Relaxation Matrix Calculations Research Articles

Fast Quantitative Validation of 3D Models of Low-Affinity Protein-Ligand Complexes by STD NMR Spectroscopy.

Low-affinity protein-ligand interactions are important for many biological processes, including cell communication, signal transduction, and immune responses. Structural characterization of these complexes is also critical for the development of new drugs through fragment-based drug discovery (FBDD), but it is challenging due to the low affinity of fragments for the binding site. Saturation transfer difference (STD) NMR spectroscopy has revolutionized the study of low-affinity receptor-ligand interactions enabling binding detection and structural characterization. Comparison of relaxation and exchange matrix calculations with 1H STD NMR experimental data is essential for the validation of 3D structures of protein-ligand complexes. In this work, we present a new approach based on the calculation of a reduced relaxation matrix, in combination with funnel metadynamics MD simulations, that allows a very fast generation of experimentally STD-NMR-validated 3D structures of low-affinity protein-ligand complexes.

Combining weak affinity chromatography, NMR spectroscopy and molecular simulations in carbohydrate–lysozyme interaction studies

By examining the interactions between the protein hen egg-white lysozyme (HEWL) and commercially available and chemically synthesized carbohydrate ligands using a combination of weak affinity chromatography (WAC), NMR spectroscopy and molecular simulations, we report on new affinity data as well as a detailed binding model for the HEWL protein. The equilibrium dissociation constants of the ligands were obtained by WAC but also by NMR spectroscopy, which agreed well. The structures of two HEWL-disaccharide complexes in solution were deduced by NMR spectroscopy using (1)H saturation transfer difference (STD) effects and transferred (1)H,(1)H-NOESY experiments, relaxation-matrix calculations, molecular docking and molecular dynamics simulations. In solution the two disaccharides β-d-Galp-(1→4)-β-D-GlcpNAc-OMe and β-D-GlcpNAc-(1→4)-β-D-GlcpNAc-OMe bind to the B and C sites of HEWL in a syn-conformation at the glycosidic linkage between the two sugar residues. Intermolecular hydrogen bonding and CH/π-interactions form the basis of the protein-ligand complexes in a way characteristic of carbohydrate-protein interactions. Molecular dynamics simulations with explicit water molecules of both the apo-form of the protein and a ligand-protein complex showed structural change compared to a crystal structure of the protein. The flexibility of HEWL as indicated by a residue-based root-mean-square deviation analysis indicated similarities overall, with some residue specific differences, inter alia, for Arg61 that is situated prior to a flexible loop. The Arg61 flexibility was notably larger in the ligand-complexed form of HEWL. N,N'-Diacetylchitobiose has previously been observed to bind to HEWL at the B and C sites in water solution based on (1)H NMR chemical shift changes in the protein whereas the disaccharide binds at either the B and C sites or the C and D sites in different crystal complexes. The present study thus highlights that protein-ligand complexes may vary notably between the solution and solid states, underscoring the importance of targeting the pertinent binding site(s) for inhibition of protein activity and the advantages of combining different techniques in a screening process.

Group epitope mapping considering relaxation of the ligand (GEM-CRL): Including longitudinal relaxation rates in the analysis of saturation transfer difference (STD) experiments

In the application of saturation transfer difference (STD) experiments to the study of protein–ligand interactions, the relaxation of the ligand is one of the major influences on the experimentally observed STD factors, making interpretation of these difficult when attempting to define a group epitope map (GEM). In this paper, we describe a simplification of the relaxation matrix that may be applied under specified experimental conditions, which results in a simplified equation reflecting the directly transferred magnetisation rate from the protein onto the ligand, defined as the summation over the whole protein of the protein–ligand cross-relaxation multiplied by with the fractional saturation of the protein protons. In this, the relaxation of the ligand is accounted for implicitly by inclusion of the experimentally determined longitudinal relaxation rates. The conditions under which this “group epitope mapping considering relaxation of the ligand” (GEM-CRL) can be applied were tested on a theoretical model system, which demonstrated only minor deviations from that predicted by the full relaxation matrix calculations (CORCEMA-ST) [7]. Furthermore, CORCEMA-ST calculations of two protein–saccharide complexes (Jacalin and TreR) with known crystal structures were performed and compared with experimental GEM-CRL data. It could be shown that the GEM-CRL methodology is superior to the classical group epitope mapping approach currently used for defining ligand–protein proximities. GEM-CRL is also useful for the interpretation of CORCEMA-ST results, because the transferred magnetisation rate provides an additional parameter for the comparison between measured and calculated values. The independence of this parameter from the above mentioned factors can thereby enhance the value of CORCEMA-ST calculations.

