with very little effect on these other functions. Interestingly, however, perturbation of this helical motif also perturbs the protein's ability to augment viral replication in primary human macrophages indicating that the integrity of this secondary structure is essential for optimal infection in these non-dividing cells.     

Nuclear import, virion incorporation, and cell cycle arrest/differentiation are mediated by distinct functional domains of human immunodeficiency virus type 1 Vpr

The vpr gene product of human immunodeficiency virus type 1 (HIV-1) is a virion-associated protein that is essential for efficient viral replication in monocytes/macrophages. Vpr is primarily localized in the nucleus when expressed in the absence of other viral proteins. Vpr is packaged efficiently into viral particles through interactions with the p6 domain of the Gag precursor polyprotein p55gag. We developed a panel of expression vectors encoding Vpr molecules mutated in the amino-terminal helical domain, leucine-isoleucine (LR) domain, and carboxy-terminal domain to map the different functional domains and to define the interrelationships between virion incorporation, nuclear localization, cell cycle arrest, and differentiation functions of Vpr. We observed that substitution mutations in the N-terminal domain of Vpr impaired both nuclear localization and virion packaging, suggesting that the helical structure may play a vital role in modulating both of these biological properties. The LR domain was found to be involved in the nuclear localization of Vpr. In contrast, cell cycle arrest appears to be largely controlled by the C-terminal domain of Vpr. The LR and C-terminal domains do not appear to be essential for virion incorporation of Vpr. Interestingly, we found that two Vpr mutants harboring single amino acid substitutions (A30L and G75A) retained the ability to translocate to the nucleus but were impaired in the cell cycle arrest function. In contrast, mutation of Leu68 to Ser resulted in a protein that localizes in the cytoplasm while retaining the ability to arrest host cell proliferation. We speculate that the nuclear localization and cell cycle arrest functions of Vpr are not interrelated and that these functions are mediated by separable putative functional domains of Vpr.     

Diversity of HIV-1 Vpr interactions involves usage of the WXXF motif of host cell proteins

Targeting protein or RNA moieties to specific cellular compartments may enhance their desired functions and specificities. Human immunodeficiency virus type I (HIV-1) encodes proteins in addition to Gag, Pol, and Env that are packaged into virus particles. One such retroviral-incorporated protein is Vpr, which is present in all primate lentiviruses. Vpr has been implicated in different roles within the HIV-1 life cycle. In testing a new hypothesis in which viral proteins are utilized as docking sites to incorporate protein moieties into virions, we used the peptide phage display approach to search for Vpr-specific binding peptides. In the present studies, we demonstrate that most of the peptides that bind to Vpr have a common motif, WXXF. More importantly, we demonstrate that the WXXF motif of uracil DNA glycosylase is implicated in the interaction of uracil DNA glycosylase with Vpr intracellularly. Finally, a dimer of the WXXF motif was fused to the chloramphenicol acetyl transferase (CAT) gene, and it was demonstrated that the WXXF dimer-CAT fusion protein construct produces CAT activity within virions in the presence of Vpr as a docking protein. This study provides a novel potential strategy in the targeting of anti-viral agents to interfere with HIV-1 replication.     

Variability in function measurements of three sensory foot nerves in neuropathic diabetic patients

Although most short, linear peptide fragments of proteins are unstructured in aqueous solution, a number of immunogenic and antigenic peptides have been shown to have conformational preferences for structured forms. By using mainly NMR and CD spectroscopy, it has been possible to detect and quantify quite small populations of beta-turn, helical, and nascent helical conformations. Recent studies have been published indicating that the presence of structured forms is correlated with the location of T cell and/or B cell epitopes in peptide sequences. X-ray crystal structures of complexes between peptides and anti-peptide antibodies frequently show the peptides bound in beta-turn conformations, and the presence of helix in one peptide-antibody complex has been shown by NMR spectroscopy. Studies of peptides free in solution and bound to anti-peptide antibodies in the crystal indicate that the structure of the principal neutralizing determinant of HIV-1 probably includes at least one beta-turn in a highly conserved region. These results can potentially be used in the design of peptide-based vaccines.     

