Circular Dichroism Studies of the Interaction of Tat Analogues Substituted in the Arg52 Position with TAR RNA HIV-1

The Tat wild-type fragment of sequence Arg⁴⁹-Lys-Lys-Arg⁵²-Arg-Gln-Arg-Arg-Arg⁵⁷-NH₂ (labelled as Tat1) and three analogues of this fragment with the substitution Arg⁵² D-Arg⁵² (labelled as Tat2) or L-citrulline (Cit) (labelled as Tat3) or L-ornithine (Orn) (labelled as Tat4) were synthesized to study Tat-TAR RNA HIV-1 (27-nucleotide fragment of sequence 5-AGAUCUGAGCCUGGAGCUCUCU-3) interactions by circular dichroism. -helical structure was the most readily adopted by the Tat3 analogue with Arg⁵² Cit substitution. All the peptides investigated caused conformational changes in the TAR structure. The most dramatic changes were observed for the Tat2-TAR complex.     

Two-Dimensional 1H NMR Study of a Tetradecapeptide with the Consensus Sequence Arg5-Asp-Val-Arg-Gly9: Structural Effects of the Outside Substitution Ser12 by Ala12

Abstract

Conformation of a tetradecapeptide with a RXVRG consensus sequence, Arg^s-Asp-Val-Arg-Gly⁹, found in several precursors of antibacterian peptides, was investigated in dimethylsulfoxide solution by proton NMR spectroscopy. Complete resonance assignments and conformational parameters were obtained through correlated (COSY) and nuclear Overhauser (NOESY) techniques. The ³J(αH, βH) coupling constants and the intramolecular NOE, NH…βH, were used to analyse the conformers around the Cα-Cβ bond and, in four cases, to obtain stereospecific assignments.

Use of restraints derived from NOE connectivities and ³J(NH, αH) coupling constants allows the determination of a range of φ and ψ dihedral angles for all the residues in the sequence. The present NMR results provide favourable evidence for the formation of two bends in the consensus sequence of the tetradecapeptide. The first one has most of the features of a Glu⁴- Val⁷ β–turn (low temperature coefficient of the Val⁷NH chemical shift, Arg⁵αH…Val⁷NH and Asp⁶NH.-.Val⁷NH NOE correlations). The second one exhibits only the Asp⁶αH…Arg⁷NH and Val⁷NH…Arg⁸NH NOE interactions. These consensus sequence organizations proposed were confirmed by molecular modeling based on low potential energy structure on the [4–9] fragment with high agreement of NOE data.

Overall, the substitution of Ser¹² by Ala¹² shifts the conformation of the hydrophobic moiety [10–14] towards a quite random coil structure in this fragment and strongly destabilizes the folded structures of the consensus domain where only one NH (Val⁷) is solvent-shielded opposed to three (Asp⁶ to Arg⁸) in the [Ser¹²] tetradecapeptide. These conformational changes could be related to the processing enzyme activities on these model oligopeptides.     

Two amino acid changes in the R3 repeat cause functional divergence of two clustered <Emphasis Type="Italic">MYB10</Emphasis> genes in peach

R2R3-MYB genes play a pivotal role in regulating anthocyanin accumulation. Here, we report two tandemly duplicated R2R3-MYB genes in peach, PpMYB10.1 and PpMYB10.2, with the latter showing lower ability to induce anthocyanin accumulation than the former. Site-directed mutation assay revealed two amino acid changes in the R3 repeat, Arg/Lys⁶⁶ and Gly/Arg⁹³, responsible for functional divergence between these two PpMYB10 genes. Anthocyanin-promoting activity of PpMYB10.2 was significantly increased by a single amino acid replacement of Arg⁹³ with Gly⁹³. However, either the Gly⁹³ → Arg⁹³ or Arg⁶⁶ → Lys⁶⁶ substitutions alone showed little impact on anthocyanin-promoting activity of PpMYB10.1, but simultaneous substitutions caused a significant decrease. Reciprocal substitution of Arg/Gly⁹³ could significantly alter binding affinity to PpbHLH3, while the Arg⁶⁶ → Lys⁶⁶ substitution is predicted to affect the folding of the MYB DNA-binding domain, instead of PpbHLH3-binding affinity. Overall, the change of anthocyanin-promoting activity was accompanied with that of bHLH-binding affinity, suggesting that DNA-binding affinity of R2R3-MYBs depends on their bHLH partners. Our study is helpful for understanding of functional evolution of R2R3-MYBs and their interaction with DNA targets.     