Specific Methyl Group Protonation for the Measurement of Pharmacophore-Specific Interligand NOE Interactions

Pharmacophore signature at glance: INPHARMA NOE interactions can be measured between two ligands that bind competitively to a common macromolecular receptor, and can be interpreted quantitatively to derive the binding mode of the two ligands. This approach relies on computationally demanding full relaxation matrix calculations. Here we demonstrate that the INPHARMA NOEs measured in the presence of a selectively-protonated receptor (see figure) can be interpreted in a semiquantitative way to discriminate between binding poses, thus relieving for the need of demanding computations.

Protein NMR Recall, Precision, and F-measure Scores (RPF Scores): Structure Quality Assessment Measures Based on Information Retrieval Statistics

One of the most important challenges in modern protein NMR is the development of fast and sensitive structure quality assessment measures that can be used to evaluate the "goodness-of-fit" of the 3D structure with NOESY data, to indicate the correctness of the fold and accuracy of the resulting structure. Quality assessment is especially critical for automated NOESY interpretation and structure determination approaches. This paper describes new NMR quality assessment scores, including Recall, Precision, and F-measure scores (referred to here are "NMR RPF" scores), which quickly provide global measures of the goodness-of-fit of the 3D structures with NOESY peak lists using methods from information retrieval statistics. The sensitivity of the F-measure is improved using a scaled Fold Discriminating Power (DP) score. These statistical RPF scores are quite rapid to compute since NOE assignments and complete relaxation matrix calculations are not required. A graphical method for site-specific assessment of structure quality based on the Precision statistic is also described. These statistical measures are demonstrated to be valuable for assessing protein NMR structure accuracy. Their relationships to other proposed NMR "R-factors" and structure quality assessment scores are also discussed.

Effect of bulky lesions on DNA: solution structure of a DNA duplex containing a cholesterol adduct.

The three-dimensional solution structure of two DNA decamers of sequence d(CCACXGGAAC)-(GTTCCGGTGG) with a modified nucleotide containing a cholesterol derivative (X) in its C1 '(chol)alpha or C1 '(chol)beta diastereoisomer form has been determined by using NMR and restrained molecular dynamics. This DNA derivative is recognized with high efficiency by the UvrB protein, which is part of the bacterial nucleotide excision repair, and the alpha anomer is repaired more efficiently than the beta one. The structures of the two decamers have been determined from accurate distance constraints obtained from a complete relaxation matrix analysis of the NOE intensities and torsion angle constraints derived from J-coupling constants. The structures have been refined with molecular dynamics methods, including explicit solvent and applying the particle mesh Ewald method to properly evaluate the long range electrostatic interactions. These calculations converge to well defined structures whose conformation is intermediate between the A- and B-DNA families as judged by the root mean square deviation but with sugar puckerings and groove shapes corresponding to a distorted B-conformation. Both duplex adducts exhibit intercalation of the cholesterol group from the major groove of the helix and displacement of the guanine base opposite the modified nucleotide. Based on these structures and molecular dynamics calculations, we propose a tentative model for the recognition of damaged DNA substrates by the UvrB protein.

Synthesis and conformational analysis of a pentasaccharide corresponding to the cell-wall polysaccharide of the Group A Streptococcus