Synthesis and conformational study of peptides possessing helically arranged delta ZPhe side chains

Z-Dehydrophenylalanine (delta ZPhe) possessing four oligopeptides, Boc-(L-Ala-delta ZPhe-Aib)n-OCH3 (n = 1-4: Boc, t-butoxycarbonyl; Aib, alpha-aminoisobutyric acid), were synthesized, and their solution conformations were investigated by 1H-nmr, ir, uv, and CD spectroscopy and theoretical CD calculation. 1H-nmr (the solvent accessibility of NH groups) and ir studies indicated that all the NH groups except for those belonging to the N-terminal L-Ala-delta ZPhe moiety participate in intramolecular hydrogen bonding in chloroform. This suggests that the peptides n = 2-4 have a 4-->1 hydrogen-bonding pattern characteristic of 3(10)-helical structures. The uv spectra of all these peptides recorded in chloroform and in trimethyl phosphate showed an intense maximum around 276 nm assigned to the delta ZPhe chromophores. The corresponding CD spectra of the peptides n = 2-4 showed exciton couplets with a negative peak at longer wavelengths, whereas that of the peptide n = 1 showed only weak signals. Theoretical CD spectra were calculated for the peptides n = 2-4 of several helical conformations, on the basis of exciton chirality method. This calculation indicated that the three peptides form a helical conformation deviating from the perfect 3(10)-helix that contains three residues per turn, and that their side chains of delta ZPhe residues are arranged regularly along the helix. The center-to-center distance between the nearest phenyl pair(s) was estimated to be approximately 5.5 A. The chemical shifts of the delta ZPhe side-chain protons (H beta and aromatic H) for the peptides n = 2-4 indicated anisotropic shielding effect of neighboring phenyl group (s); the effect also supports a regular arrangement of the delta ZPhe side chains along the helical axis.     

A role of myofilament Ca2+ sensitivity in enhanced vascular reactivity in cardiomyopathic hamsters

Conformational studies of the salivary peptides histatin 3 (H3) and histatin 5 (H5) were performed by NMR and circular dichroism (CD) in aqueous and nonaqueous solutions. Histatin 5 has no defined structure in H2O but adopts a more helical conformation in dimethyl sulfoxide and aqueous trifluoroethanol. This is in agreement with the CD analysis, which shows no secondary structure in H2O but increasing helical content in the presence of trifluoroethanol. CD analysis shows that H3 has less propensity to form a helical structure than H5 in similar conditions. The NMR analysis of H3 in H2O at pH 7.4 reveals that its conformational mobility is less than that of H5 as indicated by the observation of backbone cross peaks alphaN (i, i + 1) and NN (i, i + 1) and the slow exchanging amide protons in the C-terminus. However, H3 remains essentially unordered as suggested by the lack of longer range nuclear Overhauser effects (NOEs) in the NOESY spectrum. H3 becomes much more ordered in a mixture of 50:50 H2O-dimethyl sulfoxide as indicated by the numerous NOEs, including several side chain to side chain and side chain to backbone connectivities. Our data suggest that in these conditions H3 contains a turn in the region of K13 to K17 and possibly a 3(10) helix at the C-terminus. This study demonstrates that H3 and H5 are both conformationally mobile and that each adopt different types of conformations in aqueous and nonaqueous solutions.     

NMR structure of the (52-96) C-terminal domain of the HIV-1 regulatory protein Vpr: molecular insights into its biological functions

