Sequence-dependent variation in DNA minor groove width dictates orientational preference of Hoechst 33258 in A-tract recognition: solution NMR structure of the 2:1 complex with d(CTTTTGCAAAAG)(2).

The solution structure of the dodecamer duplex d(CTTTTGCAAAAG)(2)and its 2:1 complex with the bis -benzimidazole Hoechst 33258 has been investigated by NMR and NOE-restrained molecular dynamics (rMD) simulations. Drug molecules are bound in each of the two A-tracts with the bulky N-methylpiperazine ring of each drug located close to the central TG (CA) step, binding essentially to the narrow minor groove of each A-tract. MD simulations over 1 ns, using an explicit solvation model, reveal time-averaged sequence-dependent narrowing of the minor groove from the 3'-end towards the 5'-end of each TTTT sequence. Distinct junctions at the TpG (CpA) steps, characterised by large positive roll, low helical and propeller twists and rapid AT base pair opening rates, add to the widening of the groove at these sites and appear to account for the bound orientation of the two drug molecules with the N-methylpiperazine ring binding in the wider part of the groove close to the junctions. Comparisons between the free DNA structure and the 2:1 complex (heavy atom RMSD 1.55 A) reveal that these sequence-dependent features persist in both structures. NMR studies of the sequence d(GAAAAGCTTTTC)(2), in which the A-tracts have been inverted with the elimination of the TpG junctions, results in loss of orientational specificity of Hoechst 33258 and formation of multiple bound species in solution, consistent with the drug binding in a number of different orientations.

The structure of an asymmetric dimer relevant to the mode of action of the glycopeptide antibiotics.

BACKGROUND: Glycopeptide antibiotics of the vancomycin group are of crucial clinical importance in the treatment of methicillin resistant Staphylococcus aureus (MRSA)--the often lethal 'super-bug'--characterized by its resistance to a wide range of antibiotics in common use. The antibiotics exert their physiological action by blocking cell wall synthesis through recognition of nascent cell wall mucopeptides terminating in the sequence -D-Ala-D-Ala. Evidence suggests that the antibiotics are able to enhance their biological activity by the formation of homodimers, and this is supported by the observation that dimerization and peptide binding in vitro are cooperative phenomena. The basis of this enhancement is not understood at the molecular level. RESULTS: The first detailed structure of a dimeric glycopeptide antibiotic, that of eremomycin, is presented based upon solution NMR data. The overall structure of the dimer complex is asymmetric. The source of this asymmetry--a parallel alignment and mutual interaction of the disaccharides--appears to promote dimerization through specific sugar-sugar recognition. CONCLUSIONS: A molecular basis for the observed cooperativity of cell wall peptide binding by eremomycin is evident from these studies of the dimer. The carboxylate anion of the cell wall component, which is crucial to binding, forms an amide-mediated ion-pair interaction to the alkylammonium ion of the ring 6 sugar in the other half of the dimer making the structure and positioning of this sugar important in mediating cooperativity.

Sequence specific conformation of a DNA decamer containing an adenine tract studied in solution by H-NMR spectroscopy.

The decanucleotide duplex d(AAAACGTTTT)2 and a variety of phase-sensitive two-dimensional (2D) NMR experiments have been used to investigate the solution conformation of an adenine-tract and its junction with another DNA sequence. 2D nuclear Overhauser effect data confirm that the oligonucleotide has a general B-type DNA morphology but an array of unusual correlations implies that the adenine tract and the 5'-ApC junction have conformations more compatible with the modified X-ray structures recently reported for DNAs of similar sequence (Nelson, H.C.M., Finch, J.T., Luisi, B.F. and Klug, A. (1987) Nature 330, 221-226). The pattern and magnitude of interstrand NOEs from the adenine H2s to the sugar H1's of the complementary base to the 5'-neighbouring residue indicate that the A-T basepairs are highly propeller twisted and that the minor groove is narrowed, showing its greatest compression at the 3'-end of the tract at the 5'-ApC step. Quantifying spin-coupling interactions within the deoxyribose rings by analysing both 1D and high-resolution 2D DQF-COSY data reveals that the conformation of the purines is predominantly C2'-endo, with the pseudorotation phase angle P lying in the range 140-180 degrees. For the pyrimidines, however, there are distortions away from this standard B-type geometry with the data being best described by P values lying in the range 90-130 degrees (i.e., O4'-endo, C1'-exo). The sugar puckers of A1, T9 and T10 are dynamically distorted no doubt as a consequence of their positions at, or close to, the ends of the duplex. Thus the conformation of the adenine and thymine sugars within the oligo(dA) and oligo(dT) strands are different with an abrupt change in sugar puckering occurring at the 5'-ApC (5'-GpT) step. Peculiar chemical shifts values for A4H2, T7CH3 and sugar C5 H1', H2' and H2", together with a number of interresidue NOEs with unusual intensities, imply that there are also substantial modifications to basepair stacking interactions at this step. Taken as a whole, our data are consistent with the view that the conformational dislocation at the 5'-ApC dinucleotide results from a combination of slide and roll manoeuvres and that the junction between the AAAA and CG sequences is a potential nucleation site for DNA bending.

