Sequence specific molecular recognition by a monocationic lexitropsin of the decadeoxyribonucleotide d-[CATGGCCATG]2: structural and dynamic aspects deduced from high field 1H-NMR studies.

All 1H-NMR resonances of d-[CATGGCCATG]2 and the 1:1 complex of lexitropsin 1 and the DNA were assigned by the NOE difference, COSY and NOESY methods. Addition of 1 causes the base and imino protons for the sequence 5'-CCAT to undergo the most marked drug-induced chemical shift changes, thereby indicating that 1 is located in this base pair sequence. NOEs confirmed the location and orientation of the drug in the 1:1 complex, with the amino terminus oriented to C(6). The van der Waals interaction between H12a,b of 1 and AH2(8) may be responsible for reading of the 3' A.T base pair in the 5'-CCAT sequence. Exchange NMR effects allow an estimate of approximately equal to 62 s-1 for the intramolecular "slide-swing" exchange of the lexitropsin between two equivalent binding sites with delta G = 58 +/- 5 kJ mol-1 at 301 degrees K.     

Sequence specific molecular recognition and binding by a GC recognizing Hoechst 33258 analogue to the decadeoxyribonucleotide d-[CATGGCCATG]2: structural and dynamic aspects deduced from high field 1H-NMR studies.

The non-exchangeable and imino proton NMR resonances have been assigned of the 1:1 complex of an analogue 2 of Hoechst 33258 1 bound to the decadeoxyribonuycleotide d-[CATGGCCATG]2 by a combination of NOE difference, COSY and NOESYPH techniques. In contrast to Hoechst 33258 which recognizes 5'-AATT sequences exclusively, analogue 2 possesses structural features designed to permit the recognition of GC sites. The NOESY and 1D-NOE experiments place the drug in the minor groove and it is located on the 5'-CCAT sequence. The orientation of the drug in the groove is such as to place the N-methylpiperazine terminus at a GC site. Cross-correlation peaks in the NOESY experiment show that the DNA duplex retains its right-handed B form, similar to that in the free decamer. Specific NOEs locate the benzoxazole moiety on the 5'-CCAT and are consistent with the pyridine nitrogen forming a new hydrogen bond to G(4)-2NH2 at 5'-CCAT. The drug appears to undergo rotation around the C9-C10 bond, at a rate comparable with NMR time scale, even after binding. Variable temperature 1H-NMR studies established that the DNA is thermally stabilized as a result of the drug binding. The drug binding is a dynamic process involving exchange between the equivalent 5'-CCAT sites at approximately 60s-1 with delta G degree of 65 kJ mol-1 at 308K. The experimental evidence is in accord with a slide-swing mechanism for this process.     

Structural and dynamic aspects of the sequence specific binding of netropsin and its bis-imidazole analogue on the decadeoxyribonucleotide d-[CGCAATTGCG]2

High field 1H-NMR techniques have been used to examine the sequence dependent binding of a lexitropsin, the bis-imidazole analogue of netropsin 1, to the decadeoxyribonucleotide d-[CGCAATTGCG]2. The non-exchangeable and imino protons of the 1:1 lexitropsin:DNA complex are assigned by 1D-NOE difference and COSY methods. Addition of 1 to the DNA resulted in marked drug induced chemical shift changes of both the non-exchangeable and imino protons of A(4,5) and T(6,7). These results suggest that the lexitropsin is located in the minor groove along A(4) to T(7) of the DNA. Weaker chemical shift changes are observed for C(3) and G(8) which suggest that the bisimidazole moiety of 1 can also accept G.C sites. Specific NOEs seen between the lexitropsin (H2, H14 and H15) and DNA (AH2(4) and AH2(5] confirmed that the N to C-terminii of 1 is, on average, bound centrally to the sequence in the direction 5'-AATT-3'. However, netropsin 2 is shown to bind tightly only to the AATT sequence. Exchange NMR effects permit the estimate of the rate of exchange of the lexitropsin 1 between the two equivalent sites on the DNA to be approximately 160s and 24s for netropsin under comparable conditions. Several factors contributing to the sequence specificity of lexitropsin binding are discussed.     