The synthesis and conformational analysis of a pentasaccharide corresponding to a fragment of the cell-wall polysaccharide (CWPS) of the bacteria Streptococcus Group A are described. The polysaccharide consists of alternating α-(1→2)- and α-(1→3)-linked l-rhamnopyranose (Rha p) residues with branching 2-acetamido-2-deoxy- d-glucopyranose (Glc pNAc) residues linked β-(1→3) to alternate rhamnose rings. The pentasaccharide is of interest as a possible terminal unit on the CWPS, for use in a vaccine. The syntheses employed a trichloroacetimidate glycosyl donor. Molecular dynamics ( md) calculations of the pentasaccharide with the force fields cvff and parm22, both in gas phase and with explicit water present, gave different predictions for the flexibility and preferred conformational space. Metropolis Monte Carlo ( mmc) calculations with the hsea force field were also performed. Experimental data were obtained from 1D transient NOE measurements. Complete build-up curves were compared to those obtained by full relaxation matrix calculations in order to derive a model of the conformation. Overall, the best fit between experimental and calculated data was obtained with mmc simulations using the hsea force field. Molecular dynamics and mmc simulations of a tetrasaccharide corresponding to the Group A-variant polysaccharide, which differs in structure from Group A in lacking the Glc pNAc residues, were also performed for purposes of comparison.

Structure of a biologically active fragment of human serum apolipoprotein C-II in the presence of sodium dodecyl sulfate and dodecylphosphocholine

We have studied the three-dimensional structure of a biologically active peptide of apolipoprotein C-II (apoC-II) in the presence of lipid mimetics by CD and NMR spectroscopy. This peptide, corresponding to residues 44–79 of apoC-II, has been shown to reverse the symptoms of genetic apoC-II deficiency in a human subject. A comparison of α-proton secondary shifts and CD spectroscopic data indicates that the structure of apoC-II(44–79) is similar in the presence of dodecylphosphocholine and sodium dodecyl sulfate. The three-dimensional structure of apoC-II(44–79) in the presence of sodium dodecyl sulfate, determined by relaxation matrix calculations, contains two amphipathic helical domains formed by residues 50–58 and 67–75, separated by a non-helical linker centered at Tyr63. The C-terminal helix is terminated by a loop formed by residues 76–79. The C-terminal helix is better defined and has a larger hydrophobic face than the N-terminal helix, which leads us to propose that the C-terminal helix together with the non-helical Ile66 constitute the primary lipid binding domain of apoC-II(44–79). Based on our structure we suggest a new mechanism of lipoprotein lipase activation in which both helices of apoC-II(44–79) remain lipid bound, while the seven-residue interhelical linker extends away from the lipid surface in order to project Tyr63 into the apoC-II binding site of lipoprotein lipase.

Intercalation of the (-)-(1R,2S,3R, 4S)-N6-[1-benz[a]anthracenyl]-2'-deoxyadenosyl adduct in an oligodeoxynucleotide containing the human N-ras codon 61 sequence.

The solution structure of the (-)-(1R,2S,3R,4S)-N6-[1-(1,2,3, 4-tetrahydroxy-benz[a]anthracenyl)]-2'-deoxyadenosyl adduct at X6 of 5'-d(CGGACXAGAAG)-3'.5'-d(CTTCTTGTCCG)-3', incorporating codons 60, 61(italic), and 62 of the human N-ras protooncogene, was determined. This adduct results from the trans opening of 1S,2R,3R,4S-1, 2-epoxy-1,2,3,4-tetrahydro-benz[a]anthracenyl-3,4-diol by the exocyclic N6 of adenine. Molecular dynamics simulations were restrained by 509 NOEs from 1H NMR. The precision of the refined structures was monitored by pairwise root-mean-square deviations which were <1.2 A; accuracy was measured by complete relaxation matrix calculations, which yielded a sixth root R factor of 9.1 x 10(-)2 at 250 ms. The refined structure was a right-handed duplex, in which the benz[a]anthracene moiety intercalated from the major groove between C5.G18 and R,S,R,SA6.T17. In this orientation, the saturated ring of BA was oriented in the major groove of the duplex, with the aromatic rings inserted into the duplex such that the terminal ring of BA threaded the duplex and faced toward the minor groove direction. The duplex suffered localized distortion at and immediately adjacent to the adduct site, evidenced by the increased rise of 8.8 A as compared to the value of 3.5 A normally observed for B-DNA between base pairs C5.G18 and R,S,R,SA6.T17. These two base pairs also buckled in opposite directions away from the intercalated BA moiety. The refined structure was similar to the (-)-(7S,8R,9S,10R)-N6-[10-(7,8,9, 10)-tetrahydrobenzo[a]pyrenyl)]-2'-deoxyadenosyl adduct of corresponding stereochemistry at X6 of the same oligodeoxynucleotide [Zegar, I. S., Kim, S. J., Johansen, T. N., Horton, P. J., Harris, C. M., Harris, T. M., and Stone, M. P. (1996) Biochemistry 35, 6212-6224]. Both adducts intercalated toward the 5'-direction from the site of adduction. The similarities in solution structures were reflected in similar biological responses, when repair-deficient AB2480 Escherichia coli were transformed with M13mp7L2 DNA site-specifically modified with these two adducts.

