Optical and x-ray diffraction studies of polynucleotides and their interaction with drugs

The interactions in solution and the solid state oT the antibiotic drugs ethidium bromide, adriamvcin and daunomycin with deoxyribonucleic acid have been investigated with the aim of understanding at a molecular level the basis of their chemotherapeutic activity. Both ethidium bromide and the two related anthracycline drugs adriamycin and daunomycin possess planar aromatic chromophores by virtue of which they can intercalate into the deoxyribonucleic acid helix. A study of the spectroscopic properties o^ the drugs in solution as a function of concentration, solvent and ionic strength gave an insight into the properties of the drug chromonhores, Which helped in understanding the spectral changes which take place when complexes form between the drugs and polynucleotides or polyelectrolytes. A microspectrophotoneter was specially constructed to measure the linear dichroism (in the visible range) and the birefringence of oriented fibres and fibre sections of drug-deoxyribonucleic acid complexes and an X-ray diffraction study of the same specimens was carried out in parallel. It was found that a change in the degree of hydration of the nucleic acid helices results in a redistribution of the drug molecules between the intercalated and non-intercalated states. The intercalations driven by hydrophobic forces (additional stabilization being gained from specific electrostatic and hydrogen bonding interactions) and is opposed by the hydrophilic nature of some features of the drugs. The helix pitch increases with increasing relative humidity up to a maximum value which may represent the situation in which no nett increase in the fraction of intercalated drug is possible because these two effects counterbalance each other. An estimate of the lower limit to the values of the unending angle of the deoxyribonucleic acid helix at the site of intercalation was made on the basis of the maximim pitch values. This angle (12° - 3°) fitted the data obtained from both ethidium bromide and the anthracyclines. Ethidium bromide dissociates completely from the intercalated state at low humidities, whereas a large fraction of the anthracvcline molecules remain intercalated even at very low relative humidities. The enhanced chemotherapeutic activity of adriamycin, as compared to daunomycin, was associated with the observed increased stability of the intercalated state of adriamycin under conditions of low hydration and could be justified in terns of an additional hydrogen bond formed by adriamycin rath the ITM backbone. While the X-ray diffraction technique provides information mainly on the drug soecies which is intercalated, the linear dichroism and birefringence reflect the state of both intercalated and non- intercalated soecies present in the fibre. A quantitative analysis of the combined X-ray diffraction and optical data, coupled rath computer simulations of the fibre dichroism and birefringence, produced on one hand, a picture of the degree of internal alignment of the helices in the fibre and, on the other hand, an estimate of the orientation of the non-intercalated species. It was concluded that in the case of ethidium bromide, and possibly, in the case of the anthracyclines, the non-intercalated drug is externally bound, essentially perpendicular to the deoxyribonucleic acid helix axis. Limits to the possible tilt angles and orientational freedom of the externally bound species were derived

An Unusual Helix Turn Helix Motif in the Catalytic Core of HIV-1 Integrase Binds Viral DNA and LEDGF

Background: Integrase (IN) of the type 1 human immunodeficiency virus (HIV-1) catalyzes the integration of viral DNA into host cellular DNA. We identified a bi-helix motif (residues 149–186) in the crystal structure of the catalytic core (CC) of the IN-Phe185Lys variant that consists of the a 4 and a 5 helices connected by a 3 to 5-residue turn. The motif is embedded in a large array of interactions that stabilize the monomer and the dimer. Principal Findings: We describe the conformational and binding properties of the corresponding synthetic peptide. This displays features of the protein motif structure thanks to the mutual intramolecular interactions of the a4 and a5 helices that maintain the fold. The main properties are the binding to: 1- the processing-attachment site at the LTR (long terminal repeat) ends of virus DNA with a Kd (dissociation constant) in the sub-micromolar range; 2- the whole IN enzyme; and 3- the IN binding domain (IBD) but not the IBD-Asp366Asn variant of LEDGF (lens epidermal derived growth factor) lacking the essential Asp366 residue. In our motif, in contrast to the conventional HTH (helix-turn-helix), it is the N terminal helix (a 4) which has the role of DNA recognition helix, while the C terminal helix (a 5) would rather contribute to the motif stabilization by interactions with the a4 helix. Conclusion: The motif, termed HTHi (i, for inverted) emerges as a central piece of the IN structure and function. It coul