Sequence Specific Molecular Recognition and Binding of a Monocationic Bis-Imidazole Lexitropsin to the Decadeoxyribonucleotide d-[(GATCCGTATG) (CATACGGATC)]: Structural and Dynamic Aspects of Intermolecular Exchange Studied by 1H-NMR

Abstract

The non-exchangeable and imino proton NMR resonances of the non self-complementary decadeoxyribonucleotide d-[(GATCCGTATG) · (GATACGGATC)] as well as those of the 1:1 complex of the monocatonic bis-imidazole lexitropsin 1 to this sequence have been assigned by using a combination of NOE difference, COSY and NOESY techniques. Confirmation of complete annealing of the two non self-complementary decamer strands to give the duplex decadeoxyribonucleotide is obtained by the detection of ten imino protons. It is established that the sugar-base orientations of all the bases in the duplex decamer are anti. From NOE studies, it is concluded that the duplex oligomer is right-handed and adopts a conformation in solution that belongs to the B family. A population analysis reveals that the sugar moieties exist predominantly in the S-form (2′-endo-3′-exo). Addition of 1 to the DNA solution leads to doubling of the resonances for CH6(4,5), GH8(6), TH6(7) and T-CH₃(7). The base, anomeric H1′ and imino proton signals for the base sequence 5′-CCGT undergo the most marked drug-induced chemical shift changes. These results provide evidence that the lexitropsin is bound to the sequence 5′-CCGT in the minor groove of the DNA NOE measurements between the amide protons (NH1 and NH4) and the imino proton (IV and V) signals confirmed the location and orientation of 1 in the 1:1 complex, with the amino terminus oriented to C(4). The specific binding of 1 to the sequence 5′-CCGT-3′ deduced in this study is in agreement with the footprinting data obtained using the Hind III/Nci I fragment from pBR322 DNA [Kissinger et al. 1987 (13)]. Intramolecular NOEs observed between H4 and H9 of the lexitropsin suggest that the molecule is not planar, but subjected to propeller twisting, in both the free and bound forms. Furthermore, NOE measurements permit assignment of the DNA duplex in the 1:1 complex to the B-form, which is similar to that of the free DNA The [(T₇A₈T₉)· (A₁₂T₁₃A₁₄)] segment of the DNA shows better stacking, by propeller twisting, compared to the rest of the molecule in the free as well as the complex forms. The intermolecular rate of exchange of 1 between the equivalent 5′-CCGT sites, at a concentration of 12 mM, is estimated to be ～88s^?1 at 308°K with ΔG≠ of 63±5 K.J mol^?1.     

Structural and dynamic aspects of non-intercalative (1:1) binding of a thiazole-lexitropsin to the decadeoxyribonucleotide d-[CGCAATTGCG]2: An 1H-NMR and molecular modeling study.

The location, orientation and dynamics of a thiazole-containing analogue of distamycin 1 bound to the decadeoxyribonucleotide d-[CGCAATTGCG]2 have been studied by non-exchangable and imino proton NMR resonances of the 1:1 complex. Using NOE difference, COSY and NOESY experiments, lexitropsin (1) was located in the minor groove of DNA at 5'-CAAT sequence. This was concluded by an intermolecular NOE between the ligand and a minor groove A4H2 proton. The NOE cross-correlations in the NOESY map confirmed that the DNA decamer duplex in the 1:1 complex remains in a right-handed B-conformation similar to that in the free decamer. Experiments on non-exchangeable and exchangeable proton NMR resonances placed the N-formylamino terminus of drug 1 on the 5'-C3 nucleotide, while the rest of the molecule extends onto the 5'-AAT sequence. The structural evidence for sequence preferential binding at 5'CAAT rather than 5'AATT suggests this reflects an attempt on the part of the sterically demanding inward directed sulfur of the thiazole to minimize compression by moving part of the molecule to the somewhat wider CG base site. The lack of evidence for a 2:1 drug:DNA complex, in contrast to distamycin, is in accord with this interpretation. The lexitropsin 1 was found to be in an exchange between the equivalent 5'-CAAT sites at a rate of approximately 35S-1 with a delta G degree of 65 +/- 5 kJ mol-1 at 303 K. The experimental data suggests a slide-swing mechanism for this exchange process.     