Yeast hexokinase PII--bound nucleotide conformation at the active site.

The structure of adenine nucleotide bound at the active site of yeast hexokinase PII (PII) was studied in the complexes PII x ADPMg(II), PII x ADPMg(II) x Glc and PII x ADPMg(II) x NO3- x Glc using two-dimensional transferred NOE spectroscopy. Binding of the nucleotide ligand to the enzyme resulted in downfield shift of several ligand resonances. Changes in the chemical shift as a function of ligand concentration indicate that various resonances in the bound and free form of the ligand appear to be in fast exchange. The largest chemical shift change between the bound and the free states (delta vM = 88 +/- 9 Hz) at an NMR frequency of 500 MHz was observed for the H2 resonance of the adenine ring. A lower limit for the rate of ligand dissociation from the complex derived from these results is k(off) >> 550 s(-1). Interproton NOEs for various ligand protons in PII x ADPMg(II), PII x ADPMg(II) x Glc and PII x ADPMg(II) x NO3- x Glc complexes were measured at 500 MHz at 10 degrees C. The NOE buildup curves constructed from such measurements made at various mixing times (40, 80, 120, 160 and 200 ms) were analyzed using complete relaxation matrix calculations and various interproton distances were determined. These distances were used in restrained molecular dynamics calculations to derive the conformation of the nucleotide bound at the active site of the enzyme. The results of these calculations indicate that the nucleotide binds in an anti conformation. The glycosidic torsion angle chi (O4'-C1'-N9-C8) determined for the nucleotide in PII x ADPMg(II), PII x ADPMg(II) x Glc and PII x ADPMg(II) x NO3- x Glc complexes are 68 +/- 4 degrees, 52 +/- 4 degrees and 49 +/- 4 degrees respectively. In all these complexes, the ribose pucker is best represented by the unsymmetrical O4'-endo-C1'-exo twist ((o)T1). The observed decrease in the glycosidic torsion angle of the bound nucleotide is attributed to the glucose-induced conformational changes in the enzyme. The structure of the nucleotide derived here is at variance from the one proposed on the basis of X-ray crystallography and model building [Shoham, M. & Steitz, T. A. (1980) J. Mol. Biol. 140, 1-14].

High resolution solution structure of a DNA duplex alkylated by the antitumor agent duocarmycin SA

The three-dimensional solution structure of duocarmycin SA in complex with d-(G 1ACTAATTGAC 11)·d-(G 12TCATT A GTC 22) has been determined by restrained molecular dynamics and relaxation matrix calculations using experimental NOE distance and torsion angle constraints derived from 1H NMR spectroscopy. The final input data consisted of a total of 858 distance and 189 dihedral angle constraints, an average of 46 constraints per residue. In the ensemble of 20 final structures, there were no distance constraint violations >0.06 Å or torsion angle violations >0.8°. The average pairwise root mean square deviation (RMSD) over all 20 structures for the binding site region is 0.57 Å (average RMSD from the mean: 0.39 Å). Although the DNA is very B -like, the sugar-phosphate backbone torsion angles β, ϵ, and ζ are distorted from standard values in the binding site region. The structure reveals site-specific bonding of duocarmycin SA at the N3 position of adenine 19 in the AT-rich minor groove of the duplex and binding stabilization via hydrophobic interactions. Comparisons have been made to the structure of a closely related complex of duocarmycin A bound to an AT-rich DNA duplex. These results provide insights into critical aspects of the alkylation site selectivity and source of catalysis of the DNA alkylating agents, and the unusual stability of the resulting adducts.

Solution conformation of an abasic DNA undecamer duplex d(CGCACXCACGC) x d(GCGTGTGTGCG): the unpaired thymine stacks inside the helix.