Helical conformation of the AD1 peptide in the AML1-ETO–E-protein complex

International audienceThe AML1-ETO fusion protein is responsible for 15% of the acute myeloid leukaemias due to its interference with transcription activation by E-proteins. The eTAFH region of the AML1-ETO competes for the AD1 domain of E-protein, thereby preventing wild-type transcription activation. The structural details concerning the eTAFH–bound AD1 domain are not known. We have previously shown that the eTAFH–AD1 interaction is strong (Kd=28 nM, H. Porumb, Spectroscopy 22 (2008), 251–260). The secondary structure prediction algorithms are undecided as to the conformation of bound AD1 peptide. Here we demonstrate by circular dichroism that the bound AD1 peptide is fully helical. This will facilitate modeling of the interaction and launches a challenge as to using synthetic peptides to out compete the eTAFH–E-protein interaction

Self-association and domains of interactions of an amphipathic helix peptide inhibitor of HIV-1 integrase assessed by analytical ultracentrifugation and NMR experiments in trifluoroethanol/H(2)O mixtures.

International audienceEAA26 (VESMNEELKKIIAQVRAQAEHLKTAY) is a better inhibitor of human immunodeficiency virus, type 1, integrase than its parent Lys-159, reproducing the enzyme segment 147-175 with a nonpolar-polar/charged residue periodicity defined by four helical heptads (abcdefg) prone to collapse into a coiled-coil. Circular dichroism, nuclear magnetic resonance, sedimentation equilibrium, and chemical cross-linking were used to analyze EAA26 in various trifluoroethanol/H(2)O mixtures. In pure water the helix content is weak but increases regularly up to 50-60% trifluoroethanol. In contrast the multimerization follows a bell-shaped curve with monomers in pure water, tetramers at 10% trifluoroethanol, and dimers at 40% trifluoroethanol. All suggest that interhelical interactions between apolar side chains are required for the coiled-coil formation of EAA26 and subsist at medium trifluoroethanol concentration. The N(H) temperature coefficients measured by nuclear magnetic resonance show that at low trifluoroethanol concentration the amide groups buried in the hydrophobic interior of four alpha-helix bundles are weakly accessible to trifluoroethanol and are only weakly subject to its hydrogen bond strengthening effect. The increased accessibility of trifluoroethanol to buried amide groups at higher trifluoroethanol concentration entails the reduction of the hydrophobic interactions and the conversion of helix tetramers into helix dimers, the latter displaying a smaller hydrophobic interface. The better inhibitory activity of EAA26 compared with Lys-159 could arise from its better propensity to form a helix bundle structure with the biologically important helical part of the 147-175 segment in integrase

Abbreviations used: IP 3 R, inositol 1,4

⁎ Cytoplasmic Ca 2+ signals are highly regulated by various ion transporters, including the inositol 1,4,5-trisphosphate (IP 3 ) receptor (IP 3 R), which functions as a Ca 2+ release channel on the endoplasmic reticulum membrane. Crystal structures of the two N-terminal regulatory regions from type 1 IP 3 R have been reported; those of the IP 3 -binding core (IP 3 R CORE ) with bound IP 3 , and the suppressor domain. This study examines the structural effects of ligand binding on an IP 3 R construct, designated IP 3 R N , that contains both the IP 3 -binding core and the suppressor domain. Our circular dichroism results reveal that the IP 3 -bound and IP 3 -free states have similar secondary structure content, consistent with preservation of the overall fold within the individual domains. Thermal denaturation data show that, while IP 3 has a large effect on the stability of IP 3 R CORE , it has little effect on IP 3 R N , indicating that the suppressor domain is critical to the stability of IP 3 R N . The NMR data for IP 3 R N provide evidence for chemical exchange, which may be due to protein conformational dynamics in both apo and IP 3 -bound states: a conclusion supported by the small-angle X-ray scattering data. Further, the scattering data show that IP 3 R N undergoes a change in average conformation in response to IP 3 binding and the presence of Ca 2+ in the solution. Taken together, these data lead us to propose that there are two flexible linkers in the N-terminal region of IP 3 R that join stably folded domains and give rise to an equilibrium mixture of conformational sub-states containing compact and more extended structures. IP 3 binding drives the conformational equilibrium toward more compact structures, while the presence of Ca 2+ drives it to a more extended set

