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A M Bonvin

Publications and source records attributed to A M Bonvin.

17 recordsLinked to original sources
PubMed2001

Rapid protein fold determination using secondary chemical shifts and cross-hydrogen bond 15N-13C' scalar couplings (3hbJNC').

The possibility of generating protein folds at the stage of backbone assignment using structural restraints derived from experimentally measured cross-hydrogen bond scalar couplings and secondary chemical shift information is investigated using as a test case the small alpha/beta protein chymotrypsin inhibitor 2. Dihedral angle restraints for the phi and psi angles of 32 out of 64 residues could be obtained from secondary chemical shift analysis with the TALOS program (Corneliscu et al., 1999a). This information was supplemented by 18 hydrogen-bond restraints derived from experimentally measured cross-hydrogen bond 3hbJNC' coupling constants. These experimental data were sufficient to generate structures that are as close as 1.0 A backbone rmsd from the crystal structure. The fold is, however, not uniquely defined and several solutions are generated that cannot be distinguished on the basis of violations or energetic considerations. Correct folds could be identified by combining clustering methods with knowledge-based potentials derived from structural databases.

Crystallography, X-Ray↗
PubMed2000

Changes in dynamical behavior of the retinoid X receptor DNA-binding domain upon binding to a 14 base-pair DNA half site.

The retinoid X receptor (RXR) is a prominent member of the nuclear receptor family of ligand-inducible transcription factors. Many proteins of this family exert their function as heterodimers with RXR as a common upstream partner. Studies of the DNA-binding domains of several nuclear receptors reveal differences in structure and dynamics, both between the different proteins and between the free- and DNA-bound receptor DBDs. We investigated the differences in dynamics between RXR free in solution and in complex with a 14 base-pair oligonucleotide, using (1)H and (15)N relaxation studies. Nano- to picosecond dynamics were probed on (15)N, employing Lipari-Szabo analysis with an axially symmetric tumbling model to estimate the exchange contributions to the transverse relaxation rates. Furthermore, milli- to microsecond dynamics were estimated qualitatively for (1)H and (15)N, using CPMG-HSQC and CPMG-T(2) measurements with differential pulse spacing. RXR shows hardly any nano- to picosecond time-scale internal motion. Upon DNA binding, the order parameters show a tiny increase. Dynamics in the milli- to microsecond time scale is more prevalent. It is localized in the first and second zinc fingers of the free RXR. Upon DNA-binding, exchange associated with specific/aspecific DNA-binding of RXR is observed throughout the sequence, whereas conformational flexibility of the D-box and the second zinc finger of RXR is greatly reduced. Since this DNA-binding induced folding transition occurs remote from the DNA in a region which is involved in protein-protein interactions, it may very well be related to the cooperativity of dimeric DNA binding.

Anisotropy↗
PubMed2000

Hydration dynamics of the collagen triple helix by NMR.

The hydration of the collagen-like Ac-(Gly-Pro-Hyp)(6)-NH(2) triple-helical peptide in solution was investigated using an integrated set of high-resolution NMR hydration experiments, including different recently developed exchange-network editing methods. This approach was designed to explore the hydration dynamics in the proximity of labile groups, such as the hydroxyproline hydroxyl group, and revealed that the first shell of hydration in collagen-like triple helices is kinetically labile with upper limits for water molecule residence times in the nanosecond to sub-nanosecond range. This result is consistent with a "hopping" hydration model in which solvent molecules are exchanged in and out of solvation sites at a rate that is not directly correlated to the degree of site localization. The hopping model thus reconciles the dynamic view of hydration revealed by NMR with the previously suggested partially ordered semi-clathrate-like cylinder of hydration. In addition, the nanosecond to sub-nanosecond upper limits for water molecule residence times imply that hydration-dehydration events are not likely to be the rate-limiting step for triple helix self-recognition, complementing previous investigations on water dynamics in collagen fibers. This study has also revealed labile proton features expected to facilitate the characterization of the structure and folding of triple helices in collagen peptides.

Animals↗
PubMed2000

beta-hairpin stability and folding: molecular dynamics studies of the first beta-hairpin of tendamistat.

