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Oral presentations Background: We selected peptide ligands for the HIV-1 packaging signal PSI by screening phage displayed peptide libraries. Peptide ligands were optimized by screening spot synthesis peptide membranes. The aim of this study is the functional characterization of these peptide ligands with respect to inhibition of HIV-1 replication. Methods: Phage displayed peptide libraries were screened with PSI-RNA structures. The Trp-rich peptide motifs were optimized for specific binding on spot synthesis peptide membranes. The best binding peptide was expressed intracellularly in fusion with RFP or linked to a protein transduction domain (PTD) for intracellular delivery. The effects on virion production were analyzed using pseudotyped lentiviral particles. Results: After positive and negative selection rounds, phages binding specifically to PSI-RNA were identified by ELISA. Peptide inserts contained conserved motifs of aromatic amino acids known to be implicated in binding of PSI-RNA by the natural Gag ligand. The filter assay identified HKWPWW as the best binding ligand for PSI-RNA, which is delivered into several cell lines by addition of a PTD. Compared to a control peptide, the HKWPWW peptide inhibited HIV-1 replication as deduced from reduced titers of culture supernatants. As HKWPWW also binds to the TAR-RNA like the natural nucleocapsid PSI-RNA ligand, the effect on Tat-TAR inhibition will also be analyzed. Currently T-cell lines are established which stably express HKWPWW as well as a control peptide, which will be infected with HIV-1 to monitor the ability of HKWPWW to inhibit wild type HIV-1 replication. Conclusion: The selection of a peptide ligand for PSI-RNA able to inhibit HIV-1 replication proves the suitability of the phage display technology for the selection of peptides binding to RNA-structures. This enables the indentification of peptides serving as leads to interfere with additional targets in the HIV-1 replication cycle.
Ribosomal proteins are assumed to stabilize specific RNA structures and promote compact folding of the large rRNA. The conformational dynamics of the protein between the bound and unbound state play an important role in the binding process. We have studied those dynamical changes in detail for the highly conserved complex between the ribosomal protein L11 and the GTPase region of 23S rRNA. The RNA domain is compactly folded into a well defined tertiary structure, which is further stabilized by the association with the C-terminal domain of the L11 protein (L11ctd). In addition, the N-terminal domain of L11 (L11ntd) is implicated in the binding of the natural thiazole antibiotic thiostrepton, which disrupts the elongation factor function. We have studied the conformation of the ribosomal protein and its dynamics by NMR in the unbound state, the RNA bound state and in the ternary complex with the RNA and thiostrepton. Our data reveal a rearrangement of the L11ntd, placing it closer to the RNA after binding of thiostrepton, which may prevent binding of elongation factors. We propose a model for the ternary L11–RNA–thiostrepton complex that is additionally based on interaction data and conformational information of the L11 protein. The model is consistent with earlier findings and provides an explanation for the role of L11ntd in elongation factor binding.
Metal-ion binding and metal-ion induced folding of the adenine-sensing riboswitch aptamer domain
(2007)
Divalent cations are important in the folding and stabilization of complex RNA structures. The adenine-sensing riboswitch controls the expression of mRNAs for proteins involved in purine metabolism by directly sensing intracellular adenine levels. Adenine binds with high affinity and specificity to the ligand binding or aptamer domain of the adenine-sensing riboswitch. The X-ray structure of this domain in complex with adenine revealed an intricate RNA-fold consisting of a three-helix junction stabilized by long-range base-pairing interactions and identified five binding sites for hexahydrated Mg2+-ions. Furthermore, a role for Mg2+-ions in the ligand-induced folding of this RNA was suggested. Here, we describe the interaction of divalent cations with the RNA–adenine complex in solution as studied by high-resolution NMR spectroscopy. Paramagnetic line broadening, chemical shift mapping and intermolecular nuclear Overhauser effects (NOEs) indicate the presence of at least three binding sites for divalent cations. Two of them are similar to those in the X-ray structure. The third site, which is important for the folding of this RNA, has not been observed previously. The ligand-free state of the RNA is conformationally heterogeneous and contains base-pairing patterns detrimental to ligand binding in the absence of Mg2+, but becomes partially pre-organized for ligand binding in the presence of Mg2+. Compared to the highly similar guanine-sensing riboswitch, the folding pathway for the adenine-sensing riboswitch aptamer domain is more complex and the influence of Mg2+ is more pronounced.