The HIV-1 regulatory protein Vpr (96 amino acid residues) is incorporated into the virus particle through a mechanism involving its interaction with the C-terminal portion of Gag. Vpr potentiates virus replication by interrupting cell division in the G2 phase and participates in the nuclear transport of proviral DNA. The domain encompassing the 40 C-terminal residues of Vpr was shown to be involved in cell cycle arrest and binding of nucleocapsid protein NCp7, and suggested to promote nuclear provirus transfer. Accordingly, we show here that the synthetic 52-96 but not 1-51 sequences of Vpr interact with HIV-1 RNA. Based on these results, the structure of (52-96)Vpr was analysed by two-dimensional 1H-NMR in aqueous TFE (30%) solution and refined by restrained molecular dynamics. The structure is characterized by a long (53-78) amphipathic alpha-helix, followed by a less defined (79-96) C-terminal domain. The Leu60 and Leu67 side-chains are located on the hydrophobic side of the helix, suggesting their involvement in Vpr dimerization through a leucine zipper-type mechanism. Accordingly, their replacement by Ala eliminates Vpr dimerization in the two hybrid systems, while mutations of Ile74 and Ile81 have no effect. This was confirmed by gel filtration measurements and circular dichroism, which also showed that the alpha-helix still exists in (52-96)Vpr and its Ala60, Ala67 mutant in the presence and absence of TFE. Based on these results, a model of the coiled-coil Vpr dimer has been described, and its biological relevance as well as that of the structural characteristics of the 52-96 domain for the different functions of Vpr, including HIV-1 RNA binding, are discussed.     

Structure effects of double D-amino acid replacements: a nuclear magnetic resonance and circular dichroism study using amphipathic model helices

D-Amino acid replacements and the determination of resulting structural changes are a useful tool to recognize amphipathic helices in biologically active peptides such as neuropeptide Y and corticotropin-releasing factor. In this paper the secondary structures of one amphipathic alpha-helical peptide and its double D-amino acid analog have been determined by means of 1H NMR and CD spectroscopies under equivalent conditions. The chemical shifts (NH and C alpha H) and the analysis of nuclear Overhauser effects show a split of the continuous helix for the all-L peptide into two helices at the position of double D-amino acid replacement. Hydrogen exchange rates correlate with water accessibilities in the hydrophobic/hydrophilic face and confirm the amphipathic helical structure in the all-L peptide as well as in its double D-amino acid analog. A significantly accelerated hydrogen isotope exchange rate is observed for the D-Ala9 backbone proton, implying an increased flexibility at that position. These results show that the incorporation of an adjacent pair of D-amino acids only causes a local change in structure and flexibility, which makes the double D replacement interesting as a tool for specific helix-disturbing modifications to search for helical conformations in biologically active peptides.     

CD of proline-rich polypeptides: application to the study of the repetitive domain of maize glutelin-2

An overview of CD of proline-rich peptides is reported. First, structural characteristics, theoretical CD studies, and the biological relevance of polyproline II structure in such peptides are discussed. Second, a CD study of peptides belonging to the repetitive domain of maize glutelin-2, H-(Val-His-Leu-Pro-Pro-Pro)n-OH (n = 3, 5, 8), is described. This series of peptides displayed the CD features of polyproline II structure in water (5 degrees C, pH 5). Moreover, it was shown that the addition of increasing amounts of the polyanionic molecule heparin forced a displacement of the conformational equilibrium of those peptides toward higher proportions of the polyproline II structure. In contrast, when the temperature is raised such a structure gradually disappears, leading to more disordered conformations.     

Folding propensities of peptide fragments of myoglobin

Myoglobin has been studied extensively as a paradigm for protein folding. As part of an ongoing study of potential folding initiation sites in myoglobin, we have synthetized a series of peptides covering the entire sequence of sperm whale myoglobin. We report here on the conformation preferences of a series of peptides that cover the region from the A helix to the FG turn. Structural propensities were determined using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution, trifluoroethanol, and methanol. Peptides corresponding to helical regions in the native protein, namely the B, C, D, and E helices, populate the alpha region of (phi, psi) space in water solution but show no measurable helix formation except in the presence of trifluoroethanol. The F-helix sequence has a much lower propensity to populate helical conformations even in TFE. Despite several attempts, we were not successful in synthesizing a peptide corresponding to the A-helix region that was soluble in water. A peptide termed the AB domain was constructed spanning the A- and B-helix sequences. The AB domain is not soluble in water, but shows extensive helix formation throughout the peptide when dissolved in methanol, with a break in the helix at a site close to the A-B helix junction in the intact folded myoglobin protein. With the exception of one local preference for a turn conformation stabilized by hydrophobic interactions, the peptides corresponding to turns in the folded protein do not measurably populate beta-turn conformations in water, and the addition of trifluoroethanol does not enhance the formation of either helical or turn structure. In contrast to the series of peptides described here, either studies of peptides from the GH region of myoglobin show a marked tendency to populate helical structures (H), nascent helical structures (G), or turn conformations (GH peptide) in water solution. This region, together with the A-helix and part of the B-helix, has been shown to participate in an early folding intermediate. The complete analysis of conformational properties of isolated myoglobin peptides supports the hypothesis that spontaneous secondary structure formation in local regions of the polypeptide may play an important role in the initiation of protein folding.     

