Photochemical deoxyribose C2' oxidation in 5-iodouracil-containing hexanucleotide.

To investigate photochemistry of 5-iodouracil (IU) in DNA, photoreaction of IU-containing oligonucleotides was examined. It was found that d(GCAIU G C) 2 undergoes selective photochemical C1' and C2' oxidation at the 5' side of IU residue to provide ribonolactone-containing hexamer 1 and erythrose-containing hexamer 2. Upon heating under alkaline conditions, erythrose-containing hexamer 2 was found to undergo retro aldol condensation to provide two fragments having glycolaldehyde termini.     

Photoreactivity of indirubin derivatives

Twenty-nine analogs of indirubin, an isomer of indigo, have been synthesized to optimize its promising kinase inhibitory scaffold. These compounds being also pigmented, have been tested for their photoreactivity. Absorption maxima were between 485 nm and 560 nm. Addition of fetal calf serum induced fluorescence and time dependent absorption modifications. Appropriate illumination induced Reactive Oxygen Species (ROS) production for nineteen compounds out of twenty-nine. The relationship between fluorescence and ROS production is discussed. Six compounds showed an important toxicity on F98 cells, a murine glioma cell line. Three of these were found to be also phototoxic, as four other non-toxic compounds. All but one phototoxic compounds were detected as ROS producers by in vitro tests. Photoreactivity assessment is important to anticipate adverse reactions for compounds that might be clinically developed. The experimental assay was found to be the only way to evaluate the photoreactivity of this family of compounds since no predictive criteria on structures could be found. Combining the vascular tumor growth inhibition induced by kinase inhibitors with the massive local blood flow arrest following photodynamic treatment may be an efficient anti-cancer strategy. These data could orientate further syntheses of either non-photoreactive compounds or compounds displaying both kinase inhibitory activity and strong phototoxicity.     

Molecular dynamics simulation of the DNA triplex d(TC)5.d(GA)5.d(C+T)5.

A molecular dynamics simulation of the DNA triple helix d(TC)5.d(GA)5.d(C+T)5 is described (C+ represents a protonated cytosine residue). The simulation has been performed using the program AMBER 3.1 and includes counterions and explicit solvent under periodic boundary conditions. Both the dynamic and time-averaged behaviour of the system has been analysed. Considerable deviations from the fibre-diffraction model for DNA triple helix structure are observed, including the repuckering of the purine strand sugars that has been identified in some nuclear magnetic resonance (n.m.r.) studies. The simulation suggests that this conformational change may be driven by the possibility of improved interactions between the phosphate groups of this strand and both the solvent and counterions. Several examples of a particular conformational transition are observed, involving correlated changes in the backbone angles alpha and gamma. These transitions provide a possible explanation for some unusual n.m.r. data that have been reported. The structure of the triple helix major groove also suggests an explanation for the observed stabilization of DNA triplexes by polyvalent cations, and their ability to interact with drugs that bind in the minor groove of DNA duplexes.     

The solution structure of the Sac7d/DNA complex: a small-angle X-ray scattering study.

Small-angle X-ray scattering has been used to study the structure of the multimeric complexes that form between double-stranded DNA and the archaeal chromatin protein Sac7d from Sulfolobus acidocaldarius. Scattering data from complexes of Sac7d with a defined 32-mer oligonucleotide, with poly[d(GC)], and with E. coli DNA indicate that the protein binds along the surface of an extended DNA structure. Molecular models of fully saturated Sac7d/DNA complexes were constructed using constraints from crystal structure and solution binding data. Conformational space was searched systematically by varying the parameters of the models within the constrained set to find the best fits between the X-ray scattering data and simulated scattering curves. The best fits were obtained for models composed of repeating segments of B-DNA with sharp kinks at contiguous protein binding sites. The results are consistent with extrapolation of the X-ray crystal structure of a 1:1 Sac7d/octanucleotide complex [Robinson, H., et al. (1998) Nature 392, 202-205] to polymeric DNA. The DNA conformation in our multimeric Sac7d/DNA model has the base pairs tilted by about 35 degrees and displaced 3 A from the helix axis. There is a large roll between two base pairs at the protein-induced kink site, resulting in an overall bending angle of about 70 degrees for Sac7d binding. Regularly repeating bends in the fully saturated complex result in a zigzag structure with negligible compaction of DNA. The Sac7d molecules in the model form a unique structure with two left-handed helical ribbons winding around the outside of the right-handed duplex DNA.     

