A protein structure from nuclear magnetic resonance data. lac repressor headpiece

A procedure is described to determine the three-dimensional structure of biomolecules from nuclear magnetic resonance data. This procedure combines model building with a restrained molecular dynamics algorithm, in which distance information from nuclear Overhauser effects is incorporated in the form of pseudo potentials. The method has been applied to the N-terminal DNA-binding domain or headpiece (amino acid residues 1 to 51) of the lac repressor from Escherichia coli, for which no crystal structure is available. The relative orientation of the three helices of the headpiece is similar to that of the three homologous helices found in the cI repressor of bacteriophage lambda.     

Determination of protein structures from nuclear magnetic resonance data using a restrained molecular dynamics approach: the lac repressor DNA binding domain

A procedure is described to determine from NMR data the three-dimensional structure of biomolecules. This procedure combines model building with a restrained Molecular Dynamics algorithm, in which distance information from NOEs is incorporated in the form of pseudo potentials. The method has been applied to the N-terminal DNA-binding domain or "headpiece" (amino acids 1-51) of the lac repressor from E. coli, for which no crystal structure is available. The spatial structure of the headpiece is discussed in terms of known physical and biochemical data and of its DNA binding properties.     

The determination of the three-dimensional structure of barley serine proteinase inhibitor 2 by nuclear magnetic resonance, distance geometry and restrained molecular dynamics

The solution structure of the 64 residue structured domain (residues 20-83) of barley serine proteinase inhibitor 2 (BSPI-2) is determined on the basis of 403 interproton distance, 34 phi backbone torsion angle and 26 hydrogen bonding restraints derived from n.m.r. measurements. A total of 11 converged structures were computed using a metric matrix distance geometry algorithm and refined by restrained molecular dynamics. The average rms difference between the final 11 structures and the mean structure obtained by averaging their coordinates is 1.4 +/- 0.2 A for the backbone atoms and 2.1 +/- 0.1 A for all atoms. The overall structure, which is almost identical to that found by X-ray crystallography, is disc shaped and consists of a central four component mixed parallel and antiparallel beta-sheet flanked by a 13 residue alpha-helix on one side and the reactive site loop on the other.     

Solution structure of neurotoxin I from the sea anemone Stichodactyla helianthus. A nuclear magnetic resonance, distance geometry, and restrained molecular dynamics study

The three-dimensional structure of the sea anemone polypeptide Stichodactyla helianthus neurotoxin I in aqueous solution has been determined using distance geometry and restrained molecular dynamics simulations based on NMR data acquired at 500 MHz. A set of 470 nuclear Overhauser enhancement values was measured, of which 216 were used as distance restraints in the structure determination along with 15 dihedral angles derived from coupling constants. After restrained molecular dynamics refinement, the eight structures that best fit the input data form a closely related family. They describe a structure that consists of a core of twisted, four-stranded, antiparallel beta-sheet encompassing residues 1-3, 19-24, 29-34, and 40-47, joined by three loops, two of which are well defined by the NMR data. The third loop, encompassing residues 7-16, is poorly defined by the data and is assumed to undergo conformational averaging in solution. Pairwise root mean square displacement values for the backbone heavy atoms of the eight best structures are 1.3 +/- 0.2A when the poorly defined loop is excluded and 3.6 +/- 1.0A for all backbone atoms. Refinement using restrained molecular dynamics improved the quality of the structures generated by distance geometry calculations with respect to the number of nuclear Overhauser enhancements violated, the size of the total distance violations and the total potential energies of the structures. The family of structures for S. heliathus neurotoxin I is compared with structures of related sea anemone proteins that also bind to the voltage-gated sodium channel.     

