Uli Schmitz

Probability assessment of conformational ensembles: sugar repuckering in a DNA duplex in solution

Conformational flexibility of molecules in solution implies that different conformers contribute to the NMR signal. This may lead to internal inconsistencies in the 2D NOE-derived interproton distance restraints and to conflict with scalar coupling-based torsion angle restraints. Such inconsistencies have been revealed and analyzed for the DNA octamer GTATAATG.CATATTAC, containing the Pribnow box consensus sequence. A number of subsets of distance restraints were constructed and used in the restrained Monte Carlo refinement of different double-helical conformers. The probabilities of conformers were then calculated by a quadratic programming algorithm, minimizing a relaxation rate-base residual index. The calculated distribution of conformers agrees with the experimental NOE data as an ensemble better than any single structure. A comparison with the results of this procedure, which we term PARSE (Probability Assessment via Relaxation rates of a Structural Ensemble), to an alternative method to generate solution ensembles showed, however, that the detailed multi-conformational description of solution DNA structure remains ambiguous at this stage. Nevertheless, some ensemble properties can be deduced with confidence, the most prominent being a distribution of sugar puckers with minor populations in the N-region and major populations in the S-region. Importantly, such a distribution is in accord with the analysis of independent experimental data--deoxyribose proton-proton scalar coupling constants.

Structure of the phylogenetically most conserved domain of SRP RNA.

The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein required for cotranslational targeting of proteins to the membrane of the endoplasmic reticulum of the bacterial plasma membrane. Domain IV of SRP RNA consists of a short stem-loop structure with two internal loops that contain the most conserved nucleotides of the molecule. All known essential interactions of SRP occur in that moiety containing domain IV. The solution structure of a 43-nt RNA comprising the complete Escherichia coli domain IV was determined by multidimensional NMR and restrained molecular dynamics refinement. Our data confirm the previously determined rigid structure of a smaller subfragment containing the most conserved, symmetric internal loop A (Schmitz et al., Nat Struct Biol, 1999, 6:634-638), where all conserved nucleotides are involved in nucleotide-specific structural interactions. Asymmetric internal loop B provides a hinge in the RNA molecule; it is partially flexible, yet also uniquely structured. The longer strand of internal loop B extends the major groove by creating a ledge-like arrangement; for loop B however, there is no obvious structural role for the conserved nucleotides. The structure of domain IV suggests that loop A is the initial site for the RNA/protein interaction creating specificity, whereas loop B provides a secondary interaction site.

Structure of the most conserved internal loop in SRP RNA.

The signal recognition particle (SRP) directs translating ribosomes to the protein translocation apparatus of endoplasmic reticulum (ER) membrane or the bacterial plasma membrane. The SRP is universally conserved, and in prokaryotes consists of two essential subunits, SRP RNA and SRP54, the latter of which binds to signal sequences on the nascent protein chains. Here we describe the solution NMR structure of a 28-mer RNA composing the most conserved part of SRP RNA to which SRP54 binds. Central to this function is a six-nucleotide internal loop that assumes a novel Mg2+-dependent structure with unusual cross-strand interactions; besides a cross-strand A/A stack, two guanines form hydrogen bonds with opposite-strand phosphates. The structure completely explains the phylogenetic conservation of the loop bases, underlining its importance for SRP54 binding and SRP function.

Solution structure of [d(GTATATAC)]2 via restrained molecular dynamics simulations with nuclear magnetic resonance constraints derived from relaxation matrix analysis of two-dimensional nuclear Overhauser effect experiments.

Two-dimensional nuclear Overhauser effect (2D NOE) spectra have been used as the experimental basis for determining the solution structure of the duplex [d(GTATATAC)]2 employing restrained molecular dynamics (rMD) simulations. The MARDIGRAS algorithm has been employed to construct a set of 233 interproton distance constraints via iterative complete relaxation matrix analysis utilizing the peak intensities from the 2D NOE spectra obtained for different mixing times and model structures. The upper and lower bounds for each of the constraints, defining size of a flat-well potential function term used in the rMD simulations, were conservatively chosen as the largest or smallest value calculated by MARDIGRAS. Three different starting models were utilized in several rMD calculations: energy-minimized A-DNA, B-DNA, and a structure containing wrinkled D-DNA in the interior. Considerable effort was made to define the appropriate force constants to be employed with the NOE terms in the AMBER force field, using as criteria the average constraints deviation, the constraints violation energy and the total energy. Of the 233 constraints, one was generated indirectly, but proved to be crucial in defining the structure: the cross-strand A5-H2 A5-H2 distance. As those two protons resonate isochronously for the self-complementary duplex, the distance cannot be determined directly. However, the general pattern of 2D NOE peak intensities, spin-lattice relaxation time (T1) values, and 31P nuclear magnetic resonance spectra lead to use of the A3-H2 A7-H2 distance for A5-H2 A5-H2 as well. Five rMD runs, with different random number seeds, were made for each of the three starting structures with the full distance constraint set. The average structure from all 15 runs and the five-structure averages from each starting structure were all quite similar. Two rMD runs for each starting structure were made with the A5-H2 A5-H2 constraint missing. The average of these six rMD runs revealed differences in structure, compared to that with the full set of constraints, primarily for the middle two base-pairs involving the missing cross-strand constraint but global deviations also were found. Conformational analysis of the resulting structures revealed that the inner four to six base-pairs differed in structure from the termini. Furthermore, an alternating structure was suggested with features alternating for the A-T and T-A steps.