The three-dimensional structural analysis of DNA undecamer 5'd(C1G2C3A4C5X6C7A8C9G10C11)3', 3'd(G22C21G20T19G18T17G16T15G14C13G12)5' duplex in which the X residue is a modified abasic site [3-hydroxy-2-(hydroxymethyl)tetrahydrofuran] has been performed using NOESY, DQFCOSY, TOCSY, and 31P-1H HSQC-TOCSY spectra in relation with molecular dynamics simulations. A total of 249 distances and 224 dihedral angles were used for construction. The optimal distances were calculated using the complete relaxation matrix method from hybrid matrices which were built with the experimental NOE intensities and additional data derived from either standard A- or B-DNA. Six independent refined structures starting from canonical A- and B-DNA were determined on the basis of the NMR data, and all converged to a single family with average rms deviations below 0.6 A and final NOE Rx factors of 0.055 +/- 0.03. A satisfactory agreement was obtained between measured NOE intensities and those resulting from full relaxation matrix calculations. A single intrahelical form of right-handed DNA duplex is observed; the aromatic base of residue T17 opposite the abasic site is stacked inside the helix. No clear correlation was detected between the C5 and C7 residues, excluding their proximity and the looping out of the abasic site. The abasic site induces a kink of about 30 degrees in the DNA duplex. This kink allows the formation of a bifurcated hydrogen bond between the amino protons of C5 and the O4 oxygen of T17. A detailed analysis of the final structures and their comparison with previous studies of abasic site lesions are described.

Refined Structure of lacRepressor Headpiece (1-56) Determined by Relaxation Matrix Calculations from 2D and 3D NOE Data: Change of Tertiary Structure upon Binding to the lacOperator

The solution structure of the DNA binding domain of lacrepressor (headpiece 1-56; HP56) has been refined using data from 2D and 3D NMR spectroscopy. The structure was derived from 1546 restraints (giving an average of 27.6 per residue), comprising 389 intraresidual, 402 sequential, 385 medium range and 325 long range distance restraints and also 30 φ and 15 χ 1dihedral angle restraints. The structures were determined by the method of direct refinement against nuclear Overhauser enhancement peak volumes with the program DINOSAUR. The final set of 32 selected structures displayed an r.m.s. deviation from the average of 0.43(±0.08) Å (backbone) and 0.95(±0.08) Å (all heavy atoms) for the best defined region of the protein (residues 3 to 49). The ensemble R-factor was 0.35, which indicates close correspondence with the experimental data. The structures revealed good stereochemical qualities. The conformations of the NMR structures of free and DNA complexed lacrepressor headpiece were compared. The regions comprising the secondary structure elements show close correspondence for both conformations. However, the conformation of the loop between helix II and III changes considerably upon complexation of the headpiece. This change in the conformation of the loop in lacHP56 is essential for binding of the side-chains of residues Asn25 and His29 to the lacoperator DNA. Finally, the lacheadpiece residues that are intolerant to mutations were analysed. Most of these mutation-sensitive residues are important for a correct folding of the headpiece region, and a number of these residues are also involved in contacting the operator DNA.

Adduction of the human N-ras codon 61 sequence with (-)-(7S,8R,9R,10S)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene: structural refinement of the intercalated SRSR(61,2) (-)-(7S,8R,9S,10R)-N6-[10-(7,8,9,10- tetrahydrobenzo[a]pyrenyl)]-2'-deoxyadenosyl adduct from 1H NMR.