Structural and dynamic aspects of binding of a prototype lexitropsin to the decadeoxyribonucleotide d(CGCAATTGCG)2 deduced from high-resolution 1H NMR Studies

Structural and dynamic properties of the self-complementary decadeoxyribonucleotide d(CGCAATTGCG)2 and the interaction between a prototype lexitropsin, or information-reading oligopeptide, and the decadeoxyribonucleotide are deduced by using high-resolution 1H NMR techniques. The nonexchangeable and imino proton resonances of d(CGCAATTGCG)2 have been completely assigned by two-dimensional NMR studies. The decadeoxyribonucleotide exists as a right-handed B-DNA. In the 1H NMR spectrum of the 1:1 complex, the selective chemical shifts and removal of degeneracy of AH2(4), AH2(5), T-CH3(6), and T-CH3(7) due to the anisotropy effects of the heterocyclic moieties of the ligand, and with lesser effects at the flanking base sites C(3) and G(8), locate the drug centrally in the decadeoxyribonucleotide. This conclusion is supported by plots of individual chemical shift changes across the decadeoxyribonucleotide. Similarly, imino protons IV and V experience larger shifts and II and III smaller shifts in accord with this conclusion while drug complexation permits the detection of imino proton I. Strong nuclear Overhauser effects (NOEs) between pyrrole H5 and AH2(5), and weaker NOEs to AH1'(5), TH3'(6), and AH2'(5), firmly locate the ligand in the minor groove. Intraligand NOEs between the adjacent heterocyclic moieties indicate that the lexitropsin is subject to propeller twisting about the N6-C9 bond in both the bound and free forms. Nuclear Overhauser effect spectroscopy (NOESY) and correlated spectroscopy (COSY) experiments also indicate that the removal of degeneracy of the C16 methylene protons upon complexation may arise from restricted rotation about the C15-N9, C15-C16, and C16-C17 bonds. Specific hydrogen bonds between amide NH groups on the concave face of the ligand (N4H, N6H, N9H) and adenine N3 or thymine O2 on the floor of the minor groove are in accord with displacement of the hydration shell by the drug. NOE measurements on the decadeoxyribonucleotide in the 1:1 complex confirm it exists as a right-handed helix and belongs to the B family. Exchange NMR effects permit an estimate of a rate of approximately equal to 44 s-1 for the two-site exchange of the lexitropsin between two equivalent sites on the decamer with delta G++ approximately equal to 70 +/- 5 kJ mol-1 at 294 K. Alternative mechanisms for this exchange process are considered.     

Molecular recognition and binding of a GC site-avoiding thiazole-lexitropsin to the decadeoxyribonucleotide d-[CGCAATTGCG]2: 1H-NMR evidence for thiazole intercalation

The structural and dynamic aspects of the interaction of the thiazole containing lexitropsin (1) with an oligodeoxyribonucleotide were studied by high field 1H-NMR spectroscopy. Complete assignment of the 1H-NMR resonances of lexitropsin 1 was accomplished by 2D-NMR techniques. The complexation-induced chemical shifts and NOE cross peaks in the NOESY map of the 1:1 complex of lexitropsin (1) and d-[CGCAATTGCG]2 reveal that the thiazole ring of the lexitropsin (1) intercalates between dA4.A5 bases and the rest of the ligand resides in the minor groove of the AT rich core of decamer, thus occupying the 5'-AATT sequence on the DNA. Intercalation of the thiazole moiety of the drug has been detected by the presence of intermolecular NOEs both in the major and the minor groove of the decamer helix. The absence of intranucleotide NOEs between base protons and H1'/H2' protons suggested local unwinding of the binding site on the DNA. From COSY and NOESY methods of 2D-NMR, it was established that the N-formyl (amino) terminus of the thiazole lexitropsin (1) is projecting into the major groove towards A5H8 while the amidinium terminus lies in the minor groove towards the T7G8 base pairs of the opposite strand. The expected intranucleotide NOEs confirmed that the decadeoxyribonucleotide in the 1:1 complex exists in a right handed B-conformation. The presence of exchange signals along the binding site 5'-AATT indicated an exchange of the bound drug process wherein the rate of exchange between the two equivalent sites was estimated to be congruent to 130 s-1 at 30 degrees C and with delta G degrees of 62.4 kJ mol-1. Force field and Pi calculations permitted a rationalization of the experimentally observed binding mode in terms of preferred conformation of the ligand and repeat length in lexitropsins compared with the DNA receptor.     

