Interaction of porphyrin with G-quadruplex structures

Isothermal titration calorimetry (ITC) is a sensitive technique for probing bimolecular processes and can provide direct information about the binding affinity and stoichiometry and the key thermodynamic parameters involved. ITC has been used to investigate the interaction of the ligand H2TMPyP to the two DNA quadruplexes, [d(AGGGT)]4 and [d(TGGGGT)]. Analysis of the ITC data reveals that porphyrin/quadruplex binding stoichiometry under saturating conditions is 1:2 for [d(AGGGT)]4 and 2:1 for [d(TGGGGT)], respectively.     

Interaction with C4b-binding protein contributes to nontypeable Haemophilus influenzae serum resistance

Complement evasion by various mechanisms is important for microbial virulence and survival in the host. One strategy used by some pathogenic bacteria is to bind the complement inhibitor of the classical pathway, C4b-binding protein (C4BP). In this study, we have identified a novel interaction between nontypeable Haemophilus influenzae (NTHi) and C4BP, whereas the majority of the typeable H. influenzae (a-f) tested showed no binding. One of the clinical isolates, NTHi 506, displayed a particularly high binding of C4BP and was used for detailed analysis of the interaction. Importantly, a low C4BP-binding isolate (NTHi 69) showed an increased deposition of C3b followed by reduced survival as compared with NTHi 506 when exposed to normal human serum. The main isoform of C4BP contains seven identical alpha-chains and one beta-chain linked together with disulfide bridges. Each alpha-chain is composed of eight complement control protein (CCP) modules and we have found that the NTHi 506 strain did not interact with rC4BP lacking CCP2 or CCP7 showing that these two CCPs are important for the binding. Importantly, C4BP bound to the surface of H. influenzae retained its cofactor activity as determined by analysis of C3b and C4b degradation. Taken together, NTHi interferes with the classical complement activation pathway by binding to C4BP.     

Interaction of Wnt-1 proteins with the binding protein BiP.

The mouse Wnt-1 gene, a target for insertional activation in mouse mammary tumor virus-induced mammary tumors, encodes poorly secreted, cysteine-rich glycoproteins required for proper central nervous system development. We have been analyzing the biosynthesis of Wnt-1 proteins in several cell lines that express Wnt-1 cDNA from heterologous promoters. A protein of 78 kDa was found to be associated with the intracellular forms of Wnt-1 proteins in mammalian and avian cells by using multiple antisera against Wnt-1 proteins. We have identified p78 as the binding protein BiP with anti-BiP antisera and by its release from Wnt-1 immunoprecipitates upon incubation with MgCl2 and ATP. Experiments with a Wnt-1 mutant that lacks the sequence encoding the signal peptide indicates that Wnt-1 proteins must enter the secretory pathway in order to interact with BiP. We demonstrate that Wnt-1 proteins are associated with BiP in cells in which active Wnt-1 proteins are produced, such as a cultured mammary epithelial cell line and Wnt-1 transgenic mouse mammary tumor cells. The association of Wnt-1 proteins with BiP may be a factor in determining the efficiency of secretion of Wnt-1 gene products.     

Interaction of the endocytic scaffold protein Pan1 with the type I myosins contributes to the late stages of endocytosis

The yeast endocytic scaffold Pan1 contains an uncharacterized proline-rich domain (PRD) at its carboxy (C)-terminus. We report that the pan1-20 temperature-sensitive allele has a disrupted PRD due to a frame-shift mutation in the open reading frame of the domain. To reveal redundantly masked functions of the PRD, synthetic genetic array screens with a pan1DeltaPRD strain found genetic interactions with alleles of ACT1, LAS17 and a deletion of SLA1. Through a yeast two-hybrid screen, the Src homology 3 domains of the type I myosins, Myo3 and Myo5, were identified as binding partners for the C-terminus of Pan1. In vitro and in vivo assays validated this interaction. The relative timing of recruitment of Pan1-green fluorescent protein (GFP) and Myo3/5-red fluorescent protein (RFP) at nascent endocytic sites was revealed by two-color real-time fluorescence microscopy; the type I myosins join Pan1 at cortical patches at a late stage of internalization, preceding the inward movement of Pan1 and its disassembly. In cells lacking the Pan1 PRD, we observed an increased lifetime of Myo5-GFP at the cortex. Finally, Pan1 PRD enhanced the actin polymerization activity of Myo5-Vrp1 complexes in vitro. We propose that Pan1 and the type I myosins interactions promote an actin activity important at a late stage in endocytic internalization.     