Membrane-induced secondary structures of neuropeptides: a comparison of the solution conformations adopted by agonists and antagonists of the mammalian tachykinin NK1 receptor

We present what we believe to be the first documented example of an inducement of distinctly different secondary structure types onto agonists and antagonists selective for the same G-coupled protein receptor using the same membrane-model matrix wherein the induced structures are consistent with those suggested to be biologically active by extensive analogue studies and conventional binding assays. 1H NMR chemical shift assignments for the mammalian NK1 receptor-selective agonists alpha-neurokinin (NKA) and beta-neurokinin (NKB) as well as the mammalian NK1 receptor-selective antagonists [d-Pro2,d-Phe7,d-Trp9]SP and [d-Arg1, d-Pro2,d-Phe7,d-His9]SP have been determined at 600 MHz in sodium dodecyl sulfate (SDS) micelles. The SDS micelle system simulates the membrane-interface environment the peptide experiences when in the proximity of the membrane-embedded receptor, allowing for conformational studies that are a rough approximation of in vivo conditions. Two-dimensional NMR techniques were used to assign proton resonances, and interproton distances were estimated from the observed nuclear Overhauser effects (NOEs). The experimental distances were used as constraints in a molecular dynamics and simulated annealing protocol using the modeling package DISCOVER to generate three-dimensional structures of the two agonists and two antagonists when present in a membrane-model environment to determine possible prebinding ligand conformations. It was determined that (1) NKA is helical from residues 6 to 9, with an extended N-terminus; (2) NKB is helical from residues 4 to 10, with an extended N-terminus; (3) [d-Pro2,d-Phe7,d-Trp9]SP has poorly defined helical properties in the midregion and a beta-turn structure in the C-terminus (residues 6-9); and (4) [d-Arg1,d-Pro2, d-Phe7,d-His9]SP has a helical structure in the midregion (residues 4-6) and a well-defined beta-turn structure in the C-terminus (residues 6-10). Attempts have been made to correlate the observed conformational differences between the agonists and antagonists to their binding potencies and biological activity.     

Uracil DNA glycosylase specifically interacts with Vpr of both human immunodeficiency virus type 1 and simian immunodeficiency virus of sooty mangabeys, but binding does not correlate with cell cycle arrest

The Vpr protein encoded by human immunodeficiency virus type 1 (HIV-1) is important for growth of virus in macrophages and prevents infected cells from passing into mitosis (G2 arrest). The cellular target for these functions is not known, but Vpr of HIV-1 and the related Vpr from simian immunodeficiency virus of sooty mangabeys (SIV(SM)) bind the DNA repair enzyme UNG, while the Vpx protein of SIV(SM) does not. Nonetheless, a mutational analysis of Vpr showed that binding to UNG is neither necessary nor sufficient for the effect of Vpr on the cell cycle.     