The stability and (un)folding of the 19-residue peptide, SCVTLYQSWRYSQADNGCA, corresponding to the first beta-hairpin (residues 10 to 28) of the alpha-amylase inhibitor tendamistat (PDB entry 3AIT) has been studied by molecular dynamics simulations in explicit water under periodic boundary conditions at several temperatures (300 K, 360 K and 400 K), starting from various conformations for simulation lengths, ranging from 10 to 30 ns. Comparison of trajectories of the reduced and oxidized native peptides reveals the importance of the disulphide bridge closing the beta-hairpin in maintaining a proper turn conformation, thereby insuring a proper side-chain arrangement of the conserved turn residues. This allows rationalization of the conservation of those cysteine residues among the family of alpha-amylase inhibitors. High temperature simulations starting from widely different initial configurations (native beta-hairpin, alpha and left-handed helical and extended conformations) begin sampling similar regions of the conformational space within tens of nanoseconds, and both native and non-native beta-hairpin conformations are recovered. Transitions between conformational clusters are accompanied by an increase in energy fluctuations, which is consistent with the increase in heat capacity measured experimentally upon protein folding. The folding events observed in the various simulations support a model for beta-hairpin formation in which the turn is formed first, followed by hydrogen bond formation closing the hairpin, and subsequent stabilization by side-chain hydrophobic interactions.

Amino Acid Sequence↗
PubMed2000

Localisation and dynamics of sodium counterions around DNA in solution from molecular dynamics simulation.

The localisation and dynamics of sodium counterions around the DNA duplex d(AGCGTACTAGTACGCT)2 corresponding to the trp operator fragment used in the crystal structure of the half site complex has been studied by a 1.4 ns molecular dynamics simulation in explicit solvent. A continuous and well-defined counterion density is shown to be present around the minor groove, while density patches are found in the major groove in regions where DNA bending is observed. A residence time analysis reveals the dynamic nature of these distributions. The resulting picture agrees with previous theoretical and experimental studies of A-tract DNA sequences, and is consistent with the polyelectrolyte condensation model.

Base Sequence↗
PubMed1999

The solution structure of Lac repressor headpiece 62 complexed to a symmetrical lac operator.

BACKGROUND: Lactose repressor protein (Lac) controls the expression of the lactose metabolic genes in Escherichia coli by binding to an operator sequence in the promoter of the lac operon. Binding of inducer molecules to the Lac core domain induces changes in tertiary structure that are propagated to the DNA-binding domain through the connecting hinge region, thereby reducing the affinity for the operator. Protein-protein and protein-DNA interactions involving the hinge region play a crucial role in the allosteric changes occurring upon induction, but have not, as yet, been analyzed in atomic detail. RESULTS: We have used nuclear magnetic resonance (NMR) spectroscopy and restrained molecular dynamics (rMD) to determine the structure of the Lac repressor DNA-binding domain (headpeice 62; HP62) in complex with a symmetrized lac operator. Analysis of the structures reveals specific interactions between Lac repressor and DNA that were not found in previously investigated Lac repressor-DNA complexes. Important differences with the previously reported structures of the HP56-DNA complex were found in the loop following the helix-turn-helix (HTH) motif. The protein-protein and protein-DNA interactions involving the hinge region and the deformations in the DNA structure could be delineated in atomic detail. The structures were also used for comparison with the available crystallographic data on the Lac and Pur repressor-DNA complexes. CONCLUSIONS: The structures of the HP62-DNA complex provide the basis for a better understanding of the specific recognition in the Lac repressor-operator complex. In addition, the structural features of the hinge region provide detailed insight into the protein-protein and protein-DNA interactions responsible for the high affinity of the repressor for operator DNA.

Bacterial Proteins↗
PubMed1998

Water molecules in DNA recognition I: hydration lifetimes of trp operator DNA in solution measured by NMR spectroscopy.

The present NMR study investigates the residence times of the hydration water molecules associated with uncomplexed trp operator DNA in solution by measuring intermolecular nuclear Overhauser effects (NOE) between water and DNA protons, and the nuclear magnetic relaxation dispersion (NMRD) of the water 2H and 17O resonances. Both methods indicate that the hydration water molecules exchange with bulk water on the sub-nanosecond time scale at 4 degreesC. No evidence was obtained for water molecules bound with longer residence times. In particular, the water molecules at the sites of interfacial hydration in the trp repressor/operator complex do not seem kinetically stabilized in the uncomplexed DNA. Analysis of the crystal structures of two different trp repressor/operator complexes shows very similar structural environments for the water molecules mediating specific contacts between the protein and the DNA, whereas much larger variations are observed for the location of corresponding water molecules detected in the crystal structure of an uncomplexed trp operator DNA duplex. Therefore, it appears unlikely that the hydration characteristics of the uncomplexed DNA target would be a major determinant of trp repressor/operator recognition.

Bacterial Proteins↗
PubMed1998

Water molecules in DNA recognition II: a molecular dynamics view of the structure and hydration of the trp operator.