Unlike in other monocotyledonous plants such as wheat and rice, the region containing the tRNA (UCC), tRNA (UCU) and CF1 ATPase α-subunit genes has not undergone recombination in the leek chloroplast DNA

The nucleotide sequence of a 1.1 kbp BamHI fragment of the leek chloroplast DNA (Allium porrum., fam. Liliaceae) has been determined. The fragment contains the 3' part of the tRNA^Gly (UCC) gene and the tRNA^Arg (UCU) gene on the same strand, and the 3' end of the atpA gene encoding the CF₁ ATPase α-subunit which is located on the opposite strand. The gene arrangement and nucleotide sequence of this fragment are similar to those of the corresponding region in the tobacco chloroplast DNA but differ significantly from what has been observed in other monocotyledonous plants such as wheat and rice, in which the region containing these genes has undergone intensive rearrangement.     

High-Field NMR and Circular Dichroism Solvent-Dependent Conformational Studies of the Bradykinin C-Terminal Tetrapeptide Ser-Pro-Phe-Arg

Abstract

The conformational properties of the tetrapeptide Ser¹-Pro²-Phe³-Arg⁴, the C-terminal fragment of the nonapeptide hormone bradykinin, have been studied by circular dichroism and two-dimensional NMR techniques. Measurements of coupling constants, NH temperature dependence rates and nuclear Overhauser effects (performed with rotating frame nuclear Overhauser spectroscopy, ROESY) in H₂O and CD₃OH/D₂O (80/20, v/v) reveal different conformations in the corresponding solvent. In aqueous solution the molecule exists in a random conformation or as an average of several conformations in rapid exchange. In CD₃OH/D₂O, however, the conformation is well-defined. The backbone of the peptide is extended, and the side-chains of Phe³ and Arg⁴ exhibit unusual rigidity for a peptide of this size. Evidently, the secondary structure is stabilized by a charge interaction between the guanidino group of Arg⁴ and the terminal carboxyl group, since experiments at various pH's show clearly that the definition of conformation decreases strongly upon protonation of the carboxyl function. A NH₃ ⁺(Ser¹)-COO^?(Arg⁴) salt bridge, as well as any form of turn stabilized by hydrogen bonds can be ruled out with certainty.     

The amino acid sequence of goat alpha-lactalbumin

The amino acid sequence of goat α-lactalbumin has been established from the structures of peptides isolated from trypsin and thermolysin digests of the reduced aminoethylated protein and from a chymotrypsin digest of the reduced carboxamidomethylated protein. The amino-terminal sequence was confirmed by automatic sequencer analysis. Of the previously sequenced species variants of α-lactalbumin, the bovine protein is most similar to the goat, differing in only 12 amino acid substitutions. One difference between these proteins corresponds to a substitution found in the bovine A genetic variant (Arg¹⁰ → Gln). The relevance of the structure to the evolutionary relationships in the α-lactalbumin-lysozyme family of proteins is discussed.     

Biophysical Investigations of Complement Receptor 2 (CD21 and CR2)-Ligand Interactions Reveal Amino Acid Contacts Unique to Each Receptor-Ligand Pair