Ligand-induced conformational changes via flexible linkers in the amino-terminal region of the inositol 1,4,5-trisphosphate receptor

Cytoplasmic Ca2+ signals are highly regulated by various ion transporters, including the inositol 1,4,5-trisphosphate (IP3) receptor (IP3), which functions as a Ca2+ release channel on the endoplasmic reticulum membrane. Crystal structures of the two N-terminal regulatory regions from type 1 IP3R have been reported; those of the IP3-binding core (IP3RCORE) with bound IP3, and the suppressor domain. This study examines the structural effects of ligand binding on an IP3R construct, designated IP3RN, that contains both the IP3-binding core and the suppressor domain. Our circular dichroism results reveal that the IP3- bound and IP3-free states have similar secondary structure content, consistent with preservation of the overall fold within the individual domains. Thermal denaturation data show that, while IP3 has a large effect on the stability of IP3RCORE, it has little effect on IP3RN, indicating that the suppressor domain is critical to the stability of IP3RN. The NMR data for IP3RN provide evidence for chemical exchange, which may be due to protein conformational dynamics in both apo and IP3-bound states: a conclusion supported by the small-angle X-ray scattering data. Further, the scattering data show that IP3RN undergoes a change m average conformation in response to IP3-binding and the presence of Ca2+, in the solution. Taken together, these data lead us to propose that there are two flexible linkers in the N-terminal region of lP(3)R that join stably folded domains and give rise to an equilibrium mixture of conformational sub-states containing compact and more extended structures. IP3 binding drives the conformational equilibrium toward more compact structures, while the presence of Ca2+ drives it to a more extended set. © 2007, Elsevier Ltd

Circular dichroism.

<p>Spectra of peptide HTHi at 6 µM (^__, red) and at 25 µM (—, black), and difference spectrum of the complex [HTHi (6 µM)+LTR34 (10 µM)] minus the CD spectrum of LTR34 (10 µM) (……‥, blue). Insert: CD spectrum of HTHi compared to the spectra of its component peptides, α₄ (after Figure 6 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004081#pone.0004081-Zargarian1" target="_blank">[43]</a>) and INH5 (after Figure 3b in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004081#pone.0004081-Maroun1" target="_blank">[30]</a>). Recall that INH5 comprises both the α₅ helix and the turn linking α₅ to α₄ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004081#pone-0004081-t001" target="_blank">Table 1a</a>).</p

Identification of an “inverted” HTH motif (HTHi) at the catalytic core surface of integrase (PDB ID 1BIU [20]).

<p>a). Crystal structure of the catalytic core domain, associated into a dimer. b). Representation of the HTHi motif, with the loop residues shown by van der Waals spheres. c). The side chain residues involved in intramolecular contacts, shown by sticks and van der Waals spheres. d). The electrostatic potential at the solvent-accessible surface; the Lys-156, Lys-159 and Lys-160 residues are shown by sticks. e). HTHi motif of IN, superimposed onto the “classical” HTH motif of the HMG (highly mobile group) protein LEF-1 (lymphoid enhancer binding factor, PDB ID 2LEF, brown). f). HTHi motif of IN, superimposed onto the HTHi motif of the Signal Recognition Particle (PDB ID 2FFH, green).</p