The structure of the (-)-(7S,8R,9S,10R)-N6-[10-(7,8,910-tetrahydrobenzo [a]pyrenyl)]-2'-deoxyadenosyl adduct at X6 of 5'-d(CGGACXAGAAG)-3'-5'-d(CTTCTTGTCCG)-3', derived from trans addition of the exocyclic N6-amino group of dA to (-)-(7S,8R,9R,10S)-7, 8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-DE2], was determined using molecular dynamics simulations restrained by 369 NOEs from 1H NMR. This was named the SRSR(61,2) adduct, derived from the N-ras protooncogene at and adjacent to the nucleotides encoding amino acid 61 (underlined) of the p21 gene product. NOEs between C5, S.R.S.R A6, and A7 were disrupted, as were those between T17 and G18. NOEs between benzo[a]pyrene and DNA protons were localized on the two faces of the pyrenyl ring. The benzo[a]pyrene H3-H6 protons showed NOEs to T17 CH3, while H1, H2, and H3 showed NOEs to T17 deoxyribose; the latter protons and H4 showed NOEs to T17 H2', H2" and to T17 H6. Noes were observed between H11 and H12 and C5 H]',H2', H2". G18 N1H showed NOEs to both faces of benzo[a]pyrene. Upfield shifts of 2.6 ppm for T17 N3H and 1.8 ppm for G18 N1H. 1 ppm for T17 H6 and CH3, and 0.75 ppm for C5 H5, with a smaller shift for C5 H6, and a 1.5 ppm dispersion of the pyrenyl protons suggested that benzo[a]pyrene intercalated above the 5'-face of S.R.S.R A6. The precision of the refined structures was monitored by pairwise root mean square deviations. which were < 1.5 A; accuracy was measured by complete relaxation matrix calculations, which yielded a sixth root R factor of 8.1 x 10(-2). Interstrand stacking between the pyrenyl ring and the T17 pyrimidine and G18 purine rings was enhanced by the bay ring. Changes of +30 degrees and -25 degrees in buckle for C5.G18 and S.R.S.R A6.T17, respectively, were calculated, as was a -40 degrees change in propeller twist for C5.G18. The rise between C5.G18 and S.R.S.R A6.T17 was calculated to be 7 A. The work extended the pattern for adenine N6 benzo[a]pyrene adducts, in which the R stereochemistry at C10 predicted 5'-intercalation of the pyrenyl moiety.

Interproton distance bounds from 2D NOE intensities: Effect of experimental noise and peak integration errors

The effect of experimental and integration errors on the calculation of interproton distances from NOE intensities is examined. It is shown that NOE intensity errors can have a large impact on the distances determined. When multiple spin ('spin diffusion') effects are significant, the calculated distances are often underestimated, even when using a complete relaxation matrix analysis. In this case, the bias of distances to smaller values is due to the random errors in the NOE intensities. We show here that accurate upper and lower bounds of the distances can be obtained if the intensity errors are properly accounted for in the complete relaxation matrix calculations, specifically the MARDIGRAS algorithm. The basic MARDIGRAS algorithm has been previously described [Borgias, B.A. and James, T.L. (1990) J. Magn. Reson., 87, 475-487]. It has been shown to provide reasonably good interproton distance bounds, but experimental errors can compromise the quality of the resulting restraints, especially for weak cross peaks. In a new approach introduced here, termed RANDMARDI (random error MARDIGRAS), errors due to random noise and integration errors are mimicked by the addition of random numbers from within a specified range to each input intensity. Interproton distances are then calculated for the modified intensity set using MARDIGRAS. The distribution of distances that define the upper and lower distance bounds is obtained by using N randomly modified intensity sets. RANDMARDI has been used in the solution structure determination of the interstrand cross-link (XL) formed between 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and the DNA oligomer d(5'-GCGTACGC-3')2 [Spielmann, H.P. et al. (1995) Biochemistry, 34, 12937-12953]. RANDMARDI generates accurate distances bounds from the experimental NOESY cross-peak intensities for the fixed (known) interproton distances in XL. This provides an independent internal check for the ability of RANDMARDI to accurately fit the experimental data. The XL structure determined using RANDMARDI-generated restraints is in good agreement with other biophysical data that indicate that there is no bend introduced into the DNA by the cross-link. In contrast, isolated spin-pair approximation calculations give distance restraints that, when applied in a restrained molecular dynamics protocol, produce a bent structure.

Solution structure of the Na+ form of the dimeric guanine quadruplex [d(G3T4G3)]2.