Metropolis Monte Carlo calculations of DNA structure using internal coordinates and NMR distance restraints: An alternative method for generating a high-resolution solution structure

SummaryA new method, a restrained Monte Carlo (rMC) calculation, is demonstrated for generating high-resolution structures of DNA oligonucleotides in solution from interproton distance restraints and bounds derived from complete relaxation matrix analysis of two-dimensional nuclear Overhauser effect (NOE) spectral peak intensities. As in the case of restrained molecular dynamics (rMD) refinement of structures, the experimental distance restraints and bounds are incorporated as a pseudo-energy term (or penalty function) into the mathematical expression for the molecular energy. However, the use of generalized helical parameters, rather than Cartesian coordinates, to define DNA conformation increases efficiency by decreasing by an order of magnitude the number of parameters needed to describe a conformation and by simplifying the potential energy profile. The Metropolis Monte Carlo method is employed to simulate an annealing process. The rMC method was applied to experimental 2D NOE data from the octamer duplex d(GTA-TAATG)·d(CATTATAC). Using starting structures from different locations in conformational space (e.g. A-DNA and B-DNA), the rMC calculations readily converged, with a root-mean-square deviation (RMSD) of <0.3 Å between structures generated using different protocols and starting structures. Theoretical 2D NOE peak intensities were calculated for the rMC-generated structures using the complete relaxation matrix program CORMA, enabling a comparison with experimental intensities via residual indices. Simulation of the vicinal proton coupling constants was carried out for the structures generated, enabling a comparison with the experimental deoxyribose ring coupling constants, which were not utilized in the structure determination in the case of the rMC simulations. Agreement with experimental 2D NOE and scalar coupling data was good in all cases. The rMC structures are quite similar to that refined by a traditional restrained MD approach (RMSD<0.5 Å) despite the different force fields used and despite the fact that MD refinement was conducted with additional restraints imposed on the endocyclic torsion angles of deoxyriboses. The computational time required for the rMC and rMD calculations is about the same. A comparison of structural parameters is made and some limitations of both methods are discussed with regard to the average nature of the experimental restraints used in the refinement.

The dynamic NMR structure of the TψC-loop: Implications for the specificity of tRNA methylation

AbstracttRNA (m5U54)-methyltransferase (RUMT) catalyzes the S-adenosylmethionine-dependentmethylation of uridine-54 in the TΨC-loop of all transfer RNAs in E. coli to form the 54-ribosylthymine residue. However, in all tRNA structures, residue 54 is completely buried andthe question arises as to how RUMT gains access to the methylation site. A 17-mer RNAhairpin consisting of nucleotides 49–65 of the TΨ-loop is a substrate for RUMT.Homonuclear NMR methods in conjunction with restrained molecular dynamics (MD)methods were used to determine the solution structure of the 17-mer T-arm fragment. Theloop of the hairpin exhibits enhanced flexibility which renders the conventional NMR averagestructure less useful compared to the more commonly found situation where a molecule existsin predominantly one major conformation. However, when resorting to softer refinementmethods such as MD with time-averaged restraints, the conflicting restraints in the loop canbe satisfied much better. The dynamic structure of the T-arm is represented as an ensembleof 10 time-clusters. In all of these, U54 is completely exposed. The flexibility of the TΨ-loop in solution in conjunction with extensive binding studies of RUMT with the TΨC-loop and tRNA suggest that the specificity of the RUMT/tRNA recognition is associated withtRNA tertiary structure elements. For the methylation, RUMT would simply have to breakthe tertiary interactions between the D- and T-loops, leading to a melting of the T-armstructure and making U54 available for methylation.

Small structural ensembles for a 17-nucleotide mimic of the tRNA TΨC-loop via fitting dipolar relaxation rates with the quadratic programming algorithm

Solution structures are typically average structures determined with the help of nmr-derived distance and torsion angle information. However, when a biomolecule populates significantly different conformations, the average structure might be prone to artifacts, and other refinement strategies are necessary. For example, when experimental restraints are used in molecular dynamics simulations in a time-averaged fashion (MDtar), the experimental structural information does no longer need to be satisfied at each step of the simulation; instead, the whole trajectory must agree with the restraints. However, the resulting structural ensembles are large and not unique and it is not trivial to extract the essential dynamic features for a system. Here we demonstrate that large MDtar ensembles can be simplified substantially by reducing the number of members to just a few on the basis of adjusting the individual probabilities of the members with the PDQPRO program [N. B. Ulyanov et al. Biophysical Journal (1995), Vol. 68, p. 13]. This algorithm finds the global minimum for a search function that represents the best match of a given ensemble with the experimental dipolar interproton relaxation rates. We have applied this strategy to a 17-residue RNA hairpin, whose loop exhibited considerable flexibility evident from nmr data. This 17mer is a mimic of the T psi C-loop of tRNA, where nucleotide 54 is usually a ribosylthymidine. The methylation of U54, which is completely buried in transfer ribonucleic acid, is administered by tRNA (m5 U54)-methyltransferase (RUMT). Since the 17mer is a good substrate for RUMT, we previously concluded that the flexibility of the 17mers loop is a key to how RUMT gains access to the methylation site [L. J. Yao et al. Journal of Biomolecular NMR (1996) Vol. 9. p. 229]. Application of the PDQPRO algorithm to the previously acquired MDtar trajectories allowed us to reduce the number of conformations from several hundred to one major and five or six minor conformations with individual populations from approximately 5% to approximately 50% without any deterioration in the match with the experimental data. The major conformation exhibits a continuation of A-form helicity through part of the loop, involving C60 and U59. In this and most other conformations the methylation site in U54 is no longer buried.