Use of restrained molecular dynamics in water to determine three-dimensional protein structure: prediction of the three-dimensional structure of Ecballium elaterium trypsin inhibitor II

Refinement of distance geometry (DG) structures of EETI-II (Heitz et al.: Biochemistry 28:2392-2398, 1989), a member of the squash family trypsin inhibitor, have been carried out by restrained molecular dynamics (RMD) in water. The resulting models show better side chain apolar/polar surface ratio and estimated solvation free energy than structures refined "in vacuo." The consistent lower values of residual NMR constraint violations, apolar/polar surface ratio, and solvation free energy for one of these refined structures allowed prediction of the 3D folding and disulfide connectivity of EETI-II. Except for the few first residues for which no NMR constraints were available, this computer model fully agreed with X-ray structures of CMTI-I (Bode et al.: FEBS Lett. 242:285-292, 1989) and EETI-II complexed with trypsin that appeared after the RMD simulation was completed. Restrained molecular dynamics in water is thus proved to be highly valuable for refinement of DG structures. Also, the successful use of apolar/polar surface ratio and of solvation free energy reinforce the analysis of Novotny et al. (Proteins 4:19-30, 1988) and shows that these criteria are useful indicators of correct versus misfolded models.     

Solution conformation of a heptadecapeptide comprising the DNA binding helix F of the cyclic AMP receptor protein of Escherichia coli. Combined use of 1H nuclear magnetic resonance and restrained molecular dynamics

A nuclear magnetic resonance study on a heptadecamer (17-mer) peptide comprising the DNA binding helix F of the cyclic AMP receptor protein of Escherichia coli is presented under solution conditions (viz. 40% (v/v) trifluorethanol) where it adopts an ordered helical structure as judged by circular dichroism. Using a combination of two-dimensional nuclear magnetic resonance techniques, complete resonance assignments are obtained in a sequential manner. From the two-dimensional nuclear Overhauser enhancement spectra, a set of 87 approximate distance restraints is derived and used as the basis for three-dimensional structure determination with a restrained molecular dynamics algorithm in which the interproton distances are incorporated into the total energy function of the system in the form of an additional effective potential term. The convergence properties of this approach are tested by starting from three different initial structures, namely an alpha-helix, a beta-strand and a 3-10 helix. In all three cases, convergence to an alpha-helical structure is achieved with a root mean square difference of less than 3 A for all atoms and less than 2 A for the backbone atoms.     

Ab initio RNA folding by discrete molecular dynamics: from structure prediction to folding mechanisms

RNA molecules with novel functions have revived interest in the accurate prediction of RNA three-dimensional (3D) structure and folding dynamics. However, existing methods are inefficient in automated 3D structure prediction. Here, we report a robust computational approach for rapid folding of RNA molecules. We develop a simplified RNA model for discrete molecular dynamics (DMD) simulations, incorporating base-pairing and base-stacking interactions. We demonstrate correct folding of 150 structurally diverse RNA sequences. The majority of DMD-predicted 3D structures have <4 A deviations from experimental structures. The secondary structures corresponding to the predicted 3D structures consist of 94% native base-pair interactions. Folding thermodynamics and kinetics of tRNA(Phe), pseudoknots, and mRNA fragments in DMD simulations are in agreement with previous experimental findings. Folding of RNA molecules features transient, non-native conformations, suggesting non-hierarchical RNA folding. Our method allows rapid conformational sampling of RNA folding, with computational time increasing linearly with RNA length. We envision this approach as a promising tool for RNA structural and functional analyses.     

Solution structure of a DNA octamer containing the Pribnow box via restrained molecular dynamics simulation with distance and torsion angle constraints derived from two-dimensional nuclear magnetic resonance spectral fitting.