Interaction of a peptide corresponding to the loop domain of the S2 SARS-CoV virus protein with model membranes

The severe acute respiratory syndrome coronavirus (SARS-CoV) envelope spike (S) glycoprotein is responsible for the fusion between the membranes of the virus and the target cell. In the case of the S2 domain of protein S, it has been found a highly hydrophobic and interfacial domain flanked by the heptad repeat 1 and 2 regions; significantly, different peptides pertaining to this domain have shown a significant leakage effect and an important plaque formation inhibition, which, similarly to HIV-1 gp41, support the role of this region in the fusion process. Therefore, we have carried out a study of the binding and interaction with model membranes of a peptide corresponding to segment 1073–1095 of the SARS-CoV S glycoprotein, peptide SARS_L in the presence of different membrane model systems, as well as the structural changes taking place in both the lipid and the peptide induced by the binding of the peptide to the membrane. Our results show that SARS_L strongly partitions into phospholipid membranes and organizes differently in lipid environments, displaying membrane activity modulated by the lipid composition of the membrane. These data would support its role in SARS-CoV mediated membrane fusion and suggest that the region where this peptide resides could be involved in the merging of the viral and target cell membranes.     

Engineering the loop region of a homeodomain-leucine zipper protein promotes efficient binding to a monomeric DNA binding site

Plant homeodomain-leucine zipper (HD-Zip) proteins, unlike many animal homeodomains (HDs), are unable to bind DNA as monomers. To investigate the molecular basis of their different behavior, we have constructed chimeras between the HD of the sunflower HD-Zip protein Hahb-4 and that of Drosophila engrailed (EN). Analysis of the interaction of these proteins with the pseudopalindromic Hahb-4 binding site and the monomeric EN binding site suggests that the loop located between helix I and helix II (amino acids 21-28) of EN is enough to confer efficient DNA binding activity to the Hahb-4 HD. Accordingly, the combined mutation of residues 24 and 25 of Hahb-4 to those present in EN (S24R/R25Y) originated an HD able to interact with the EN binding site, while single mutations were ineffective. We have also determined that a protein with the leucine zipper and helix III of Hahb-4 fused to the rest of the EN HD binds to the Hahb-4 pseudopalindomic binding site with increased affinity and shows extended contacts with DNA respective to Hahb-4. We conclude that the loop located between helix I and helix II of the HD must be regarded as one of the segments that contribute to the present-day diversity in the properties of different HDs.     

Interaction energy based protein structure networks

The three-dimensional structure of a protein is formed and maintained by the noncovalent interactions among the amino-acid residues of the polypeptide chain. These interactions can be represented collectively in the form of a network. So far, such networks have been investigated by considering the connections based on distances between the amino-acid residues. Here we present a method of constructing the structure network based on interaction energies among the amino-acid residues in the protein. We have investigated the properties of such protein energy-based networks (PENs) and have shown correlations to protein structural features such as the clusters of residues involved in stability, formation of secondary and super-secondary structural units. Further we demonstrate that the analysis of PENs in terms of parameters such as hubs and shortest paths can provide a variety of biologically important information, such as the residues crucial for stabilizing the folded units and the paths of communication between distal residues in the protein. Finally, the energy regimes for different levels of stabilization in the protein structure have clearly emerged from the PEN analysis.     

Arginine 197 of lac repressor contributes significant energy to inducer binding. Confirmation of homology to periplasmic sugar binding proteins.

Based on primary sequence homology between the lactose repressor protein and periplasmic sugar-binding proteins (Müller-Hill, B. (1983) Nature 302, 163-164), a hypothetical sugar-binding site for the lac repressor was proposed using the solved x-ray crystallographic structure of the arabinose-binding protein (ABP) (Sams, C. F., Vyas, N. K., Quiocho, F. A., and Matthews, K. S. (1984) Nature 310, 429-430). By analogy to Arg151 in the ABP sugar site, Arg197 is predicted to play an important role in lac repressor binding to inducer sugars. Hydrogen bonding occurs between Arg151 and the ring oxygen and 4-hydroxyl of the sugar ligand, two backbone carbonyls, and a side chain in ABP, and similar interactions in the lac repressor would be anticipated. To test this hypothesis, Arg197 in the lac repressor protein was altered by oligonucleotide-directed site-specific mutagenesis to substitute Gly, Leu, or Lys. Introduction of these substitutions at position 197 had no effect on operator binding parameters of the isolated mutant proteins, whereas the affinity for inducer was dramatically decreased, consistent with in vivo phenotypic behavior obtained by suppression of nonsense mutations at this site (Kleina, L. G., and Miller, J. H. (1990) J. Mol. Biol. 212, 295-318). Inducer binding affinity was reduced approximately 3 orders of magnitude for Leu, Gly, or Lys substitutions, corresponding to a loss of 50% of the free energy of binding. The pH shift characteristic of wild-type repressor is conserved in these mutants. Circular dichroic spectra demonstrated no significant alterations in secondary structure for these mutants. Thus, the primary effect of substitution for Arg197 is a very significant decrease in the affinity for inducer sugars. Arginine is uniquely able to make the multiple contacts found in the ABP sugar site, and we conclude that this residue plays a similar role in sugar binding for lactose repressor protein. These results provide experimental validation for the proposed homology between ABP and the lac repressor and suggest that homology with ABP may be employed to generate additional insight into the structure and function of this regulatory protein.     