We have investigated the mechanism responsible for half-of-the-sites activity in the dimeric cytochrome bc(1) complex from Paracoccus denitrificans by characterizing the kinetics of inhibitor binding to the ubiquinol oxidation site at center P. Both myxothiazol and stigmatellin induced a 2-3 nm shift of the visible absorbance spectrum of the b(L) heme. The shift generated by myxothiazol was symmetric, with monophasic kinetics that indicate equal binding of this inhibitor to both center P sites. In contrast, stigmatellin generated an asymmetric shift in the b(L) spectrum, with biphasic kinetics in which each phase contributed approximately half of the total magnitude of the spectral change. The faster binding phase corresponded to a more symmetrical shift of the b(L) spectrum relative to the slower binding phase, indicating that approximately half of the center P sites bound stigmatellin more slowly and in a different position relative to the b(L) heme, generating a different effect on its electronic environment. Significantly, the slow stigmatellin binding phase was lost as the inhibitor concentration was increased. This implies that a conformational change is transmitted from one center P site in the dimer to the other upon stigmatellin binding to one monomer, rendering the second site less accessible to the inhibitor. Because the position that stigmatellin occupies at center P is considered to be analogous to that of the quinol substrate at the moment of electron transfer, these results indicate that the productive enzyme-substrate configuration is prevented from occurring in both monomers simultaneously.
Bovine mitochondrial ATP synthase commonly is isolated as a monomeric complex that contains 16 protein subunits and the natural IF1 inhibitor protein in substoichiometric amounts. Alternatively ATP synthase can be isolated in dimeric and higher oligomeric states using digitonin for membrane solubilization and blue native or clear native electrophoresis for separation of the native mitochondrial complexes. Using blue native electrophoresis we could identify two ATP synthase-associated membrane proteins with masses smaller than 7 kDa and isoelectric points close to 10 that previously had been removed during purification. We show that in the mitochondrial membrane both proteins are almost quantitatively bound to ATP synthase. Both proteins had been identified earlier in a different context, but their association with ATP synthase was unknown. The first one had been named 6.8-kDa mitochondrial proteolipid because it can be isolated by chloroform/methanol extraction from mitochondrial membranes. The second one had been denoted as diabetes-associated protein in insulin-sensitive tissue (DAPIT), which may provide a clue for further functional and clinical investigations.
In a combined NMR/MD study, the temperature-dependent changes in the conformation of two members of the RNA YNMG-tetraloop motif (cUUCGg and uCACGg) have been investigated at temperatures of 298, 317 and 325 K. The two members have considerable different thermal stability and biological functions. In order to address these differences, the combined NMR/MD study was performed. The large temperature range represents a challenge for both, NMR relaxation analysis (consistent choice of effective bond length and CSA parameter) and all-atom MD simulation with explicit solvent (necessity to rescale the temperature). A convincing agreement of experiment and theory is found. Employing a principle component analysis of the MD trajectories, the conformational distribution of both hairpins at various temperatures is investigated. The ground state conformation and dynamics of the two tetraloops are indeed found to be very similar. Furthermore, both systems are initially destabilized by a loss of the stacking interactions between the first and the third nucleobase in the loop region. While the global fold is still preserved, this initiation of unfolding is already observed at 317 K for the uCACGg hairpin but at a significantly higher temperature for the cUUCGg hairpin.
Mitochondrial complex I (NADH:ubiquinone oxidoreductase) undergoes reversible deactivation upon incubation at 30–37 °C. The active/deactive transition could play an important role in the regulation of complex I activity. It has been suggested recently that complex I may become modified by S-nitrosation under pathological conditions during hypoxia or when the nitric oxide:oxygen ratio increases. Apparently, a specific cysteine becomes accessible to chemical modification only in the deactive form of the enzyme. By selective fluorescence labeling and proteomic analysis, we have identified this residue as cysteine-39 of the mitochondrially encoded ND3 subunit of bovine heart mitochondria. Cysteine-39 is located in a loop connecting the first and second transmembrane helix of this highly hydrophobic subunit. We propose that this loop connects the ND3 subunit of the membrane arm with the PSST subunit of the peripheral arm of complex I, placing it in a region that is known to be critical for the catalytic mechanism of complex I. In fact, mutations in three positions of the loop were previously reported to cause Leigh syndrome with and without dystonia or progressive mitochondrial disease.