The DNA octamer [d(GTATAATG].[(CATATTAC)], containing the prokaryotic upstream consensus recognition sequence, has been examined via proton homonuclear two-dimensional nuclear Overhauser effect (2D NOE) and double-quantum-filtered correlation (2QF-COSY) spectra. All proton resonances, except those of H5' and H5" protons, were assigned. A temperature dependence study of one-dimensional nuclear magnetic resonance (NMR) spectra, rotating frame 2D NOE spectroscopy (ROESY), and T1 rho measurements revealed an exchange process that apparently is global in scope. Work at lower temperatures enabled a determination of structural constraints that could be employed in determination of a time-averaged structure. Simulations of the 2QF-COSY cross-peaks were compared with experimental data, establishing scalar coupling constant ranges of the individual sugar ring protons and hence pucker parameters for individual deoxyribose rings. The rings exhibit a dynamic equilibrium of N and S-type conformers with 80 to 100% populations of the latter. A program for iterative complete relaxation matrix analysis of 2D NOE spectral intensities, MARDIGRAS, was employed to give interproton distances for each mixing time. According to the accuracy of the distance determination, upper and lower distance bounds were chosen. The distance bounds define the size of a flat-well potential function term, incorporated into the AMBER force-field, which was employed for restrained molecular dynamics calculations. Torsion angle constraints in the form of a flat-well potential were also constructed from the analysis of the sugar pucker data. Several restrained molecular dynamics runs of 25 picoseconds were performed, utilizing 184 experimental distance constraints and 80 torsion angle constraints; three different starting structures were used: energy minimized A-DNA, B-DNA, and wrinkled D-DNA, another member of the B-DNA family. Convergence to similar structures obtained with root-mean-square deviations between resulting structures of 0.37 to 0.92 A for the central hexamer of the octamer. The average structure from the nine different molecular dynamics runs was subjected to final restrained energy minimization. The resulting final structure was in good agreement with the structures derived from different molecular dynamics runs and exhibited a substantial improvement in the 2D NOE sixth-root residual index in comparison with the starting structures. An approximation of the structure in the terminal base-pairs, which displayed experimental evidence of fraying, was made by maintaining the structure of the inner four base-pairs and performing molecular dynamics simulations with the experimental structural constraints observed for the termini.(ABSTRACT TRUNCATED AT 400 WORDS)     

A molecular dynamics study of the 41-56 beta-hairpin from B1 domain of protein G.

The structural and dynamical behavior of the 41-56 beta-hairpin from the protein G B1 domain (GB1) has been studied at different temperatures using molecular dynamics (MD) simulations in an aqueous environment. The purpose of these simulations is to establish the stability of this hairpin in view of its possible role as a nucleation site for protein folding. The conformation of the peptide in the crystallographic structure of the protein GB1 (native conformation) was lost in all simulations. The new equilibrium conformations are stable for several nanoseconds at 300K (>10 ns), 350 K (>6.5 ns), and even at 450 K (up to 2.5 ns). The new structures have very similar hairpin-like conformations with properties in agreement with available experimental nuclear Overhauser effect (NOE) data. The stability of the structure in the hydrophobic core region during the simulations is consistent with the experimental data and provides further evidence for the role played by hydrophobic interactions in hairpin structures. Essential dynamics analysis shows that the dynamics of the peptide at different temperatures spans basically the same essential subspace. The main equilibrium motions in this subspace involve large fluctuations of the residues in the turn and ends regions. Of the six interchain hydrogen bonds, the inner four remain stable during the simulations. The space spanned by the first two eigenvectors, as sampled at 450 K, includes almost all of the 47 different hairpin structures found in the database. Finally, analysis of the hydration of the 300 K average conformations shows that the hydration sites observed in the native conformation are still well hydrated in the equilibrium MD ensemble.     

Structural elucidation of 4'-epiadriamycin by nuclear magnetic resonance spectroscopy and comparison with adriamycin and daunomycin using quantum mechanical and restrained molecular dynamics approach

The structural and electronic properties of 4′-epiadriamycin, adriamycin, and daunomycin have been studied using density functional theory (DFT) employing B3LYP exchange correlation. The chemical shifts of ¹H and ¹³C resonances in nuclear magnetic resonance spectra have been calculated using Gauge-Invariant Atomic Orbital (GIAO) method as implemented in Gaussian 98 and compared with experimental spectra recorded at 500 MHz. ¹³C resonances of drugs have been assigned for the first time. A restrained molecular dynamics approach was used to get the optimized solution structure of drugs using inter-proton distance constraints obtained from 2D NOESY spectra. The glycosidic angle C7-O7-C1′-C2′ is found to show considerable flexibility by adopting 156°-161° (I), 142°-143° (II), and 38°-78° (III) conformations, of which the biological relevant structure appears to be the conformer II. The observed different conformations of the three drugs are correlated to the differential anticancer activity and the available biochemical evidence exhibited by these drugs.     