Structure of segments of a G protein-coupled receptor: CD and NMR analysis of the Saccharomyces cerevisiae tridecapeptide pheromone receptor

Peptides representing both loop and the sixth transmembrane regions of the alpha-factor receptor of Saccharomyces cerevisiae were synthesized by solid-phase procedures and purified to near homogeneity. CD, nmr, and modeling analysis indicated that in aqueous media the first extracellular loop peptide E1(107-125), the third intracellular loop peptide I3(231-243), and the carboxyl terminus peptide I4(350-372) were mostly disordered. In contrast, the second extracellular loop peptide E2(191-206) assumed a well-defined structure in aqueous medium and the sixth transmembrane domain peptide receptor M6(252-269, C252A) was highly helical in trifluoroethanol/water (4:1), exhibiting a kink at Pro258. A synthetic peptide containing a sequence similar to that of the sixth transmembrane domain of a constitutively active alpha-factor receptor M6(252-269, C252A, P258L) in which Leu replaces Pro258 exhibited significantly different biophysical properties than the wild-type sequence. In particular, this peptide had very low solubility and gave CD resembling that of a beta-sheet structure in hexafluoroacetone/water (1:1) whereas the wild-type peptide was partially helical under identical conditions. These results would be consistent with the hypothesis that the constitutive activity of the mutant receptor is linked to a conformational change in the sixth transmembrane domain. The study of the receptor segments also indicate that peptides corresponding to loops of the alpha-factor receptor do not appear to assume turn structures.     

Molecular and cellular actions of chronic electroconvulsive seizures

Reducing a CO to a CH2 moiety in a peptide bond destroys the ability of the peptide link to act as a proton acceptor in a hydrogen-bonded structure. Here, this modification is introduced into different positions of the helical peptide, acetyl-WGG(RAAAA)4R-amide, and the melting of these peptides is followed using CD. Effects of this modification on helical peptides are compared to our previous N-methylation studies [C. F. Chang and M. H. Zehfus (1996) Biopolymers, Vol. 40, pp. 609-616]. While the experiments were designed to remove the same hydrogen bond from the peptide, no consistent results are obtained between these two modifications. This result suggests that these modifications not only break the backbone hydrogen bonds, but also involve other destabilizing effects. When our data is analyzed using different helix-coil transition models, the results show that as the models increase in complexity the energy associated with a single residue modification increases. Unfortunately, the most detailed dichroic model, which should best describe this system, works for only one peptide. Apparently, the models need to be further improved to better mimic our system.     

Structure-function relationships in side chain lactam cross-linked peptide models of a conserved N-terminal domain of apolipoprotein E

Bioactive peptides have multiple conformations in solution but adopt well-defined conformations at lipid surfaces and in interactions with receptors. We have used side chain lactam cross-links to stabilize secondary structures in the following peptide models of a conserved N-terminal domain of apolipoprotein E (cross-link periodicity in parentheses): I, H2N-GQTLSEQVQEELLSSQVTQELRAG-COOH (none); III, [sequence; see text] (i to i + 3); IV,[sequence; see text] (i to i + 4); IVa, [sequence, see text] (i to i + 4) (lactams above the sequence, potential salt bridges below the sequence). We previously demonstrated [Luo et al. (1994) Biochemistry 33, 12367-12377; Braddock et al. (1996) Biochemistry 35, 13975-13984] that peptide III, containing lactam cross-links between the i and i + 3 side chains, enhances specific binding of LDL via a receptor other than the LDL-receptor. Peptide III in solution consists of two short alpha helices connected by a non alpha helical segment. Here we examine the hypothesis that the domain modeled by peptide III is one antipode of a conformational switch. To model another antipode of the switch, we introduced two strategic modifications into peptide III to examine structure-function relationships in this domain: (1) the spacing of the lactam cross-links was changed (i to i + 4 in peptides IV and IVa) and (2) peptides IV and IVa contain the two alternative sequences at a site of a possible end-capping interaction in peptide III. The structure of peptide IV, determined by 2D-NMR, is alpha helical across its entire length. Despite the remarkable degree of structural order, peptide IV is biologically inactive. In contrast, peptides III and possibly IVa contain a central interruption of the alpha helix, which appears necessary for biological activity. These and other studies support the hypothesis that this domain is a conformational switch which, to the extent that it models apolipoprotein E itself, may modulate interactions between apo E and its various receptors.     