Preferential binding of a G-quadruplex ligand to human chromosome ends

The G-overhangs of telomeres are thought to adopt particular conformations, such as T-loops or G-quadruplexes. It has been suggested that G-quadruplex structures could be stabilized by specific ligands in a new approach to cancer treatment consisting in inhibition of telomerase, an enzyme involved in telomere maintenance and cell immortality. Although the formation of G-quadruplexes was demonstrated in vitro many years ago, it has not been definitively demonstrated in living human cells. We therefore investigated the chromosomal binding of a tritiated G-quadruplex ligand, ³H-360A (2,6-N,N′-methyl-quinolinio-3-yl)-pyridine dicarboxamide [methyl-³H]. We verified the in vitro selectivity of ³H-360A for G-quadruplex structures by equilibrium dialysis. We then showed by binding experiments with human genomic DNA that ³H-360A has a very potent selectivity toward G-quadruplex structures of the telomeric 3′-overhang. Finally, we performed autoradiography of metaphase spreads from cells cultured with ³H-360A. We found that ³H-360A was preferentially bound to chromosome terminal regions of both human normal (peripheral blood lymphocytes) and tumor cells (T98G and CEM1301). In conclusion, our results provide evidence that a specific G-quadruplex ligand interacts with the terminal ends of human chromosomes. They support the hypothesis that G-quadruplex ligands induce and/or stabilize G-quadruplex structures at telomeres of human cells.     

Drosophila damaged DNA binding protein 1 contributes to genome stability in somatic cells

The damaged DNA-binding protein (DDB) complex consists of a heterodimer of p127 (DDB1) and p48 (DDB2) subunits and is believed to have a role in nucleotide excision repair (NER). We used the GAL4-UAS targeted expression system to knock down DDB1 in wing imaginal discs of Drosophila. The knock-down was achieved in transgenic flies using over-expression of inverted repeat RNA of the D-DDB1 gene [UAS-D-DDB1(650)-dsRNA]. As a consequence of RNA interference (RNAi), the fly had a shrunken wing phenotype. The wing spot test showed induced genome instability in transgenic flies with RNAi knock-down of D-DDB1 in wing imaginal discs. When Drosophila larvae with RNAi knock-down of D-DDB1 in wing imaginal discs were treated with the chemical mutagen methyl methanesulfonate (MMS), the frequency of flies with a severely shrunken wing phenotype increased compared to non-treated transgenic flies. These results suggested that DDB1 plays a role in the response to DNA damaged with MMS and in genome stability in Drosophila somatic cells.     

Small GTP binding protein Ras contributes to norepinephrine-induced mitogenesis of vascular smooth muscle cells

Norepinephrine stimulates release of arachidonic acid from tissue lipids. Arachidonic acid metabolites generated through the lipoxygenase and cytochrome P-450 pathways but not cyclooxygenase stimulate mitogen activated protein (MAP) kinase activity and proliferation of vascular smooth muscle cells (VSMC). Moreover, norepinephrine has been shown to activate the Ras/MAP kinase pathway through generation of cytochrome P450 metabolite of arachidonic acid, 20-hydroxyeicosatetraenoic acid (20-HETE). The purpose of this study was to investigate the contribution of Ras in norepinephrine-induced mitogenesis in aortic VSMC. Farnesylation of Ras by farnesyl transferase is required for its full activation. Norepinephrine-induced DNA synthesis, as measured by [3H]-thymidine incorporation, was attenuated by inhibitors of Ras farnesyl transferase FPT III and BMS-191563. These agents also inhibited 20-HETE-stimulated [3H]-thymidine incorporation. In cells transiently transfected with dominant negative Ras (RasN17), norepinephrine, and 20-HETE-induced proliferation of VSMC was attenuated. Both norepinephrine and 20-HETE increased localization of Ras to plasma membrane and MAP kinase activity; FPT III attenuated these effects. These data suggest that VSMC proliferation induced by norepinephrine and 20-HETE is mediated by Ras/MAP kinase pathway.     