The solution structure of the DNA quadruplex formed by the association of two strands of the DNA oligonucleotide, d(G3T4G3), in NaCl solution has been determined by 1H two-dimensional NMR techniques, full relaxation matrix calculations and restrained molecular dynamics. The refined structure incorporates the sequences 5'-G1sG2AG3AT4AT5AT6AT7AG8sG9AG10A-3' and 5'-G11sG12AG13AT14AT15AT16AT17AG18sG19sG20A-3' (where S and A denote syn and anti, respectively) in a three-quartet, diagonal-looped structure that we [Strahan, G. D., Shafer, R. H. & Keniry, M. A. (1994) Nucleic Acids Res. 22, 5447-5455] and others [Smith, F. W., Lau, F. W. & Feigon, J. (1994) Proc. Natl. Acad. Sci. USA 91, 10546-10550] have described. The loop structure is compact and incorporates many of the features found in duplex hairpin loops including base stacking, intraloop hydrogen bonding and extensive van der Waals' interactions. The first and third loop thymines stack over the outermost G-quartet and are also associated by hydrogen bonding. The second and the fourth loop thymines fold inwards in order to enhance van der Waals' interactions. The unexpected sequential syn-syn deoxyguanosines in the quadruplex stem appear to be a direct consequence of the way DNA oligonucleotides fold and the subsequent search for the most stable loop structure. The implications of loop sequence and length on the structure of quadruplexes are discussed.

Refined solution structure of 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 opposite CpA in the complementary strand of an oligodeoxynucleotide duplex as determined by 1H NMR.

The solution structure of d(CCATCAFBGATCC).d(GGATCAGATGG), containing the 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct, was refined using molecular dynamics restrained by NOE data obtained from 1H NMR. The modified guanosine was positioned opposite cytosine, while the aflatoxin moiety was positioned opposite adenosine in the complementary strand. Sequential 1H NOEs were interrupted between C5 and AFBG6, but intrastrand NOEs were traced through the aflatoxin moiety, via H6a of aflatoxin and H8 of the modified guanine. Opposite the lesion, the NOE between A16 H1' and G17 H8 was weak. A total of 43 NOEs were observed between DNA protons and aflatoxin protons. Molecular dynamics calculations restrained with 259 experimental and empirical distances, and using sp2 hybridization at AFBG6 N7, refined structures with pairwise rms differences < 0.85 A, excluding terminal base pairs. Relaxation matrix calculations yielded a sixth root rms difference between refined structures and NOE intensity data of 7.3 x 10(-2). The aflatoxin moiety intercalated on the 5'-face of the modified guanine. The extra adenine A16 was inserted between base pair AFBG6.C15 and the aflatoxin moiety. A 36 degree bending between the plane of base pair AFBG6.C15 and the plane of the aflatoxin moiety was predicted. The aflatoxin moiety stacked below the top domain of the oligodeoxynucleotide, which consisted of base pairs C1.G21, C2.G20, A3.T19, T4.A18, and C5.G17. The bottom domain consisted of base pairs AFBG6.C15, A7.T14, T8.A13, C9.G12, and C10.G11. The average winding angle between base pair C5.G17, the intercalated aflatoxin moiety, A16, and base pair AFBG6.C15 was reduced to 10 degrees. The preponderance of base pair substitutions in the aflatoxin B1 mutational spectrum, particularly G-->T transversions, suggests that the stability of this modified oligodeoxynucleotide, which models a templated +1 addition mutation, does not reliably predict the frequency of frame shifts.

Major groove (R)-alpha-(N6-adenyl)styrene oxide adducts in an oligodeoxynucleotide containing the human N-ras codon 61 sequence: conformations of the R(61,2) and R(61,3) sequence isomers from 1H NMR.

Conformations of (R)-alpha-(N6-adenyl)styrene oxide adducts at positions X6 in d(CGGACXAGAAG).d(CTTCTTGTCCG) and X7 in d(CGGACAXGAAG).d(CTTCTTGTCCG), incorporating codons 60, 61 (underlined), and 62 of the human n-ras protooncogene, were refined from 1H NMR data. These were the R(61,2) and R(61,3) adducts. Chemical shift perturbations were in the 5'-direction from the sites of adduction; large changes were observed for C5 H5 and H6 in the R(61,2) adduct. The styrene moieties were only partially defined by NOE data. Spectral overlap, particularly for the R(61,2) adduct, prevented complete assignments of the aromatic resonances; likewise, there were insufficient data to orient the CH2OH moieties. Ring flips were slow on the NMR time scale. For the R(61,2) adduct 260 restraints were obtained from NOE data at three mixing times using relaxation matrix analysis; for the R(61,3) adduct 230 restraints were obtained. Structures emergent from molecular dynamics/simulated annealing for the R(61,2) adduct converged to average and maximum pairwise rms differences of 1.3 and 1.7 A, respectively, while those for the R(61,3) adduct converged to average and maximum pairwise rms differences of 1.2 and 1.6 A. Sixth root residual indices of 7.5 and 6.8 x 10(-2) were measured between the refined structures and NOE intensities using relaxation matrix calculations for the R(61,2) and R(61,3) adducts, respectively. The styrene rings were in the 5'-direction from the lesion sites in the major groove. The preferred orientation calculated for the R(61,2) adduct placed the styrene ring edgewise and approximately orthogonal to C5, while that calculated for the R(61,3) duplex had the styrene ring approximately orthogonal to the major groove edges of base pairs A6.T17 and R-SOA7.T16.