Structure, dynamics and hydration of the nogalamycin-d(ATGCAT)2Complex determined by NMR and molecular dynamics simulations in solution.

The structure of the 1:1 nogalamycin:d(ATGCAT)2 complex has been determined in solution from high-resolution NMR data and restrained molecular dynamics (rMD) simulations using an explicit solvation model. The antibiotic intercalates at the 5'-TpG step with the nogalose lying along the minor groove towards the centre of the duplex. Many drug-DNA nuclear Overhauser enhancements (NOEs) in the minor groove are indicative of hydrophobic interactions over the TGCA sequence. Steric occlusion prevents a second nogalamycin molecule from binding at the symmetry-related 5'-CpA site, leading to the conclusion that the observed binding orientation in this complex is the preferred orientation free of the complication of end-effects (drug molecules occupy terminal intercalation sites in all X-ray structures) or steric interactions between drug molecules (other NMR structures have two drug molecules bound in close proximity), as previously suggested. Fluctuations in key structural parameters such as rise, helical twist, slide, shift, buckle and sugar pucker have been examined from an analysis of the final 500 ps of a 1 ns rMD simulation, and reveal that many sequence-dependent structural features previously identified by comparison of different X-ray structures lie within the range of dynamic fluctuations observed in the MD simulations. Water density calculations on MD simulation data reveal a time-averaged pattern of hydration in both the major and minor groove, in good agreement with the extensive hydration observed in two related X-ray structures in which nogalamycin is bound at terminal 5'-TpG sites. However, the pattern of hydration determined from the sign and magnitude of NOE and ROE cross-peaks to water identified in 2D NOESY and ROESY experiments identifies only a few "bound" water molecules with long residence times. These solvate the charged bicycloaminoglucose sugar ring, suggesting an important role for water molecules in mediating drug-DNA electrostatic interactions within the major groove. The high density of water molecules found in the minor groove in X-ray structures and MD simulations is found to be associated with only weakly bound solvent in solution.

Dissecting the stability of a beta-hairpin peptide that folds in water: NMR and molecular dynamics analysis of the beta-turn and beta-strand contributions to folding.

NMR studies of the folding and conformational properties of a beta-hairpin peptide, several peptide fragments of the hairpin, and sequence-modified analogues, have enabled the various contributions to beta-hairpin stability in water to be dissected. Temperature and pH-induced unfolding studies indicate that the folding-unfolding equilibrium approximates to a two-state model. The hairpin is highly resistant to denaturation and is still significantly folded in 7 M urea at 298 K. Thermodynamic analysis shows the hairpin to fold in water with a significant change in heat capacity, however, DeltaCp degrees in 7 M urea is reduced. V/Y-->A mutations on one strand of the hairpin reduce folding to <10 %, consistent with a hydrophobic stabilisation model. We show that in a truncated peptide (residues 6-16) lacking the hydrophobic residues on one beta-strand, the type I' Asn-Gly turn in the sequence SINGKK is significantly populated in water in the absence of interstrand hydrophobic contacts. Unrestrained molecular dynamics simulations of unfolding, using an explicit solvation model, show that the conformation of the NG turn persists for longer than the AG analogue, which has a much lower propensity for type I' turn formation from a data base analysis of preferred turns. The origin of the high stability of the Asn-Gly turn is not entirely clear; data base analysis of 66 NG turns, together with molecular dynamics simulations, reveals no participation of the Asn side-chain in turn-stabilising interactions with the peptide backbone. However, hydration analysis of the molecular dynamics simulations reveals a pocket of "high density" water bridging between the Asn side-chain and peptide main-chain that suggests solvent-mediated interactions may play an important role in modulating phi,psi propensities in the NG turn region.