Molecular recognition between oligopeptides and nucleic acids. Specificity of binding of a monocationic bis-furan lexitropsin to DNA deduced from footprinting and 1H NMR studies

MPE-Fe(EDTA) footprinting of a novel monocationic bis-furan lexitropsin 6 on a HindIII/EcoRI restriction fragment of pBR322 DNA revealed a series of four-base binding sites (all 5'----3') of (primary) TGTA, TGAA, AAAT, ACAA, TTAT, and (secondary) CTAA, TCGT, TGTA, GTCA, and GGTT. Thus 6 can accept a GC pair at positions 1, 2 or 3 of the binding site with a strict 3' (4 position) AT requirement. Marked enhancement of cleavage, particularly at GC rich sequences, is observed at regions flanking or even up to 18 base pairs remote from a given binding site. The non-exchangeable and imino 1H NMR resonances of the 1:1 complex and d-[CATGGCCATG]2 were assigned using a combination of NOE differences, NOESY and COSY techniques. 1H NMR studies (ligand induced chemical shifts and NOE differences) of Lexitropsin 6 with d-[CATGGCCATG]2 show unambiguously the location and orientation of the N to C termini of 6 on the sequence 5'-G5C6C7A8-3', with the C terminus oriented to A8. This orientation of 6 in the minor groove of 5'-GCCA is confirmed by an NOE observed between H1 2a of 6 and AH8(8). This preference for binding of 6 to the sequence 5'-GCCA when challenged with d-[CATGGCCATG]2 is in accord with the conclusions of the footprinting experiments wherein GC base pairs can be accepted in the first three positions and with a strict 3' terminus AT reading requirement. Collectively the data support the inference of a GC recognizing capacity for a 2,5-substituted furan moiety within a lexitropsin. The 1H NMR data indicate that the decadeoxyribonucleotide duplex exists in the B conformation in both the 1:1 complex and the free form. The apparent binding constant of 6 to calf thymus DNA is 1.68 X 10(5) M-1 whereas netropsin under similar conditions gives a value of 1.85 X 10(7) M-1. This suggests that if advantage is to be taken of the GC recognizing property of a 2,5-substituted furan in longer lexitropsins it should be flanked by more strongly bound moieties.     

Theoretical aspects of specific and non-specific equilibrium binding of proteins to DNA as studied by the nitrocellulose filter binding assay. Co-operative and non-co-operative binding to a one-dimensional lattice

The analysis of equilibrium binding isotherms obtained by methods such as the nitrocellulose filter binding assay, which measure the fraction. θ, of DNA to which at least one protein molecule is bound, as a function of the free protein concentration (L_F) require a different type of theoretical framework from that required for analysis of conventional equilibrium binding data, in which the number of moles of protein bound per mole of DNA, θ_c is measured as a function of L_F. The theoretical framework required to analyse equilibrium binding data generated by measuring θ(L_F) is developed for co-operative and non-co-operative binding of a protein to a large number of non-specific sites and to a specific sites(s) in the presence of a large number of non-specific sites on a DNA molecule. The theory is simple to apply, equations for θ(L_F) being easy to derive and evaluate, and is suitable for least-squares analysis. Two examples of the application of the theory to the analysis of experimental data are provided for the specific and non-specific binding of the EcoRI restriction endonuclease to bacteriophage λ DNA, and for the specific and non-specific binding of the enzyme dihydrofolate reductase from Lactobacillus casei to pBR322 and pWDLcB1 DNA, the latter differing from the former only in a 2.9 × 10³ base-pair insert containing the L. casei dihydrofolate reductase structural gene. The theoretical and experimental advantages and disadvantages of measuring θ(L_F) rather than θ_c(L_F) are discussed.     