Calorimetric and spectroscopic investigations of the binding of metallated porphyrins to G-quadruplex DNA

BackgroundThe human telomere contains tandem repeat of (TTAGG) capable of forming a higher order DNA structure known as G-quadruplex. Porphyrin molecules such as TMPyP4 bind and stabilize G-quadruplex structure.MethodsIsothermal titration calorimetry (ITC), circular dichroism (CD), and mass spectroscopy (ESI/MS), were used to investigate the interactions between TMPyP4 and the Co(III), Ni(II), Cu(II), and Zn(II) complexes of TMPyP4 (e.g. Co(III)-TMPyP4) and a model human telomere G-quadruplex (hTel22) at or near physiologic ionic strength ([Na⁺] or [K⁺] ≈ 0.15 M).ResultsThe apo-TMPyP4, Ni(II)-TMPyP4, and Cu(II)-TMPyP4 all formed complexes having a saturation stoichiometry of 4:1, moles of ligand per mole of DNA. Binding of apo-TMPyP4, Ni(II)-TMPyP4, and Cu(II)-TMPyP4 is described by a “four-independent-sites model”. The two highest-affinity sites exhibit a K in the range of 10⁸ to 10¹⁰ M^− 1 with the two lower-affinity sites exhibiting a K in the range of 10⁴ to 10⁵ M^− 1. Binding of Co(III)-TMPyP4, and Zn(II)-TMPyP4, is best described by a “two-independent-sites model” in which only the end-stacking binding mode is observed with a K in the range of 10⁴ to 10⁵ M^− 1.ConclusionsIn the case of apo-TMPyP4, Ni(II)-TMPyP4, and Cu(II)-TMPyP4, the thermodynamic signatures for the two binding modes are consistent with an “end stacking” mechanism for the higher affinity binding mode and an “intercalation” mechanism for the lower affinity binding mode. In the case of Co(III)-TMPyP4 and Zn(II)-TMPyP4, both the lower affinity for the “end-stacking” mode and the loss of the intercalative mode for forming the 2:1 complexes with hTel22 are attributed to the preferred metal coordination geometry and the presence of axial ligands.General significanceThe preferred coordination geometry around the metal center strongly influences the energetics of the interactions between the metallated-TMPyP4 and the model human telomeric G-quadruplex. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Interaction between the yellow fever virus nonstructural protein NS3 and the host protein Alix contributes to the release of infectious particles

The ESCRT (endosomal sorting complex required for transport) machinery normally executes cargo sorting and internalization during multivesicular body biogenesis, but is also utilized by several enveloped viruses to facilitate their budding from cellular membranes. Although the mechanisms of flavivirus infectious particle assembly and release are poorly understood, the nonstructural protein NS3 has been reported to have an essential role via an undescribed mechanism. Here, we shed light on the role of NS3 by connecting it to the host factor Alix, a protein intimately connected with the ESCRT machinery. We demonstrate that NS3 and Alix interact and show that dominant negative versions of Alix inhibit YFV release. Furthermore, we show that NS3 supplied in trans rescues this effect. We propose that the interaction between NS3 and Alix contributes to YFV release.     

Studies on the binding of 5-N-methylated quindoline derivative to human telomeric G-quadruplex

Quindoline derivatives as telomeric quadruplex ligands have shown good biological activity for telomerase inhibition. In the present study, we used spectroscopic and calorimetric methods to investigate the interactions between a quindoline derivative (5-methyl-11-(2-morpholinoethylamino)-10-H-indolo-[3,2-b]quinolin-5-ium iodide, compound 1) and human telomeric G-quadruplex. The thermodynamic studies using isothermal titration calorimetry (ITC) indicated that their binding process was temperature-dependent and enthalpy–entropy co-driven. The significant negative heat capacity was obtained experimentally from the temperature dependence of enthalpy changes, which was consistent with that from theoretical calculation, and all suggesting significant hydrophobic contribution to the molecular recognition process. Based on the results from UV–vis, ITC, steady-state and time-resolved fluorescence, their binding mode was determined as two ligand molecules stacking on the quartets on both ends of the quadruplex. These results shed light on rational design and development of quindoline derivatives as G-quadruplex binding ligands.