Dihydrofolate reductase. 1H resonance assignments and coenzyme-induced conformational changes.

Lactobacillus casei dihydrofolate reductase has been studied in solution by one and two-dimensional 1H nuclear magnetic resonance (n.m.r.) spectroscopy at 500 MHz. By using a combination of n.m.r. methods in conjunction with the crystal structure of the enzyme-methotrexate-NADPH complex, resonances have been assigned for 32 of the 162 residues of the enzyme. These are widely distributed throughout the structure of the protein, and include all the histidine and tyrosine residues, as well as several valine, leucine, isoleucine and phenylalanine residues. The assignments have been made for the enzyme-methotrexate and enzyme-methotrexate-NADP+ complexes as well as the enzyme-methotrexate-NADPH complex. Comparison of assigned resonances in the spectra of the three complexes has permitted a preliminary assessment of structural differences between them. The beta-sheet "core" of the protein is unaffected by coenzyme binding, but two regions of the structure that undergo coenzyme-induced conformation changes have been identified. These are the loop comprising residues 13 to 23, and alpha-helix C (residues 42 to 49).

Modulation of intrinsic phi,psi propensities of amino acids by neighbouring residues in the coil regions of protein structures: NMR analysis and dissection of a beta-hairpin peptide.

Analysis of residues in coil regions of protein structures presents a novel approach to deconvoluting the various competing factors which determine the intrinsic phi,psi propensities of amino acids free from the regular interactions associated with beta-strands and alpha-helices. We have considered the role of context on phi,psi preferences by examining the effects of neighbouring residues in modulating coil propensities within a data base of 512 high-resolution, low-homology structures. In the general case, when flanking residues are beta-branched or aromatic (Val, Ile, Tyr and Phe) the beta-propensity (Pbeta) increases significantly, largely due to steric effects between flanking residues. More subtle residue-specific effects are apparant when Pbeta values are examined in detail, showing "random coil" conformations to be highly sequence-dependent. The effects of flanking residues on phi distributions have been used to calculate context-dependent average 3JNH-Halpha coupling constants. We have examined these findings in the context of the folding of a model 16-residue beta-hairpin peptide, "mutant" hairpin (VSI-->KSK sequence change) and the isolated C-terminal beta-strand fragments of both hairpins. We find a better correlation between 3JNH-Halpha values derived from the data base model and those determined experimentally when context-dependent phi distributions are considered. The individual C-terminal beta-strand sequences (GKKITVSI versus GKKITKSK) of the two hairpins are predisposed to different extents to formation of an extended beta-like conformation. Conformational "predisposition" in this context may contribute significantly to beta-hairpin stability.

Hoogsteen versus Watson-Crick A-T basepairing in DNA complexes of a new group of 'quinomycin-like' antibiotics.

The interaction of a new group of 'quinomycin-like' antibiotics with the DNA duplexes d(ACGT)2 and d(GACGTC)2 has been investigated in solution by 1H NMR spectroscopy. By monitoring the intensity of intranucleotide base H6/H8 to deoxyribose H1'NOE cross-peaks we conclude that the terminal A-T basepairs flanking the CG bisintercalation site in the d(ACGT)2 complex adopt the Hoogsteen bonding scheme, with the purine base in a syn conformation. By comparison in the d(GACGTC)2 complex all glycosidic bond angles are anti, consistent with a preferred Watson-Crick basepairing scheme. Both DNA duplexes appear to be significantly unwound compared with the ligand-free DNAs. The data illustrate the influence of helical constraints on the stability of the Hoogsteen bonding scheme adjacent to the drug binding sites.

31P NMR investigation of the backbone conformation and dynamics of the hexamer duplex d(5'-GCATGC)2 in its complex with the antibiotic nogalamycin.