Culture and early infancy among central African foragers and farmers

A 27-residue stretch of amino acids encompassing two putative 13-residue amphiphilic helical segments is an important determinant of activity in the 47-residue antibacterial peptide bovine seminalplasmin. Synthetic peptides corresponding to the 27-residue stretch (P27) SLSRYAKLANRLANPKLLETFLSKWIG as well as the 13-residue segments PKLLETFLSKWIG (SPF),exhibit antimicrobial activity. An analog of SPF where E has been replaced by K(SPFK) showed improved antimicrobial properties as compared to SPF. The peptides have the ability to bind and permeabilize membranes. We have modeled helical bundles of P27 and the two 13-residue peptides SPF and SPFK using simulated annealing via molecular dynamics. Octameric but not hexameric aggregates of P27 can form channels which would allow the passage of ions. In the case of 13-residue peptides, aggregates formed by 6 monomers can conceivably form ion conducting channels. Since the ability to form channels which would allow the passage of ions across the membranes is an important determinant of the biological activities of these peptides, knowledge of the pore forming structures should help in the design of analogs with improved activities.     

Arginine residues in the C-terminus of HIV-1 Vpr are important for nuclear localization and cell cycle arrest

HIV-1 viral protein R (Vpr) is predominantly localized to the nucleus and plays an important role for viral preintegration complex import into the nucleus. In this study, we investigated the influence on subcellular localization of Arg residues in the C-terminus of Vpr. Consistent with previous studies, about 90% of the cells manifested diffuse nuclear staining in the Vpr-expressed cells. Besides diffuse nuclear staining, punctate perinuclear staining, and punctate cytoplasmic staining were also observed in the immunofluorescence studies. Deletion of the Ser-Arg-lle-Gly residues (amino acids 79-82; SRIG) had no effect on the Vpr localization. However, deletion of the Arg-Gln-Arg-Arg residues (amino acids 85-88; RQRR) resulted in a smooth perinuclear staining pattern. Substitution of five Arg residues with Asn (amino acids 80, 85, 87, 88, and 90; R-->N5) resulted in a diffuse cytoplasmic staining. Subcellular fractionation analyses support the immunofluorescence staining results. These findings indicate that the C-terminal Arg residues of HIV-1 Vpr play an important role for Vpr nuclear localization. All the Vpr mutants were appropriately expressed, exhibited no significant defect on the protein stability, and were incorporated efficiently into virus-like particles. Both SRIG and R-->N5 mutants lost their cell cycle arrest activities and the RQRR deletion only exhibited a low level of cell arrest activity. Therefore, the Arg residues in the HIV-1 Vpr C-terminus are important for Vpr nuclear localization and cell cycle arrest, but had no effect on protein stability or Vpr incorporation into virus-like particles.     

Crystal structure of the simian immunodeficiency virus (SIV) gp41 core: conserved helical interactions underlie the broad inhibitory activity of gp41 peptides

The gp41 subunit of the envelope protein complex from human and simian immunodeficiency viruses (HIV and SIV) mediates membrane fusion during viral entry. The crystal structure of the HIV-1 gp41 ectodomain core in its proposed fusion-active state is a six-helix bundle. Here we have reconstituted the core of the SIV gp41 ectodomain with two synthetic peptides called SIV N36 and SIV C34, which form a highly helical trimer of heterodimers. The 2.2 A resolution crystal structure of this SIV N36/C34 complex is very similar to the analogous structure in HIV-1 gp41. In both structures, three N36 helices form a central trimeric coiled coil. Three C34 helices pack in an antiparallel orientation into highly conserved, hydrophobic grooves along the surface of this coiled coil. The conserved nature of the N36-C34 interface suggests that the HIV-1 and SIV peptides are functionally interchangeable. Indeed, a heterotypic complex between HIV-1 N36 and SIV C34 peptides is highly helical and stable. Moreover, as with HIV-1 C34, the SIV C34 peptide is a potent inhibitor of HIV-1 infection. These results identify conserved packing interactions between the N and C helices of gp41 and have implications for the development of C peptide analogs with broad inhibitory activity.