Indole ring binds to 7-methylguanine base by pi-pi stacking interaction. Crystal structure of 7-methylguanosine 5'-monophosphate-tryptamine complex

Strong pi-pi stacking interaction between the indole ring and 7-methylguanine base was shown by X-ray crystal analysis of the 7-methylguanosine 5'-monophosphate-tryptamine complex. This interaction appears to be strengthened by the attachment of ribose and phosphate groups to the base.     

The structure of human complement component C7 and the C5b-7 complex

The molecular architecture of human complement component C7 was elucidated at several structural levels. The complete primary structure of C7 was derived from the cDNA sequence of clones isolated from a human liver library. C7 is a mosaic protein that consists of 821 amino acids. The amino-terminal two-thirds of C7 has 23-30% homology with complement components C8 and C9. In addition, the carboxyl-terminal third contains four cysteine-rich segments that have overlapping internal homology. The protein is a single polypeptide chain with 28 disulfide bonds and is glycosylated at two sites. Virtually all the cysteines are found in small units of 35-77 amino acids that exhibit homology with those of various proteins including the low density lipoprotein receptor, epidermal growth factor precursor, thrombospondin, and blood coagulation factors IX and X. The secondary structural analysis, estimated by circular dichroism, suggested a high content of beta-sheet (38%) and beta-turns (24%). The tertiary structure, visualized by transmission electron microscopy, indicated a flexible elongated molecule with dimensions of 151 X 59 X 43 A. The quaternary structure of the C5b-7 complex bound to lipid vesicles was observed to be in the form of monomers or dimers. The monomer C5b-7 consists of a leaflet and a long flexible stalk, and the dimer has two leaflets linked through a supercoiled stalk. Membrane binding is mediated by the stalk part of the complexes. Using a radioiodinated photoreactive cross-linking reagent bound to the polar head group of phosphatidylethanolamine, the stalk part of the C5b-7 complex could be labeled preferentially, and it was found to consist mainly of C6 and C7. Thus, C7 plays a major role in bringing about the hydrophilic-amphiphilic transition during the formation of the membrane attack complex, and it serves as a membrane anchor for the C5b-7 complex.     

Molecular mechanical studies of base-pair opening in d(CGCGC):d(GCGCG), dG5·dC5, d(TATAT):d(ATATA), and dA5·dT5 in the B and Z forms of DNA

Molecular mechanical energy refinement of double-helical pentanucleotide tetra-phosphates, d(CGCGC):d(GCGCG), dG₅·dC₅, d(TATAT):d(ATATA), and dA₅ ·dT₅ geometries, are presented in order to examine the energy required to open the Nl(purine) …? N3(pyrimidine) distance (base-pair opening) of a Watson-Crick base pair from its normal value of 3 Å to a value of 6 Å. The structural consequences of forcing base-pair opening is sequence dependent. For both dA₅ ·dT₅ and d(TATAT):d(ATATA), forcing the Nl (AdeKN3 (Thy) distance of the central base pair to a value of 6 Å slides the bases perpendicular to the helix axis forming a low-energy non-Watson-Crick base pair having an adenine amine hydrogen …? thymine carbonyl oxygen hydrogen bond. The two GC sequences behave differently from both AT sequences and differently from each other. Forcing the Nl(Gua) …? N3(Cyt) distance to 6 Å leads to unconventional structures in which hydrogen bonds are formed between the separated bases and the bases above or below them. These structures appear to be trapped in true local minima 6–10 kcal/mol higher in energy than the Watson-Crick structures. Preliminary simulations on d(CGCGC):d(GCGCG) in the Z geometry suggest the reason the Z form may be more refractory to proton exchange than the B form, consistent with experimental observations.     