Heteronuclear chemical shift correlation experiments confirm that the two down-field shifted 31P resonances in the spectrum of the (nogalamycin)2-d(GCATGC)2 complex correspond to the phosphodiesters CpA and TpG at the intercalation sites. 31P relaxation measurements (R1, R2 and [1H]-31P NOE) at 4.7 and 9.4 T permit the correlation time of each phosphate to be determined together with their chemical shift anisotropies. Significant differences in deoxyribose H3'-31P coupling constants and chemical shift anisotropy contributions are observed, consistent with an asymmetric DNA backbone conformation for the phosphate groups at the intercalation sites. Large amplitude internal motions of the phosphates do not appear to contribute significantly to relaxation.

Identification of the 1H resonances of valine and leucine residues in dihydrofolate reductase by using a combination of selective deuteration and two-dimensional correlation spectroscopy.

Lactobacillus casei dihydrofolate reductase (Mr 18 500) contains 16 valine and 14 leucine residues. By comparing the 2D COSY NMR spectra of normal and [gamma-2H6]valine enzyme we have been able to identify all 60 methyl resonances from these residues, and to connect the pairs arising from the same residue. This pairing of the methyl resonances was aided by the examination of the 2D RELAY spectrum which also allowed the C alpha H resonances (and hence the complete spin systems) of 14 of the valine residues to be identified. The combination of selective deuteration with 2D NMR techniques is shown to be a powerful general method for resolving 1H resonances in the complex spectra of proteins and for assigning them to amino-acid type.

15N and 1H NMR evidence for multiple conformations of the complex of dihydrofolate reductase with its substrate, folate.

The binding of folate to Lactobacillus casei dihydrofolate reductase in the presence and absence of NADP+ has been studied by 15N NMR, using [5-15N]folate. In the presence of NADP+, three separate signals were observed for the single 15N atom, in agreement with our earlier evidence from 1H and 13C NMR for multiple conformations of this complex [(1982) Biochemistry 21, 5831-5838]. The 15N spectra of the binary enzyme-folate complex provide evidence for the first time that this complex also exists in at least two conformational states. This is confirmed by the observation of two separate resonances for the 7-proton of bound folate, located by two-dimensional exchange spectroscopy.

The pattern of change in leisure activity behavior among older adults with arthritis.

This study employed a "ceasing participation" framework to examine changing leisure activity patterns. Respondents of the Living With Arthritis project were classified into four participation pattern categories. Results confirmed that older adults with arthritis are more likely to experience changes to their activity regimen than older adults without arthritis. A multi-group discriminant function analysis showed that arthritis severity distinguished those who tend to cease activity. Social network and age best distinguished those who quit activities without replacement. Results are placed in the context of coping strategies. Those who do not replace forfeited activities with other activities are least flexible in their response to their chronic condition and face challenges to their well-being.

Activity participation and well-being among older people with arthritis.

This study tests the hypotheses that (a) severity of arthritis is inversely associated with frequency of activity participation, and (b) arthritis sufferers who maintain higher levels of participation, particularly in activities which are social in nature, are less likely to experience a decline in well-being. Three activity types are considered: social, physical, and solitary. Results indicate that well-being is influenced by social activity, whereas solitary and physical activity have minimal impact. This suggests that elders with arthritis need not remove themselves from the pursuit of activity and should be encouraged to develop new interests when physical functioning fails. The study also demonstrates the utility of considering activity as multidimensional.

Survey of exercise and dietary knowledge and behaviour in persons with type II diabetes.

A survey was conducted to assess the potential for an exercise and weight control program for persons with Type II diabetes. Questionnaires were sent to 1,000 individuals with diabetes, who were randomly selected from the provincial health records office. Physicians and dietitians were the primary sources of information about both exercise and diet. Although few respondents participated in organized (7.7%) or informal (36.8%) exercise programs, or expressed an interest in participating (36.8%), the majority (84.0%) believed that they should get more exercise. This points to a gap between attitude and behaviour. Activity preferences were similar to those reported previously for all Canadians, however, barriers to participation differed in the present group. It was concluded that barriers must be assessed, and behaviour modification included, if diet and exercise programs are to be successful in this population.

Child abuse and neglect by parents with disabilities: A tale of two families.