Mechanism and binding specificity of beta-glucosidase-catalyzed hydrolysis of cellobiose analogues studied by competition enzyme kinetics monitored by 1H-NMR spectroscopy

The application of high-resolution 1H-NMR spectroscopy to monitor substrate and product time dependencies in progress curve enzyme kinetics is described with beta-glucosidase-catalyzed hydrolyses of cellobiose analogues as examples. It is demonstrated that inhibition patterns, relative binding specificities and catalytic rates can be inferred from competition experiments with two or more substrates. It could be concluded from competition experiments that substrates which form less stable enzyme-substrate complexes than methyl beta-cellobioside are hydrolyzed faster than this reference substrate when they are the sole substrate, due to a lower activation energy in the catalytic step, but that they are hydrolyzed slower than the reference compound in direct competition, due to the formation of the less stable enzyme-substrate complex in the binding step.     

Binding and interactions of L-BABP to lipid membranes studied by molecular dynamic simulations

Chicken liver bile acid-binding protein (L-BABP) is a member of the fatty acid-binding proteins super family. The common fold is a β-barrel of ten strands capped with a short helix-loop-helix motif called portal region, which is involved in the uptake and release of non-polar ligands. Using multiple-run molecular dynamics simulations we studied the interactions of L-BABP with lipid membranes of anionic and zwitterionic phospholipids. The simulations were in agreement with our experimental observations regarding the electrostatic nature of the binding and the conformational changes of the protein in the membrane. We observed that L-BABP migrated from the initial position in the aqueous bulk phase to the interface of anionic lipid membranes and established contacts with the head groups of phospholipids through the side of the barrel that is opposite to the portal region. The conformational changes in the protein occurred simultaneously with the binding to the membrane. Remarkably, these conformational changes were observed in the portal region which is opposite to the zone where the protein binds directly to the lipids. The protein was oriented with its macrodipole aligned in the configuration of lowest energy within the electric field of the anionic membrane, which indicates the importance of the electrostatic interactions to determine the preferred orientation of the protein. We also identified this electric field as the driving force for the conformational change. For all the members of the fatty acid-binding protein family, the interactions with lipid membranes is a relevant process closely related to the uptake, release and transfer of the ligand. The observations presented here suggest that the ligand transfer might not necessarily occur through the domain that directly interacts with the lipid membrane. The interactions with the membrane electric field that determine orientation and conformational changes described here can also be relevant for other peripheral proteins.     

Complete 1H assignments of the non-exchangeable protons of the non self-complementary heptadeoxyribonucleotide d[(GTCGTCA).(TGACGAC)] and its component strands by high field NMR

The non self complementary heptadeoxyribonucleotides d(GTCGTCA) and d(TGACGAC) were synthesized by the phosphotriester method. While complete 1H-NMR assignments of the former were obtained by a combination of one and two-dimensional techniques at room temperature, extensive stacking of the latter under these conditions dictated analysis at 50 degrees C when the lines were sharply resolved. The duplex form of the annealed strands under the conditions of the 1H-NMR experiment was established independently of the NMR evidence by 32P end labeling with T4 polynucleotide kinase followed by butt end joining using the absolute specificity of T4 ligase for double strand DNA. Analysis of the resulting ladder of polymers was performed using gel electrophoresis and autoradiography. Complete 1H-NMR assignments of the non-exchangeable protons in the self complementary heptamer was achieved. The assignments were confirmed using NOE differences, and two-dimensional COSY, and HH-INADEQUATE experiments at 400 and 500 MHz. The assignments are in accord with a conformation for the heptamer belonging to the B family of structures.     

Sequence specificity of the binding of 9-aminoacridine- and amsacrine-4-carboxamides to DNA studied by DNase I footprinting.