Studies on formation and stability of the d[G(AG)5]* d[G(AG)5]·d[C(TC)5] and d[G(TG)5]* d[G(AG)5]· d[C(TC)5] triple helices

We have targeted the d[G(AG)₅] · d[C(TC)₅] duplex for triplex formation at neutral pH with either d[G(AG)₅] or d[G(TG)₅]. Using a combination of gel electrophoresis, uv and CD spectra, mixing and melting curves, along with DNase I digestion studies, we have investigated the stability of the 2:1 pur*pur · pyr triplex, d[G(AG)₅] * d[G(AG)₅] · d[C(TC)₅], in the presence of MgCl₂. This triplex melts in a monophasic fashion at the same temperature as the underlying duplex. Although the uv spectrum changes little upon binding of the second purine strand, the CD spectrum shows significant changes in the wavelength range 200–230 nm and about a 7 nm shift in the positive band near 270 nm. In contrast, the 1:1:1 pur/pyr*pur · pyr triplex, d[G(TG)₅] * d[G(AG)₅] · d[C(TC)₅], is considerably less stable thermally, melting at a much lower temperature than the underlying duplex, and possesses a CD spectrum that is entirely negative from 200 to 300 nm. Ethidium bromide undergoes a strong fluorescence enhancement upon binding to each of these triplexes, and significantly stabilizes the pur/pyr*pur · pyr triplex. The uv melting and differential scanning calorimetry analysis of the alternating sequence duplex and pur*pur · pyr triplex shows that they are lower in thermodynamic stability than the corresponding 10-mer d(G₃A₄G₃) · d(C₃T₄C₃) duplex and its pur*pur · pyr triplex under identical solution conditions. © 1997 John Wiley & Sons, Inc.     

Formation and structure of the C5b-7 complex of the lytic pathway of complement.

The formation and structure of the complement cytolytic intermediary complex, C5b-7, were studied with the aim of determining the interactive regions of C5, C6, and C7. The structure of human complement component C5 was elucidated by the application of limited proteolysis which generated well characterized major polypeptide fragments of this molecule. Plasmin, thrombin, and kallikrein cleave C5b with greater facility than C5. The most useful cleavage of C5b was effected by plasmin because the fragmentation pattern was similar to the processing of C3b by factors H, I, and kallikrein. Plasmin hydrolyzes peptide bonds within the alpha'-chain of C5b, resulting in a four-chain fragment, C5c (M(r) = 142,000), and a single chain fragment, C5d (M(r) = 43,000). Circular dichroism spectroscopic analyses indicated that C5d is substantially richer in alpha-helical content than is C5c (27 versus 9%). Polyclonal antibodies directed against C5c blocked the interaction of C5b-6 with C7, whereas antibodies directed against C5d inhibited the binding of C5 with C3b. Chemical cross-linking using a cleavable radioiodinated photoreactive reagent revealed that both C6 and C7 associate preferentially with the alpha'-chain of C5b. The reversible interactions of C5 with C6, C7, and major polypeptide fragments derived from these were investigated with solid phase binding assays. The results indicate that the carboxyl-terminal domains of C6 and C7, which have cysteine-rich modules homologous to those found in factors H and I, have the capacity to link specifically with C5.     

EphrinA5-EphA7 complex induces apoptotic cell death via TNFR1

A previous study showed that the EphA7 receptor regulates apoptotic cell death during early brain development. In this study, we provide evidence that the EphA7 receptor interacts with death receptors such as tumor necrosis factor receptor 1 (TNFR1) to decrease cell viability. We showed that ephrinA5 stimulates EphA7 to activate the TNFR1-mediated apoptotic signaling pathway. In addition, a pull-down assay using biotinylated ephrinA5-Fc revealed that ephrinA5-EphA7 complexes recruit TNFR1 to form a multi-protein complex. Immunocytochemical staining analysis showed that EphA7 was co-localized with TNFR1 on the cell surface when cells were incubated with ephrinA5 at low temperatures. Finally, both the internalization motif and death domain of TNFR1 was important for interacting with an intracytoplasmic region of EphA7; this interaction was essential for inducing the apoptotic signaling cascade. This result suggests that a distinct multi-protein complex comprising ephrinA5, EphA7, and TNFR1 may constitute a platform for inducing caspase-dependent apoptotic cell death.     

The SC5b-7 complex: formation, isolation, properties, and subunit composition.

Activation of C in C8-depleted serum results in the formation of a soluble complex containing C5, C6, and C7. The complex has an electrophoretic mobility of an alpha-globulin, an s-rate of 18.5S, and a m.w. of 668,000 daltons. This complex was isolated and upon SDS polyacrylamide gel electrophoresis it was found to contain, in addition to C5b, C6 and C7, an 88,000 dalton glycoprotein. The protein was identified as the band V protein of the soluble C5b-9 complex. It is referred to as SIIIs-protein, or S-protein. Since the S-protein does not bind to C5b-6, it is concluded that it is incorporated during the fusion of C5b-6 with C7. The SC5b-7 complex exhibits the same neoantigen as the SC5b-9 complex, but compared to the C5b-6 complex it appears to contain an additionally qualitatively distinct neoantigen.     