The structure and hydration of the DNA duplex d-(AGCGTACTAGTACGCT)2 corresponding to the trp operator fragment used in the crystal structure of the half site complex (PDB entry 1TRR) was studied by a 1.4 ns molecular dynamics simulation in water. The simulation, starting from a B-DNA conformation, used a non-bonded cutoff of 1.4 nm with a reaction field correction and resulted in a stable trajectory. The average DNA conformation obtained was closer to the ones found in the crystal structures of the complexes (PDB entries 1TRO and 1TRR) than to the crystal structure of unbound trp operator (Nucleic Acid Database entry BDJ061). The DNA hydration was characterized in terms of hydrogen bond percentages and corresponding residence times. The residence times of water molecules within 0.35 nm of the DNA non-exchangeable protons were calculated for comparison with NMR measurements of intermolecular water-DNA NOEs and nuclear magnetic relaxation dispersion measurements. No significant difference was found between major and minor groove hydration. The DNA donors and acceptors were hydrogen bonded to water molecules for 77(+/-19)% of the time on average. The average residence time of the hydrogen bonded water molecules was 11(+/-11) ps with a maximum of 223 ps. When all water molecules within NOE distance (0.35 nm) of non-exchangeable protons were considered, the average residence times increased to an average of 100(+/-4) ps and a maximum of 608 ps. These results agree with the experimental NMR results of Sunnerhagen et al. which did not show any evidence for water molecules bound with more than 1 ns residence time on the DNA surface. The exchange of hydration water from the DNA occurred in the major groove primarily through direct exchange with the bulk solvent, while access to and from the minor groove frequently proceeded via pathways involving ribose O3' and O4' and phosphate O2P oxygen atoms. The most common water diffusion pathways in the minor groove were perpendicular to the groove direction. In general, water molecules visited only a limited number of sites in the DNA grooves before exiting. The hydrogen bonding sites, where hydrogen bonds could be formed with donor and acceptor groups of the DNA, were filled with water molecules with an average B-factor value of 0.58 mn2. No special values were observed at any of the sites, where water molecules were observed both in the trp repressor/operator co-crystals and in the crystal structure of unbound DNA.

Bacterial Proteins↗
PubMed1996

Refined structure of lac repressor headpiece (1-56) determined by relaxation matrix calculations from 2D and 3D NOE data: change of tertiary structure upon binding to the lac operator.

The solution structure of the DNA binding domain of lac repressor (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 phi and 15 chi 1 dihedral 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) A angstroms (backbone) and 0.95(+/-0.08) angstroms (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 lac repressor 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 lac HP56 is essential for binding of the side-chains of residues Asn25 and His29 to the lac operator DNA. Finally, the lac headpiece 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.

Crystallography, X-Ray↗
PubMed1996

Do NOE distances contain enough information to assess the relative populations of multi-conformer structures?

The feasibility of determining the relative populations of multi-conformer structures from NOE-derived distances alone is assessed. Without cross-validation of the NOE restraints, any population ratio can be refined to a similar quality of the fit. Complete cross-validation provides a less biased measure of fit and allows the estimation of the correct population ratio when used in conjunction with very tight distance restraints. With the qualitative distance restraints most commonly used in NMR structure determination, cross-validation is unsuccessful in providing the correct answer. Other experimental sources are therefore needed to determine relative populations of multi-conformer structures.

Allergens↗
PubMed1995

Conformational variability of solution nuclear magnetic resonance structures.

In structure determination by X-ray crystallography and solution NMR spectroscopy, experimental data are collected as time and ensemble-averages. Thus, in principle, appropriate time and ensemble-averaged models should be used. Refinement of an ensemble of conformers rather than one single structure against the experimental NMR data could, however, result in overfitting the data because of the significantly increased number of parameters. To avoid overfitting, complete cross-validation, which provides an unbiased measure of the fit, has been applied to nuclear Overhauser effect derived distance refinement. Using two synthetic test cases, a correlation was demonstrated between the cross-validated measure to the fit (defined in terms of root-mean-square deviations from the distance restraints and number of violations) and the number of models that best reproduce the conformational variability in solution. A new method, based on a probability map, has been used to generate good representations of the resulting ensembles of structures. The method has also been applied to observed NMR data for two proteins, interleukin 4 and interleukin 8. For interleukin 4, cross-validation indicates that a single-conformer model gives the most accurate representation of the structure, whereas conventional measures of fit between the experimental data and those calculated from the model decrease when increasing the number of conformers, indicating overfitting. For interleukin 8, complete cross-validation predicts a twin-conformer model to be the most faithful representation of the experimental data. Two distinct conformations for the loop formed by residues 16 to 22 emerge from the family of twin-conformer structures. The putative alternate conformation of the loop is not observed in the crystal structure of interleukin 8. However, because of crystal packing contacts in this region this does not necessarily exclude the presence of the alternate conformation in solution. The twin-conformer model is supported by observed chemical exchange line broadening for the amide of His18 obtained by 15N relaxation studies. This region has also been implied to be involved in receptor binding.