Two families, in which the children had been placed in foster care due to abuse and neglect by parents who had disabilities, were studied. In the first case, the mother was instructed in skills that our assessment suggested were important for her child's survival. The mother readily acquired and applied these skills, a fact reflected both in changes in her behavior and in changes in the child's well-being. In the second case, the parent's incremental resumption of child custody was made contingent upon completion of relevant parenting tasks. Initially, improvements in the completion of such tasks were evident, but over time and with the onset of militating factors, no further progress was made and all parental rights were terminated. The implications of these cases for behavior analysis and the effort to reunite and preserve families are discussed.

p62 mutations, ubiquitin recognition and Paget's disease of bone.

Functional analyses of PDB (Paget's disease of bone)-associated mutants of the p62 [also known as SQSTM1 (sequestosome 1)] signalling adaptor protein represent an interesting paradigm for understanding not only the disease mechanism in this skeletal disorder, but also the critical determinants of ubiquitin recognition by an ubiquitin-binding protein. The 11 separate PDB mutations identified to date all affect the C-terminal region of p62 containing the UBA domain (ubiquitin-associated domain), a ubiquitin-binding element. All of these mutations have deleterious effects on ubiquitin binding by p62 in vitro, and there is evidence of an inverse relationship between ubiquitin-binding function and disease severity. The effects on ubiquitin-binding function of most of the mutations can be attributed to either reduced UBA domain stability, and/or the mutations affecting the presumed ubiquitin-binding interface of the UBA domain. However, a subset of the mutations are more difficult to rationalize; several of these affect sequences of p62 outside of the minimal ubiquitin-binding region, providing insights into non-UBA domain sequences within the host protein which mediate ubiquitin-binding affinity. The p62 mutations are presumed to result in activation of (osteoclast) NF-kappaB (nuclear factor kappaB) signalling. Understanding how loss of ubiquitin-binding function of p62 impacts on signal transduction events in osteoclasts will undoubtedly further our understanding of the disease mechanism in PDB at the molecular level.

Loss of ubiquitin binding is a unifying mechanism by which mutations of SQSTM1 cause Paget's disease of bone.

Ubiquitin-associated (UBA) domain mutations of SQSTM1 are an important cause of Paget's disease of bone (PDB), which is a human skeletal disorder characterized by abnormal bone turnover. We previously showed that, when introduced into the full-length SQSTM1 protein, the disease-causing P392L, M404V, G411S, and G425R missense mutations and the E396X truncating mutation (representative of all of the SQSTM1 truncating mutations) cause a generalized loss of monoubiquitin binding and impaired K48-linked polyubiquitin binding at physiological temperature. Here, we show that the remaining three known PDB missense mutations, P387L, S399P, and M404T, have similar deleterious effects on monoubiquitin binding and K48-linked polyubiquitin binding by SQSTM1. The P387L mutation affects an apparently unstructured region at the N terminus of the UBA domain, some five residues from the start of the first helix, which is dispensable for polyubiquitin binding by the isolated UBA domain. Our findings support the proposal that the disease mechanism in PDB with SQSTM1 mutations involves a common loss of ubiquitin binding function of SQSTM1 and implicate a sequence extrinsic to the compact globular region of the UBA domain as a critical determinant of ubiquitin recognition by the full-length SQSTM1 protein.

Binding of quinomycin antibiotic UK-65,662 to DNA: 1H-n.m.r. studies of drug-induced changes in DNA conformation in complexes with d(ACGT)2 and d(GACGTC)2.