Human complement receptor type 2 (CR2 and CD21) is a cell membrane receptor, with 15 or 16 extracellular short consensus repeats (SCRs), that promotes B lymphocyte responses and bridges innate and acquired immunity. The most distally located SCRs, SCR1–2, mediate the interaction of CR2 with its four known ligands (C3d, EBV gp350, IFNα, and CD23). To ascertain specific interacting residues on CR2, we utilized NMR studies wherein gp350 and IFNα were titrated into ¹⁵N-labeled SCR1–2, and chemical shift changes indicative of specific inter-molecular interactions were identified. With backbone assignments made, the chemical shift changes were mapped onto the crystal structure of SCR1–2. With regard to gp350, the binding region of CR2 is primarily focused on SCR1 and the inter-SCR linker, specifically residues Asn¹¹, Arg¹³, Ala²², Arg²⁸, Ser³², Arg³⁶, Lys⁴¹, Lys⁵⁷, Tyr⁶⁴, Lys⁶⁷, Tyr⁶⁸, Arg⁸³, Gly⁸⁴, and Arg⁸⁹. With regard to IFNα, the binding is similar to the CR2-C3d interaction with specific residues being Arg¹³, Tyr¹⁶, Arg²⁸, Ser⁴², Lys⁴⁸, Lys⁵⁰, Tyr⁶⁸, Arg⁸³, Gly⁸⁴, and Arg⁸⁹. We also report thermodynamic properties of each ligand-receptor pair determined using isothermal titration calorimetry. The CR2-C3d interaction was characterized as a two-mode binding interaction with K_d values of 0.13 and 160 μm, whereas the CR2-gp350 and CR2-IFNα interactions were characterized as single site binding events with affinities of 0.014 and 0.035 μm, respectively. The compilation of chemical binding maps suggests specific residues on CR2 that are uniquely important in each of these three binding interactions.     

Scope and limitations of the simple 13C-n.m.r. method of structural analysis of carbohydrates: glucodisaccharides

The scope and limitations of the SIMPLE n.m.r. method (secondary isotope multiplet n.m.r. spectroscopy of partially labelled entities) has been investigated for a series of glucodisaccharies. ¹³C-SIMPLE n.m.r. measurements have been made on solutions of (1→1)- (α,α-trehalose), (1→2)- (sophorose and kojibiose), (1→3)- (laminaribiose), and (1→6)-linked (gentiobiose and isomaltose) glucodisaccharides in (CD₃)₂SO and the results combined with those previously published for (1→4)-linked analogues (maltose and cellobiose). Each linkage (and substitution) type gives rise to a unique pattern of ¹³C isotopomers which, in principle, may be used for complete assignment of the spectra and structural analysis of the molecule. The glucodisaccharides are difficult to analyse, compared with other disaccharides, because the presence of two glucose moieties leads to degeneracies of a few isotopic multiplets which cannot be differentiated by the magnitudes of the isotope effects. Assignments in aqueous solutions were obtained by using the DIS (differential isotope shift) n.m.r. method in conjunction with the results from SIMPLE n.m.r. In practice, nearly all of the signals can be assigned unequivocally and the remaining signals are choices between two possible assignments.     

Exploring the peptide 310-helix ⇆ α–helix equilibrium with double label electron spin resonance

Over the last several years we have used spin labeling as a means for exploring the structure of helical peptides. Two nitroxide labels are engineered into a peptide sequence and distances are ranked with electron spin resonance (ESR). We have found that there is a significant amount of 3₁₀–helix in 16–residue model peptides containing only L –amino acids. This review covers several facets of the methodology including spin labeling strategy, interpretation of ESR spectra and the influence of molecular dynamics on the spectral line shapes. Also covered are recent findings of a length–dependent 3_l0-helix → α-helix transition and the role of Arg⁺ in the stabilization of specific helix structures. © 1994 John Wiley & Sons, Inc.     

Synthesis and DNA-binding ability of Sp1 protein zinc finger domain and its peptidomimetics

The second zinc finger fragment of Sp1 (Sp1-ZF2), its mutant (Sp1-ZF2/HT. E²⁰ → H, R²³ → T), and two mimic analogues (ZF20 and ZF15) were synthesized by stepwise solid phase technique. The CD spectra and UV-visible spectrum with CoC1₂ indicated that the formation of zinc finger structure was affected not only by the hydrophobic amino acids but also by the change of the distance between Cys and His. Gel-retardat ion electrophoresis assays indicated that the Glu and Arg residues are very important for recognition. A single zinc finger like Sp1-ZF2 is able to bind DNA sequence specifically.     