Binding of glutamine to glutamine-binding protein from Escherichia coli induces changes in protein structure and increases protein stability

Glutamine-binding protein (GlnBP) from Escherichia coli is a monomeric protein localized in the periplasmic space of the bacterium. It is responsible for the first step in the active transport of L-glutamine across the cytoplasmic membrane. The protein consists of two similar globular domains linked by two peptide hinges, and X-ray crystallographic data indicate that the two domains undergo large movements upon ligand binding. Fourier transform infrared spectroscopy (FTIR) was used to analyze the structure and thermal stability of the protein in detail. The data indicate that glutamine binding induces small changes in the secondary structure of the protein and that it renders the structure more thermostable and less flexible. Detailed analyses of IR spectra show a lower thermal sensitivity of alpha-helices than beta-sheets in the protein both in the absence and in the presence of glutamine. Generalized two-dimensional (2D) analyses of IR spectra reveal the same sequence of unfolding events in the protein in the absence and in the presence of glutamine, indicating that the amino acid does not affect the unfolding pathway of the protein. The data give new insight into the structural characteristics of GlnBP that are useful for both basic knowledge and biotechnological applications.     

Exploring the dynamic information content of a protein NMR structure: Comparison of a molecular dynamics simulation with the NMR and X‐ray structures of Escherichia coli ribonuclease HI

The multiconformer nature of solution nuclear magnetic resonance (NMR) structures of proteins results from the effects of intramolecular dynamics, spin diffusion and an uneven distribution of structural restraints throughout the molecule. A delineation of the former from the latter two contributions is attempted in this work for an ensemble of 15 NMR structures of the protein Escherichia coli ribonuclease HI (RNase HI). Exploration of the dynamic information content of the NMR ensemble is carried out through correlation with data from two crystal structures and a 1.7‐ns molecular dynamics (MD) trajectory of RNase HI in explicit solvent. Assessment of the consistency of the crystal and mean MD structures with nuclear Overhauser effect (NOE) data showed that the NMR ensemble is overall more compatible with the high‐resolution (1.48 Å) crystal structure than with either the lower‐resolution (2.05 Å) crystal structure or the MD simulation. Furthermore, the NMR ensemble is found to span more conformational space than the MD simulation for both the backbone and the sidechains of RNase HI. Nonetheless, the backbone conformational variability of both the NMR ensemble and the simulation is especially consistent with NMR relaxation measurements of two loop regions that are putative sites of substrate recognition. Plausible side‐chain dynamic information is extracted from the NMR ensemble on the basis of (i) rotamericity and syn‐pentane character of variable torsion angles, (ii) comparison of the magnitude of atomic mean‐square fluctuations (msf) with those deduced from crystallographic thermal factors, and (iii) comparison of torsion angle conformational behavior in the NMR ensemble and the simulation. Several heterogeneous torsion angles, while adopting non‐rotameric/syn‐pentane conformations in the NMR ensemble, exist in a unique conformation in the simulation and display low X‐ray thermal factors. These torsions are identified as sites whose variability is likely to be an artifact of the NMR structure determination procedure. A number of other torsions show a close correspondence between the conformations sampled in the NMR and MD ensembles, as well as significant correlations among crystallographic thermal factors and atomic msf calculated from the NMR ensemble and the simulation. These results indicate that a significant amount of dynamic information is contained in the NMR ensemble. The relevance of the present findings for the biological function of RNase HI, protein recognition studies, and previous investigations of the motional content of protein NMR structures are discussed. Proteins 1999;36:87–110. © 1999 Wiley‐Liss, Inc.     