Quinomycin antibiotic UK-65,662 binds selectively to the 5'-CpG-binding sites of the DNA duplexes d(ACGT)2 and d(GACGTC)2; the complexes have been studied in detail by 1H-n.m.r. spectroscopy and molecular-modelling techniques employing nuclear Overhauser effect-restrained energy minimization and molecular dynamics. Whereas the terminal A.T base pairs of the tetamer duplex d(ACGT)2 adopt a stable Hoogsteen alignment (characterized by a syn glycosidic conformation of the purine base), when internalized within the hexamer duplex d(GACGTC)2, the A.T base pairs revert to anti glycosidic torsion angles characteristic of the Watson-Crick hydrogen-bonding scheme. The energetics of base-pair stacking at the terminal 5'-GpA steps of the hexamer complex, with base pairs in the Watson-Crick alignment, are concluded to be important determinants of the adopted conformation, whereas an energetic preference for stacking interactions between terminal Hoogsteen A.T base pairs and the drug quinoline chromophores is evident in the tetramer complex. The internal G.C base pairs in both complexes are highly stabilized, as indicated by the very slow exchange rates of the guanine imino protons; in contrast, the flanking A.T base pairs are no more stable than in the ligand-free DNA duplexes. A large number of intermolecular nuclear Overhauser effects are indicative of many van der Waals contacts and hydrogen-bonding between the antibiotic and the minor groove of the central G.C base pairs in both complexes, indicating that interactions with the G.C base pairs in each duplex are very similar providing the essential features for recognition and tight binding. Despite the difference in the conformation of the A.T base pairs, stacking with the quinoline rings occurs primarily with the adenine bases in both complexes. Relative intensities of intranucleotide versus internucleotide nuclear Overhauser effects indicate that both duplexes are substantially unwound by drug binding (particularly at the CpG step) and this is confirmed by the structure calculations. Both duplexes have ladder-like structures that must lead to significant local distortions of the DNA conformation in vivo.

Templating peptide folding on the surface of a micelle: nucleating the formation of a beta-hairpin.

NMR spectroscopy is used to show that a 20-residue beta-hairpin peptide sequence derived from ferredoxin I, with a Pro-Asp two-residue type I turn which is uncommon in beta-hairpins, is unstructured in aqueous solution but shows NOE evidence for partial folding in the presence of sodium dodecylsulphate micelles. The peptide has a number of lysine residues in the N-terminal beta-strand capable of interacting with the micelle surface and templating the partial folding of the hairpin by reducing the entropic cost of ordering the peptide backbone.

Conformation and dynamics of the deoxyribose rings of a (nogalamycin)2-d (5'-GCATGC)2 complex studied in solution by 1H-n.m.r. spectroscopy.

The conformation and dynamics of the deoxyribose rings of a (nogalamycin)2-d(5'-GCATGC)2 complex have been determined from an analysis of 1H-1H vicinal coupling constants and sums of coupling constants (J1'-2',J1'-2",epsilon 1', epsilon 2' and epsilon 2") measured from one-dimensional n.m.r. spectra and from H-1'-H-2' and H-1'-H-2" cross-peaks in high-resolution phase-sensitive two-dimensional correlation spectroscopy (COSY) and double-quantum-filtered correlation spectroscopy (DQF-COSY) experiments. The value of J3'-4' has also been estimated from the magnitude of H-3'-H-4' cross-peaks in DQF-COSY spectra and H-1'-H-4' coherence transfer cross-peaks in two-dimensional homonuclear Hartman-Hahn spectroscopy (HOHAHA) spectra. The data were analysed, in terms of a dynamic equilibrium between North (C-3'-endo) and South (C-2'-endo) conformers, by using the graphical-analysis methods described by Rinkel & Altona [(1987) J. Biomol. Struct. Dyn. 4,621-649]. The data reveal that the sugars of the 2C-5G and 3A-4T base-pairs, which form the drug-intercalation site, have strikingly different properties. The deoxyribose rings of the 2C-5G base-pair are best described in terms of an equilibrium heavily weighted in favour of the C-2'-endo geometry (greater than 95% 'S'), with a phase angle, P, lying in the range 170-175 degrees and amplitude of pucker between 35 and 40 degrees, as typically found for B-DNA. For the deoxyribose rings of the 3A-4T base-pair, however, the analysis shows that, for 3A, the C-2'-endo and C3'-endo conformers are equally populated, whereas a more limited data set for the 4T nucleotide restricts the equilibrium to within 65-75% C-2'-endo. The deoxyribose rings of the 1G-6C base-pair have populations of 70-80% C-2'-endo, typical of nucleotides at the ends of a duplex. Although drug-base-pair stacking interactions are an important determinant of the enhanced duplex stability of the complex [Searle, Hall, Denny, & Wakelin (1988) Biochemistry 27, 4340-4349], the current findings make it clear that the same interactions can be associated with considerable variations in the degree of local structural dynamics at the level of the sugar puckers.