The membrane attack complex of complement: relation of C7 to the metastable membrane binding site of the intermediate complex C5b-7

Isolated C7 (m.w. 120,000) in 1% deoxycholate (DOC) forms dimers with an apparent m.w. of 230,000 and a DOC-binding capacity of 82 mol per mol of dimer. Dimerization of C7 also occurs in the presence of DOC-phospholipid mixed micelles and eventuates in the insertion of C7 dimers into the lipid bilayer upon the removal of the detergent. C5b-7 complex formation in the fluid phase or on lipid vesicles likewise involves polymerization. C5b-7 sedimented with 17-40S, which suggests a dimeric to hexameric composition. In avidin-biotin binding experiments in which two differentially labeled forms of C5b,6 (biotinyl 125I-C5b,6, and 131I-C5b,6) were used in equimolar amounts to assemble C5b-7, more than 50% of the biotinyl 125I-C5b,6-containing complexes also contained 131I label; again suggesting that C5b-7 consisted of oligomers rather than monomers. The conformation of C7 in C5b-7 and in dimeric C7 appeared similar by the following criteria. On formation of C5b-7 from C5b,6 and C7, a 20% increase in beta-pleated sheet structure was observed by circular dichroism spectroscopy, and a similar change occurred on dimerization of isolated C7. Tryptic and thermolytic digests of C5b-7 and C7 dimers containing 125I-C7 were analyzed by autoradiography after SDS-polyacrylamide gel electrophoresis and were found to contain similar peptides that were distinct from those in the digests of monomeric C7. Direct evidence showing that the metastable membrane binding site of the C5b-7 complex resides in the C7 subunit was obtained by using the conjugates of C5b,6 and colloidal gold. Viewed in the electron microscope, these conjugates were aggregated upon the addition of isolated C7. In contrast, when conjugates of C7 and colloidal gold were treated with soluble C5b,6, no such aggregates occurred, but instead, individual C5b-7 complexes were observed arranged around single gold particles, resulting in star-like structures. The results strongly suggest that structures of C7 are responsible for the expression of the membrane binding site of metastable C5b-7.     

Specific inhibition of capped mRNA translation in vitro by m7G5''pppp5''G and m7G5''pppp5''m7G.

A unique set of diguanosine cap analogues containing a 5'-5' tetraphosphate linkage instead of the normal triphosphate was synthesized by chemical methylation of G5'pppp5'G. Both 7-methylguanosine products, m7G5'pppp5'G and m7G5'pppp5'm7G, acted as potent inhibitors of capped brome mosaic virus (BMV) RNA translation in the homologous wheat germ protein synthesis system. Inhibition of in vitro protein synthesis required the presence of the 7-methyl group on guanosine and was specific for capped mRNA. In comparison with the partial cap analogue, m7GTP, the methylated diguanosine tetraphosphate structures were 25-50 fold more potent inhibitors of in vitro protein synthesis. Analysis of the in vitro translation products of the four species of BMV RNA showed a differential sensitivity to inhibition by m7G5'pppp5'm7G.     

Photoreactivity of histidyl residues in subtilisins Novo and DY. Photooxidation of subtilisins

Subtilisins Novo and DY were photoinactivated in the presence of methylene blue according to first order kinetics. The competitive inhibitor N alpha-benzoyl-L-arginine protected significantly against inactivation. Under the conditions employed in this study a selective photooxidation of the active site histidine 64 was achieved. Rate constants of 0.32 X 10(-2), s-1 and 0.35 X 10(-2), s-1, were calculated for the Novo enzyme and subtilisin DY, respectively. Apparent pKa values of the catalytically important imidazole group of 7.0 +/- 0.1 (s. Novo) and 7.1 +/- 0.1 (s. DY) were directly determined. The histidyl residues in the two proteases, except the active site histidine, which is the first target of photooxidation, are "buried" in the interior of the protein globule. Conformational studies suggested that the photoreactive histidine is not involved in the stabilization of the protein conformation.     