A Sequential Mechanism for Exosite-mediated Factor IX Activation by Factor XIa

During blood coagulation, the protease factor XIa (fXIa) activates factor IX (fIX). We describe a new mechanism for this process. FIX is cleaved initially after Arg¹⁴⁵ to form fIXα, and then after Arg¹⁸⁰ to form the protease fIXaβ. FIXα is released from fXIa, and must rebind for cleavage after Arg¹⁸⁰ to occur. Catalytic efficiency of cleavage after Arg¹⁸⁰ is 7-fold greater than for cleavage after Arg¹⁴⁵, limiting fIXα accumulation. FXIa contains four apple domains (A1–A4) and a catalytic domain. Exosite(s) on fXIa are required for fIX binding, however, there is lack of consensus on their location(s), with sites on the A2, A3, and catalytic domains described. Replacing the A3 domain with the prekallikrein A3 domain increases K_m for fIX cleavage after Arg¹⁴⁵ and Arg¹⁸⁰ 25- and ≥90-fold, respectively, and markedly decreases k_cat for cleavage after Arg¹⁸⁰. Similar results were obtained with the isolated fXIa catalytic domain, or fXIa in the absence of Ca²⁺. Forms of fXIa lacking the A3 domain exhibit 15-fold lower catalytic efficiency for cleavage after Arg¹⁸⁰ than for cleavage after Arg¹⁴⁵, resulting in fIXα accumulation. Replacing the A2 domain does not affect fIX activation. The results demonstrate that fXIa activates fIX by an exosite- and Ca²⁺-mediated release-rebind mechanism in which efficiency of the second cleavage is enhanced by conformational changes resulting from the first cleavage. Initial binding of fIX and fIXα requires an exosite on the fXIa A3 domain, but not the A2 or catalytic domain.     

N Terminus Is Key to the Dominant Negative Suppression of CaV2 Calcium Channels: IMPLICATIONS FOR EPISODIC ATAXIA TYPE 2*

Expression of the calcium channels Ca_V2.1 and Ca_V2.2 is markedly suppressed by co-expression with truncated constructs containing Domain I. This is the basis for the phenomenon of dominant negative suppression observed for many of the episodic ataxia type 2 mutations in Ca_V2.1 that predict truncated channels. The process of dominant negative suppression has been shown previously to stem from interaction between the full-length and truncated channels and to result in downstream consequences of the unfolded protein response and endoplasmic reticulum-associated protein degradation. We have now identified the specific domain that triggers this effect. For both Ca_V2.1 and Ca_V2.2, the minimum construct producing suppression was the cytoplasmic N terminus. Suppression was enhanced by tethering the N terminus to the membrane with a CAAX motif. The 11-amino acid motif (including Arg⁵² and Arg⁵⁴) within the N terminus, which we have previously shown to be required for G protein modulation, is also essential for dominant negative suppression. Suppression is prevented by addition of an N-terminal tag (XFP) to the full-length and truncated constructs. We further show that suppression of Ca_V2.2 currents by the N terminus-CAAX construct is accompanied by a reduction in Ca_V2.2 protein level, and this is also prevented by mutation of Arg⁵² and Arg⁵⁴ to Ala in the truncated construct. Taken together, our evidence indicates that both the extreme N terminus and the Arg⁵², Arg⁵⁴ motif are involved in the processes underlying dominant negative suppression.     

One- and Two-Dimensional lH NMR Study of the Substance P Fragment ARG-PRO-LYS-PRO

Abstract

The Substance P fragment Arg¹Pro²-Lys³-Pro⁴ (SP_1–4) has been extensively investigated by means of proton nuclear magnetic resonance at 400 MHz. The combined application of different 2D techniques and a comparison of SP_1–4 with its derivative SPM-amide allowed the complete and unambiguous assignment of the proton NMR spectrum. Conformational data obtained from the different NMR parameters are compared with theoretical calculations. The results suggest that SP_1–4 exists, at the chosen experimental conditions, as a stretched molecule.     