DNase I footprinting has been used to probe the sequence selectivity of binding of a series of intercalating amsacrine-4-carboxamides and a related 9-aminoacridine-4-carboxamide to three DNA restriction fragments. These ligands have good experimental antileukemic activity, and for those members of the series that gave evaluable footprints, our principal finding is that they bind preferentially to GC-rich regions in agreement with the conclusion of equilibrium and kinetic measurements. The highest affinity sites generally occur in clusters of GC base pairs with runs of AT pairs being excluded from binding. It is important to appreciate that the 9-aminoacridine- and amsacrine-4-carboxamides exhibit a very high degree of selectivity for GC sites which, to our knowledge, has not been previously matched by acridine derivatives in footprinting experiments. The principal determinant of specificity appears to be the 4-carboxamide group itself since neither variations in the terminal funtionality of the 4-carboxamide sidechain nor the presence of the 9-anilino substituent modifies sequence preferences. The molecular origins of selectivity may be discerned in terms of potential hydrogen bonding interactions between the 4-carboxamide moiety and carbonyl oxygen and amino groups of GC base pairs in the DNA minor groove at CG dinucleotide sites. The related therapeutic agent amsacrine failed to inhibit cleavage by DNase I, so no conclusion can be drawn concerning its binding selectivity, save to note that amsacrine does not possess the 4-carboxamide group which appears to be the crucial determinant of GC specificity. Whether selectivity for binding to GC-rich sequences is an important element in the antitumor activity of both the 9-aminoacridine- and amsacrine-4-carboxamides remains to be determined.     

Phlorizin recognition in a C-terminal fragment of SGLT1 studied by tryptophan scanning and affinity labeling

SGLT1 as a sodium/glucose cotransporter is strongly inhibited by phlorizin, a phloretin 2'-glucoside that has strong interactions with the C-terminal loop 13. We have examined phlorizin recognition by the protein by site-directed single Trp scanning mutagenesis experiments. Six mutants (Q581W, E591W, R601W, D611W, E621W, and L630W) of truncated loop 13 (amino acids 564-638) were expressed in Escherichia coli and purified to homogeneity. Changes in Trp quenching and positions of the emission maxima were determined after addition of phlorizin. D611W displayed the largest quenching of 80%, followed by R601W (67%). It also exhibited the maximum red shift in Trp fluorescence ( approximately 14 nm), indicating an exposure of this region to a more hydrophilic environment. Titration experiments performed for each mutant showed a similar affinity for all mutants, except for D611W, which exhibited a significantly lower affinity (Kd approximately 54 microm). Also the maximum change in the collisional quenching constant by acrylamide was noted for D611W (KSV = 11 m-1 in the absence of phlorizin and 55 m-1 in its presence). Similar results were obtained with phloretin. CD measurements and computer modeling revealed that D611W is positioned in a random coil situated between two alpha-helical segments. By combining gel electrophoresis, enzymatic fragmentation, and matrix-assisted laser desorption ionization mass spectrometry, we also analyzed truncated loop 13 photolabeled with 3-azidophlorizin. The attachment site of the ortho-position of aromatic ring B of phlorizin was localized to Arg-602. Taken together, these data indicate that phlorizin binding elicits changes in conformation leading to a less ordered state of loop 13. Modeling suggests an interaction of the 4- and 6-OH groups of aromatic ring A of phlorizin with the region between amino acids 606 and 611 and an interaction of ring B at or around amino acid 602. Phloretin seems to interact with the same region of the protein.     

Structure and conformation of the duplex consensus acceptor exon:intron junction d[(CpTpApCpApGpGpT). (ApCpCpTpGpTpApG)] deduced from high-field 1H-NMR of non-exchangeable and imino protons