Solution Structure of (Cd2+)1-calbindin D9kReveals Details of the Stepwise Structural Changes along the Apo*rarr;(Ca2+)1II*rarr;(Ca2+)2I,IIBinding Pathway

The three-dimensional solution structure of (Cd2+)1-calbindin D9khas determined by distance geometry, restrained molecular dynamics and relaxation matrix calculations using experimental constraints obtained from two-dimensional1H and15N-1H NMR spectroscopy. The final input data consisted of 1055 NOE distance constraints and 71 dihedral angle constraints, corresponding to 15 constraints per residue on average. The resulting ensemble of 24 structures has no distance or dihedral angle constarints consistently violated by more than 0.07 Å and 1.8°, respectively. The structure is characteristic of an EF-hand protein, with two helix-loop-helix calcium binding motifs joined by a flexible linker, and a short anti-parallel β-type interaction between the two ion-binding sites. The four helices are well defined with a root mean square deviation from the mean coordinates of 0.35 Å for the backbone atoms. The structure of the half-saturated cadmium state was compared with the previously determined solution structures of the apo and fully calcium saturated calbindin D9k. The comparisons were aided by introducing the ensemble averaged distance difference matrix as a tool for analyzing differences between two ensembles of structures. Detailed analyses of differences between the three states in backbone and side-chain dihedral angles, hydrogen bonds, interatomic distances, and packing of the hydrophobic core reveal the reorganization of the protein that occurs upon ion binding. Overall, it was found that (Cd2+)1-calbindin D9k, representing the half-saturated calcium state with an ion in site II, is structurally more similar to the fully calcium-saturated state than the apo state. Thus, for the binding sequence apo→(Ca2+)1II→(Ca2+)2I,II, the structural changes occurring upon ion binding are most pronounced for the first binding step, an observation that bears significantly on the molecular basis for cooperative calcium binding in calbindin D9k.

Structure refinement of the glucocorticoid receptor-DNA binding domain from NMR data by relaxation matrix calculations.

The solution structure of the glucocorticoid receptor (GR) DNA-binding domain (DBD), consisting of 93 residues, has been refined from two and three-dimensional NMR data using an ensemble iterative relaxation matrix approach followed by direct NOE refinement with DINOSAUR. A set of 47 structures of the rat GR fragment Cys440-Arg510 was generated with distance geometry and further refined with a combination of restrained energy minimization and restrained molecular dynamics in a parallel refinement protocol. Distance constraints were obtained from an extensive set of NOE build-up curves in H2O and 2H2O via relaxation matrix calculations (1186 distance constraints from NOE intensities, 10 phi and 22 chi 1 dihedral angle constraints from J- coupling data were used for the calculations). The root-mean-square deviation values of the 11 best structures on the well-determined part of the protein (Cys440 to Ser448, His451 to Glu469 and Pro493 to Glu508) are 0.60 A and 1.20 A from the average for backbone and all heavy atoms, respectively. The final structures have R-factors around 0.40 and good stereochemical qualities. The first zinc-coordinating domain of the GR DBD is very similar to the crystal structure with a root-mean-square difference of 1.4 A. The second zinc-coordinating domain is still disordered in solution. No secondary structure element is found in this domain in the free state. As suggested by crystallographic studies on the estrogen receptor DBD-DNA and GR DBD-DNA complexes, part of this region will form a distorted helix and the D-box will undergo a conformational change upon cooperative binding to DNA.