Nonintercalative binding of proflavin to Z-DNA: structure of a complex between d(5BrC-G-5BrC-G) and proflavin

The crystal structure of a disordered 1:1 complex between the tetradeoxyoligomer d(5BrC-G-5BrC-G) and proflavin has been determined and refined to an R factor of 26.9% for 474 reflections initially in space group P6(5) and to an R factor of 22.2% for 475 reflections in space group P2(1), both at 2-A resolution with Fobsd greater than or equal to 4.0. The unit cell constants are a = b = 17.9 A, c = 44.5 A, and gamma = 120 degrees. The final models are essentially the same in the two space groups with greater disorder in space group P6(5). In space group P2(1), the asymmetric unit is a tetranucleotide duplex, two sandwiched proflavin molecules, and four "outside-bound" proflavins. The tetranucleotide duplex is in the Z conformation and is located at the origin of the unit cell with a pair of proflavins sandwiched between the tetranucleotides. Thus, the tetranucleotides and proflavin dimers stack alternatively forming a quasi-continuous helix with the helix axis coincident with the c axis. The structure analysis revealed the presence of outside-bound proflavins as well. It is interesting that one type of outside-bound proflavins occupies a similar environment as the cobalt hexaammines in their complex with the decadeoxyoligomer d(CGTACGTACG) [Brennan, R. G., Westhof, E., & Sundaralingam, M. (1986) J. Biomol. Struct. Dyn. 3, 649]. Crystals of the latter are isomorphous to the present complex. The outside-bound proflavins penetrate the deep minor groove, thereby closing it off, and provide a visualization of a quasi-internal mode of binding of proflavin to a nucleic acid.     

Thermodynamics of core hydrophobicity and packing in the hyperthermophile proteins Sac7d and Sso7d

The influence of core hydrophobicity and packing on the structure and stability of the hyperthermophile proteins Sac7d and Sso7d have been studied by calorimetry, circular dichroism, and NMR. Valine 30 is positioned in Sac7d to allow a cavity-filling Val --> Ile substitution which occurs naturally in the homologous more thermostable Sso7d. The cavity-filling mutation in Sac7d has been characterized and compared to the reciprocal Ile --> Val mutation in Sso7d. A detailed analysis of the stability of the proteins was obtained by globally fitting the variation of DSC parameters and circular dichroism intensities as a function of temperature (0-100 degrees C), salt (0-0.3 M), and pH (0-8). A global analysis over such a range of conditions permitted an unusually precise measure of the thermodynamic parameters, as well as the separation of the thermodynamics of the intrinsic unfolding reaction from the linked effects of protonation and chloride binding associated with acid-induced folding. The results indicate differences in the energetics of unfolding Sac7d and Sso7d that would not be apparent from an analysis of DSC data alone using conventional methods. The sign and magnitude of the changes in DeltaG, DeltaH, TDeltaS, and DeltaC(P) of unfolding resulting from core Ile/Val substitutions in the two proteins were consistent with differences in hydrophobicity of Val and Ile and negligible changes in packing (van der Waals) interactions. The benefit of increased hydrophobicity of the core increased with temperature, with maximal effect around 116 degrees C. Increased hydrophobicity of the core achieved not only an increase in the free energy of unfolding, but also a lateral shift of the temperature of maximal stability to higher temperature.     

Mode of inhibition of globin synthesis by m7G5'p and m7G5'ppp.

The mode of inhibition of rabbit globin synthesis by m7G5'p and m7G5'ppp ("cap analogs") was studied using the rabbit reticulocyte lysate system. The rate of globin synthesis was measured at various concentrations of both f[35S]Met-tRNAf and the cap analogs. The cap analogs were found to inhibit competitively the incorporation of f[35S]Met into hot trichloroacetic acid-insoluble material. Nascent chains prelabelled with f[35S]Met were released at various concentrations of m7G5'ppp. The release of nascent chains was not inhibited by m7G5'p (Suzuki, H. (1976) FEBS Lett. 72, 309) and m7G5'ppp, and it is therefore concluded that the cap analogs inhibit a step of initiation of globin synthesis. Under conditions such that the elongation of nascent chains was inhibited by sparsomycin, the formation of an 80S/fMet-tRNAf was inhibited by the cap analogs, but not that of a 40S/fMet-tRNAf complex. These data suggest that a factor which is required for the binding of globin mRNA with 40S/fMet-tRNAf complex forms an inactive complex with the cap analogs, so that the cap analogs inhibit globin synthesis.