The complementary consensus acceptor exon:intron junction d(ApCpCpTpGpTpApG) has been synthesized by a modified phosphotriester method. The non self-complementary octamer exists in the random coil form in aqueous buffer at 20 degrees C as evidenced by temperature variable 1H-NMR and NOE measurements. The non-exchangeable proton assignments were secured using a combination of techniques including two-dimensional COSY, NOESY and 1H-1H-INADEQUATE. The octamer was annealed with the primary consensus sequence d(CpTpApCpApGpGpT). Confirmation of complete duplex formation was confirmed by detection and assignment of imino protons in D2O:H2O mixtures. Assignment of the non-exchangeable proton signals in the duplex consensus junction was then secured by a combination of two-dimensional COSY correlations, NOESY and NOE experiments. Determination of individual vicinal coupling constants in the component deoxyribose moieties permitted deduction of the population of S conformations in this sequence. It is concluded that the consensus acceptor junction exists in solution in a conformation belonging to the B family, and that the bases are oriented anti. In addition the deoxyribose moieties in the 5' regions exist predominantly in the S form (2'endo-3'exo) whereas those residues on or adjacent to the junction on the primary strand show more N character (2'exo-3'endo). The contiguous bases A5-G6 (adjacent to the junction) and A15-G16 are stacked more closely than the other neighbor bases in this duplex sequence. These subtle structural and conformational differences in the exon:intron junction may serve as recognition signals for these critical sites in the genome.     

The DNA sequence at echinomycin binding sites determines the structural changes induced by drug binding: NMR studies of echinomycin binding to [d(ACGTACGT)]2 and [d(TCGATCGA)]2

The complexes formed between the cyclic octadepsipeptide antibiotic echinomycin and the two DNA octamers [d(ACGTACGT)]2 and [d(TCGATCGA)]2 have been investigated by using one- and two-dimensional proton NMR spectroscopy techniques. The results obtained for the two complexes are compared to each other, to the crystal structures of related DNA-echinomycin complexes, and to enzymatic and chemical footprinting results. In the saturated complexes, two echinomycin molecules bind to each octamer by bisintercalation of the quinoxaline moieties on either side of each CpG step. Binding of echinomycin to the octamer [d(ACGTACGT)]2 is cooperative so that only the two-drug complex is observed at lower drug-DNA ratios, but binding to [d(TCGATCGA)]2 is not cooperative. At low temperatures, both the internal and terminal A.T base pairs adjacent to the binding site in the [d(ACGTACGT)]2-2 echinomycin complex are Hoogsteen base paired (Gilbert et al., 1989) as observed in related crystal structures. However, as the temperature is raised, the internal A.T Hoogsteen base pairs are destabilized and are observed to be exchanging between the Hoogsteen base-paired and an open (or Watson-Crick base-paired) state. In contrast, in the [d(TCGATCGA)]2-2 echinomycin complex, no A.T Hoogsteen base pairs are observed, the internal A.T base pairs appear to be stabilized by drug binding, and the structure of the complex does not change significantly from 0 to 45 degrees C. Thus, the structure and stability of the DNA in echinomycin-DNA complexes depends on the sequence at and adjacent to the binding site. While we conclude that no single structural change in the DNA can explain all of the footprinting results, unwinding of the DNA helix in the drug-DNA complexes appears to be an important factor while Hoogsteen base pair formation does not.     

The structural and dynamic response of MAGI-1 PDZ1 with noncanonical domain boundaries to the binding of human papillomavirus E6

PDZ domains are protein interaction domains that are found in cytoplasmic proteins involved in signaling pathways and subcellular transport. Their roles in the control of cell growth, cell polarity, and cell adhesion in response to cell contact render this family of proteins targets during the development of cancer. Targeting of these network hubs by the oncoprotein E6 of “high-risk” human papillomaviruses (HPVs) serves to effect the efficient disruption of cellular processes. Using NMR, we have solved the three-dimensional solution structure of an extended construct of the second PDZ domain of MAGI-1 (MAGI-1 PDZ1) alone and bound to a peptide derived from the C-terminus of HPV16 E6, and we have characterized the changes in backbone dynamics and hydrogen bonding that occur upon binding. The binding event induces quenching of high-frequency motions in the C-terminal tail of the PDZ domain, which contacts the peptide upstream of the canonical X-[T/S]-X-[L/V] binding motif. Mutations designed in the C-terminal flanking region of the PDZ domain resulted in a significant decrease in binding affinity for E6 peptides. This detailed analysis supports the notion of a global response of the PDZ domain to the binding event, with effects propagated to distal sites, and reveals unexpected roles for the sequences flanking the canonical PDZ domain boundaries.