Inhibition of Tryptase TL2 from Human T4+ Lymphocytes and Inhibition of HIV-1 Replication in H9 Cells by Recombinant Aprotinin and Bikunin Homologues

The serine esterase TL₂ from human T4+ lymphocytes is a binding component to HIV-1 glycoprotein gp120 and seems to play a role in the HIV-1 infection mechanism. Recombinant variants of the Kunitz-type serine proteinase inhibitor aprotinin were investigated for their ability to inhibit tryptase TL₂ and the binding of gp120 to this enzyme. Furthermore, the viral replication of HIV-1 was investigated in H9 cell cultures under the influence of recombinant aprotinin and bikunin variants. In contrast to native aprotinin, the recombinant variant [Arg¹⁵, Phe¹⁷, Glu⁵²]aprotinin with a reactive-site sequence homologous to the V3 loop of HIV-1 gp120 showed a specific inhibition of tryptase TL₂ (>80%). However, the [Leu¹⁵, Phe¹⁷, Glu⁵²]aprotinin variant with hydrophobic subsites was the most potent inhibitor of the binding of gp120 to tryptase TL₂ (68%). Our results show that the enzyme activity of purified tryptase TL₂ is inhibited not only by variants with basic amino acids, but also those with hydrophobic residues in the reactive-site region. Therefore, tryptase TL₂ is not a typical trypsin-like or chymotrypsin-like protease. Investigations on inhibition of HIV-1 replication in H9 cell cultures showed that tryptase TL₂ is involved in the mechanism of virus internalization into human lymphocytes. The [Leu¹⁵, Phe¹⁷, Glu⁵²]aprotinin showed a significant retardation of syncytium formation over a period of 5 days in a 1 μM concentration. Similar investigations were performed with recombinant variants of bikunin, the light chain of human inter-α-trypsin inhibitor. Only the single-headed variant [Arg⁹⁴]⁸²bikunin inhibited slightly the syncytium formation over a period of 2 days in a 2.2 μM concentration. Wild-type bikunin and all full-length variants showed no effect, possibly due to steric hindrance by the second domain of the double-headed inhibitor.     

The binding of Arg- and Lys-peptides to single standed polyribonucleotides and its effect on the polymer conformation

The interactions between basic oligopeptides (Lys₂, Lys₃, Arg₂, and Arg₃) and single stranded polynucleotides (poly(A), poly(C), poly(I) and poly(U) were investigated at low ion concentration by UV spectroscopy, circular dichroism and field jump relaxation. Various domains of binding were detected: 1) High concentrations (up to 1 mM) of some peptides induce opalescencs followed by coacervation- Arg₃ causes coacervation in all polynucleotides used, yet Lys₃ only in poly(I). In the case of poly(I) the threshold concentration for coacervation is much lower for Arg₃ (150 μM) than for Lys₃ (500 μM). 2) Medium concentrations (?10 μM) of Arg₃ and Lys₃ induce helix formation in poly(U). In the case of poly(I) cooperative helix formation is only induced by Lys₃, but not by Arg₃. 3) The onset of peptide association is observed at very low peptide concentrations (?1 μM) already by using the field jump method. The association is reflected by a relaxation process, that can be described by a single exponential within experimental accuracy. Measurements of relaxation time constants as a function of the peptide concentration provide information on the association constants K, the number of nucleotide residues per binding place n and the rate constants k_R and k_D. Using a simple model with independent and “separate” binding sites, K for Arg₃ and Lys₃ is found to be in the range of 10⁶ to 10⁷ M^?1. In the case of Arg₂ and K is lower by a factor of about 10. For various polynucleotides K_Arg3 is slightly higher than K_Lys3. except in the case of poly(I), where K_Arg3/K_Lys3 ≈ 5. Similar data are obtained by application of a “sphere model” (see below). These results provide quantitative evidence for specific hydrogen bonding between the guanidino group of Arg and inosine. They also explain the absence of helix formation for poly(I) + Arg₃: Arg blocks the hydrogen bonding sites of inosine. Thus cooperative coupling leads in this case to a considerable amplification of specificity in the peptide-polynucleotide interaction Both field jump and stopped flow data demonstrate a high mobility of the peptide lisands along the polymer, resulting in a redistribution being fast compared with the overall binding step. Based on this result the relaxation data are analysed by a “sphere” model, which considers a) excluded binding under the condition of fast Ugand distribution along the lattice and b) the connection of sites into a polymer sphere. The rate constants obtained by this model are in the range of 4 × 10¹¹ M^?1 s^?1. These high values reflect the large reaction distance for polymers of chain lengths around 1000. A comparison with rate constants obtained previously for oligomer complexes indicates that the recombination rate is approximately a function of the square root of the nucleotide chain length, which is directly related to the mean radius of coiled polymers.     