Direct structure refinement of high molecular weight proteins against residual dipolar couplings and carbonyl chemical shift changes upon alignment: an application to maltose binding protein

The global fold of maltose binding protein in complex with -cyclodextrin has been determined using a CNS-based torsion angle molecular dynamics protocol involving direct refinement against dipolar couplings and carbonyl chemical shift changes that occur upon alignment. The shift changes have been included as structural restraints using a new module, CANI, that has been incorporated into CNS. Force constants and timesteps have been determined that are particularly effective in structure refinement applications involving high molecular weight proteins with small to moderate numbers of NOE restraints. Solution structures of the N- and C-domains of MBP calculated with this new protocol are within 2 Å of the X-ray conformation.     

Calculation of IR dichroic values and order parameters from molecular dynamics simulations and their application to structure determination of lipid bilayers

In polarized infrared (IR) absorption experiments, dichroic values are used to study the structure and orientation of lipid molecules. From computer simulations, we obtained angular distributions of IR transition moment (TM) orientations of the stretch vibrations of CH₂ groups of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholin (POPC) lipid bilayers in the gel (L ) and fluid (L ) phases. From these distributions, we calculated dichroic absorption values, as well as order parameters. We established a connection between the dichroic ratio R ^ATR , which is measured in IR-ATR setups, with the dichroic ratio D and the order parameter S _zz . The calculated values compare well with experimental results for the fluid phase. In addition, we computed angular distributions of transition moments with respect to the tail director orientation for the gel and the fluid phases. Only small differences were found between the distributions in the symmetric stretch orientation, the asymmetric stretch orientation, and the C-H bond orientation of CH₂ groups. The distributions of tail directors of POPC showed average tilts of 14.7° in the gel phase and 32.9° in the fluid phase. We developed a theory which makes it possible to calculate average tilt angles of tail directors in the gel phase from dichroic absorption values obtained from IR measurements for a wide range of lipids. Legendre coefficients were calculated from TM distributions. Order parameters, defined as the second Legendre polynomial, were found to closely approximate the TM distribution in lipid bilayers in the fluid phase.Abbreviations MD molecular dynamics - IR infrared - ATR attenuated total reflection - TM transition moment - POPC 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl-cholin - POPG 1-palmitoyl-2-oleoyl-sn-glycero-3-phos-phatidylglycerol - DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholin - NMR nuclear magnetic resonance     

Structure determination of UL49.5 transmembrane protein from bovine herpesvirus 1 by NMR spectroscopy and molecular dynamics

The transporter associated with antigen processing (TAP) directly participates in the immune response as a key component of the cytosolic peptide to major histocompatibility complex (MHC) class I protein loading machinery. This makes TAP an important target for viruses avoiding recognition by CD8+ T lymphocytes. Its activity can be suppressed by the UL49.5 protein produced by bovine herpesvirus 1, although the mechanism of this inhibition has not been understood so far.Therefore, the main goal of our study was to investigate the 3D structure of bovine herpesvirus 1 - encoded UL49.5 protein. The final structure of the inhibitor was established using circular dichroism (CD), 2D nuclear magnetic resonance (NMR), and molecular dynamics (MD) in membrane mimetic environments. In NMR studies, UL49.5 was represented by two fragments: the extracellular region (residues 1–35) and the transmembrane-intracellular fragment (residues 36–75), displaying various functions during viral invasion. After the empirical structure determination, a molecular docking procedure was used to predict the complex of UL49.5 with the TAP heterodimer.Our results revealed that UL49.5 adopted a highly flexible membrane-proximal helical structure in the extracellular part. In the transmembrane region, we observed two short α-helices. Furthermore, the cytoplasmic part had an unordered structure. Finally, we propose three different orientations of UL49.5 in the complex with TAP. Our studies provide, for the first time, the experimental structural information on UL49.5 and structure-based insight in its mechanism of action which might be helpful in designing new drugs against viral infections.