Allergens↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1994

Nuclear magnetic resonance solution structure of the Arc repressor using relaxation matrix calculations.

The Arc repressor of Salmonella bacteriophage P22 is a dimeric sequence-specific DNA-binding protein. The solution structure of Arc has been determined from 2D NMR data using an "ensemble" iterative relaxation matrix approach (IRMA) followed by direct NOE refinement with DINOSAUR. A set of 51 structures 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-ups in H2O and 2H2O via relaxation matrix calculations from the ensemble of structures. Methyl group rotation, aromatic ring flaps and internal mobility effects (via order parameters obtained from a free molecular dynamics run in water) were included in these calculations. The best structures were finally refined with direct NOE constraints following a slow-cooling simulated annealing protocol. In this final refinement stage, theoretical NOE intensities were directly compared with the experimental data and forces were derived using a simple two-spin approximation for the gradient of the NOE function. Dynamic assignment was applied to the peaks involving unassigned diastereotopic groups. The structure is determined to a precision (r.m.s.d. from the average excluding the ill defined C and N-terminal region) of 0.55 and 1.10 A for backbone and all atoms, respectively. The final structures, with R factor values around 0.35, have good stereochemical qualities, contain an extensive network of hydrogen bonds consistent with the secondary structure elements and structural features in concordance with genetic data. The overall folding of the solution and crystal structures is the same.

Amino Acid Sequence↗
PubMed1993

"Ensemble" iterative relaxation matrix approach: a new NMR refinement protocol applied to the solution structure of crambin.

The structure in solution of crambin, a small protein of 46 residues, has been determined from 2D NMR data using an iterative relaxation matrix approach (IRMA) together with distance geometry, distance bound driven dynamics, molecular dynamics, and energy minimization. A new protocol based on an "ensemble" approach is proposed and compared to the more standard initial rate analysis approach and a "single structure" relaxation matrix approach. The effects of fast local motions are included and R-factor calculations are performed on NOE build-ups to describe the quality of agreement between theory and experiment. A new method for stereospecific assignment of prochiral groups, based on a comparison of theoretical and experimental NOE intensities, has been applied. The solution structure of crambin could be determined with a precision (rmsd from the average structure) of 0.7 A on backbone atoms and 1.1 A on all heavy atoms and is largely similar to the crystal structure with a small difference observed in the position of the side chain of Tyr-29 which is determined in solution by both J-coupling and NOE data. Regions of higher structural variability (suggesting higher mobility) are found in the solution structure, in particular for the loop between the two helices (Gly-20 to Pro-22).

Amino Acid Sequence↗
PubMed1993

The solution structure of the human retinoic acid receptor-beta DNA-binding domain.

The three-dimensional structure of the DNA-binding domain of the human retinoic acid receptor-beta (hRAR-beta) has been determined by nuclear magnetic resonance spectroscopy in conjunction with distance geometry, restrained molecular dynamics and iterative relaxation matrix calculations. A total of 1244 distance restraints were obtained from NOE intensities, of which 448 were intra-residue and 796 inter-residue restraints. In addition 23 chi and 30 phi dihedral angle restraints were obtained from J-coupling data. The two 'zinc-finger' regions of the 80-amino acid residue protein are followed by two alpha-helices that cross each other perpendicularly. There is a short stretch of b-sheet near the N-terminus. The alpha-helical core of the protein is well determined with a backbone root-mean-square deviation (r.m.s.d.) with respect to the average of 0.18 A and 0.37 A when the side chains of residues 31, 32, 36, 61, 62, 65 and 69 are included. The r.m.s.d. for the backbone of residues 5-80 is 0.76 A. For the first finger (residues 8-28), the r.m.s.d. of the backbone is 0.79 A. For the second finger (residues 44-62) the r.m.s.d. is 0.64 A. The overall structure is similar to that of the corresponding domain of the glucocorticoid receptor, although the C-terminal part of the protein is different. The second alpha-helix is two residues shorter and is followed by a well-defined region of extended backbone structure.

Amino Acid Sequence↗
PubMed1991

Direct NOE refinement of biomolecular structures using 2D NMR data.

A set of computer programs called DINOSAUR has been developed, which allows the refinement of biomolecular structures directly from 2D NOE intensities. The NOE restraining potential implemented emphasises the weak intensities corresponding to larger distances which are more likely to determine the three-dimensional structure. An approximation based on a two-spin approximation is proposed for the gradient of the NOE intensities instead of the exact solution which is extremely time-consuming. The DINOSAUR routines have been implemented in various refinement programs (Distance bound Driven Dynamics, Molecular Dynamics and Energy Minimisation) and tested on an eight-residue model peptide.

Magnetic Resonance Spectroscopy↗