Pressure-Induced Endocytic Degradation of the Saccharomyces cerevisiae Low-Affinity Tryptophan Permease Tat1 Is Mediated by Rsp5 Ubiquitin Ligase and Functionally Redundant PPxY Motif Proteins

Cells of Saccharomyces cerevisiae express two tryptophan permeases, Tat1 and Tat2, which have different characteristics in terms of their affinity for tryptophan and intracellular localization. Although the high-affinity permease Tat2 has been well documented in terms of its ubiquitin-dependent degradation, the low-affinity permease Tat1 has not yet been characterized fully. Here we show that a high hydrostatic pressure of 25 MPa triggers a degradation of Tat1 which depends on Rsp5 ubiquitin ligase and the EH domain-containing protein End3. Tat1 was resistant to a 3-h cycloheximide treatment, suggesting that it is highly stable under normal growth conditions. The ubiquitination of Tat1 most likely occurs at N-terminal lysines 29 and 31. Simultaneous substitution of arginine for the two lysines prevented Tat1 degradation, but substitution of either of them alone did not, indicating that the roles of lysines 29 and 31 are redundant. When cells were exposed to high pressure, Tat1-GFP was completely lost from the plasma membrane, while substantial amounts of Tat1^K29R-K31R-GFP remained. The HPG1-1 (Rsp5^P514T) and rsp5-ww3 mutations stabilized Tat1 under high pressure, but any one of the rsp5-ww1, rsp5-ww2, and bul1Δ bul2Δ mutations or single deletions of genes encoding arrestin-related trafficking adaptors did not. However, simultaneous loss of 9-arrestins and Bul1/Bul2 prevented Tat1 degradation at 25 MPa. The results suggest that multiple PPxY motif proteins share some essential roles in regulating Tat1 ubiquitination in response to high hydrostatic pressure.     

Dissecting a role of a charge and conformation of Tat2 peptide in allosteric regulation of 20S proteasome

Proteasome is a ‘proteolytic factory’ that constitutes an essential part of the ubiquitin‐proteasome pathway. The involvement of proteasome in regulation of all major aspects of cellular physiology makes it an attractive drug target. So far, only inhibitors of the proteasome entered the clinic as anti‐cancer drugs. However, proteasome regulators may also be useful for treatment of inflammatory and neurodegenerative diseases. We established in our previous studies that the peptide Tat2, comprising the basic domain of HIV‐1 Tat protein: R⁴⁹KKRRQRR⁵⁶, supplemented with Q⁶⁶DPI⁶⁹ fragment, inhibits the 20S proteasome in a noncompetitive manner. Mechanism of Tat2 likely involves allosteric regulation because it competes with the proteasome natural 11S activator for binding to the enzyme noncatalytic subunits. In this study, we performed alanine walking coupled with biological activity measurements and FTIR and CD spectroscopy to dissect contribution of a charge and conformation of Tat2 to its capability to influence peptidase activity of the proteasome. In solution, Tat2 and most of its analogs with a single Ala substitution preferentially adopted a conformation containing PPII/turn structural motifs. Replacing either Asp10 or two or more adjacent Arg/Lys residues induced a random coil conformation, probably by disrupting ionic interactions responsible for stabilization of the peptides ordered structure. The random coil Tat2 analogs lost their capability to activate the latent 20S proteasome. In contrast, inhibitory properties of the peptides more significantly depended on their positive charge. The data provide valuable clues for the future optimization of the Tat2‐based proteasome regulators. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.