Type I interferons (IFNs) signal for their diverse biological effects by binding a common receptor on target cells, composed of the two transmembrane IFNAR1 and IFNAR2 proteins. We have previously differentially enhanced the antiproliferative activity of IFN by increasing the weak binding affinity of IFN to IFNAR1. In this study, we further explored the affinity interdependencies between the two receptor subunits and the role of IFNAR1 in differential IFN activity. For this purpose, we generated a panel of mutations targeting the IFNAR2 binding site on the background of the IFNalpha2 YNS mutant, which increases the affinity to IFNAR1 by 60-fold, resulting in IFNAR2-to-IFNAR1 binding affinity ratios ranging from 1000:1 to 1:1000. Both the antiproliferative and antiviral potencies of the interferon mutants clearly correlated to the in situ binding IC(50) values, independently of the relative contributions of the individual receptors, thus relating to the integral lifetime of the complex. However, the antiproliferative potency correlated throughout the entire range of affinities, as well as with prolonged IFNAR1 receptor down-regulation, whereas the antiviral potency reached a maximum at binding affinities equivalent to that of wild-type IFNalpha2. Our data suggest that (i) the specific activity of interferon is related to the ternary complex binding affinity and not to affinity toward individual receptor components and (ii) although the antiviral pathway is strongly dependent on pSTAT1 activity, the cytostatic effect requires additional mechanisms that may involve IFNAR1 down-regulation. This differential interferon response is ultimately mediated through distinct gene expression profiling.
High-resolution NMR structure of an RNA model system : the 14-mer cUUCGg tetraloop hairpin RNA
(2009)
We present a high-resolution nuclear magnetic resonance (NMR) solution structure of a 14-mer RNA hairpin capped by cUUCGg tetraloop. This short and very stable RNA presents an important model system for the study of RNA structure and dynamics using NMR spectroscopy, molecular dynamics (MD) simulations and RNA force-field development. The extraordinary high precision of the structure (root mean square deviation of 0.3 Å) could be achieved by measuring and incorporating all currently accessible NMR parameters, including distances derived from nuclear Overhauser effect (NOE) intensities, torsion-angle dependent homonuclear and heteronuclear scalar coupling constants, projection-angle-dependent cross-correlated relaxation rates and residual dipolar couplings. The structure calculations were performed with the program CNS using the ARIA setup and protocols. The structure quality was further improved by a final refinement in explicit water using OPLS force field parameters for non-bonded interactions and charges. In addition, the 2'-hydroxyl groups have been assigned and their conformation has been analyzed based on NOE contacts. The structure currently defines a benchmark for the precision and accuracy amenable to RNA structure determination by NMR spectroscopy. Here, we discuss the impact of various NMR restraints on structure quality and discuss in detail the dynamics of this system as previously determined.
We previously proposed that the dimeric cytochrome bc(1) complex exhibits half-of-the-sites reactivity for ubiquinol oxidation and rapid electron transfer between bc(1) monomers (Covian, R., Kleinschroth, T., Ludwig, B., and Trumpower, B. L. (2007) J. Biol. Chem. 282, 22289-22297). Here, we demonstrate the previously proposed half-of-the-sites reactivity and intermonomeric electron transfer by characterizing the kinetics of ubiquinol oxidation in the dimeric bc(1) complex from Paracoccus denitrificans that contains an inactivating Y147S mutation in one or both cytochrome b subunits. The enzyme with a Y147S mutation in one cytochrome b subunit was catalytically fully active, whereas the activity of the enzyme with a Y147S mutation in both cytochrome b subunits was only 10-16% of that of the enzyme with fully wild-type or heterodimeric cytochrome b subunits. Enzyme with one inactive cytochrome b subunit was also indistinguishable from the dimer with two wild-type cytochrome b subunits in rate and extent of reduction of cytochromes b and c(1) by ubiquinol under pre-steady-state conditions in the presence of antimycin. However, the enzyme with only one mutated cytochrome b subunit did not show the stimulation in the steady-state rate that was observed in the wild-type dimeric enzyme at low concentrations of antimycin, confirming that the half-of-the-sites reactivity for ubiquinol oxidation can be regulated in the wild-type dimer by binding of inhibitor to one ubiquinone reduction site.