Experimental Residual Dipolar Couplings Research Articles

Recombinant Expression and Chemical Amidation of Isotopically Labeled Native Melittin.

Post-translational modifications are ubiquitous in the eukaryotic proteome. However, these modifications are rarely incorporated in NMR studies of eukaryotic proteins, which are typically produced through recombinant expression in E. coli. Melittin is the primary peptide in honey bee venom. Its native C-terminal amide significantly affects its equilibrium structure and dynamics in solution and is thus a prerequisite for studying its native structure and function. Here, we present a method for producing triply isotopically labeled (2H, 13C, and 15N) native melittin through recombinant expression followed by chemical amidation. We then show that structural models produced with AlphaFold-Multimer are in even better agreement with experimental residual dipolar couplings than the 2.0 Å resolution X-ray crystal structure for residues G3-K23.

Enantiodiscrimination of Inherently Chiral Thiacalixarenes by Residual Dipolar Couplings.

Inherently chiral compounds, such as calixarenes, are chiral due to a nonplanar three-dimensional (3D) structure. Determining their conformation is essential to understand their properties, with nuclear magnetic resonance (NMR) spectroscopy being one applicable method. Using alignment media to measure residual dipolar couplings (RDCs) to obtain structural information is advantageous when classical NMR parameters like the nuclear Overhauser effect (NOE) or J-couplings fail. Besides providing more accurate structural information, the alignment media can induce different orientations of enantiomers. In this study, we examined the ability of polyglutamates with different side-chain moieties─poly-γ-benzyl-l-glutamate (PBLG) and poly-γ-p-biphenylmethyl-l-glutamate (PBPMLG) ─to enantiodifferentiate the inherently chiral phenoxathiin-based thiacalix[4]arenes. Both media, in combination with two solvents, allowed for enantiodiscrimination, which was, to the best of our knowledge, proved for the first time on inherently chiral compounds. Moreover, using the experimental RDCs, we investigated the calix[4]arenes conformational preferences in solution, quantitatively analyzed the differences in the alignment of the enantiomers, and discussed the pitfalls of the use of the RDC analysis.

Solution structure ensemble of human obesity-associated protein FTO reveals druggable surface pockets at the interface between the N- and C-terminal domain

The fat mass and obesity-associated FTO protein catalyzes demethylation of the N6-methyladenosine, an epigenetic mark that controls several metabolic pathways by modulating the transcription, translation, and cellular localization of RNA molecules. Since the discovery that its overexpression links to the development of obesity and cancer, FTO was the target of screening campaigns and structure-based drug design efforts. Although several FTO inhibitors were generated, these often lack potency or selectivity. Herein, we investigate the structure and dynamics of human FTO in solution. We show that the structure of the catalytic N-terminal domain is unstable in the absence of the C-terminal domain, which explains why the isolated N-terminal domain is incompetent for catalysis and suggests that the domain interaction represents a target for the development of specific inhibitors. Then, by using NMR relaxation measurements, we show that the interface between the FTO structural domains, the active site, and several peripheral loops undergo conformational dynamics on both the picosecond–nanosecond and microsecond–millisecond timescales. Consistent with this, we found that the backbone amide residual dipolar couplings measured for FTO in phage pf1 are inconsistent with the static crystal structure of the enzyme. Finally, we generated a conformational ensemble for apo FTO that satisfies the solution NMR data by combining the experimental residual dipolar couplings with accelerated molecular dynamics simulations. Altogether, the structural ensemble reported in this work provides an atomic-resolution model of apo FTO and reveals transient surface pockets at the domain interface that represent potential targets for the design of allosteric inhibitors.

Model free analysis of experimental residual dipolar couplings in small organic compounds.

Residual dipolar couplings (RDCs) contain information on the relative arrangement and dynamics of internuclear spin vectors in chemical compounds. Classically, RDC data is analyzed by fitting to structure models, while model-free approaches (MFA) directly relate RDCs to the corresponding internuclear vectors. The recently introduced software TITANIA implements the MFA and extracts structure and dynamics parameters directly from RDCs to facilitate de novo structure refinement for small organic compounds. Encouraged by our previous results on simulated data, we herein focus on the prerequisites and challenges faced when using purely experimental data for this approach. These concern mainly the fact that not all couplings are accessible in all media, leading to voids in the RDC matrix and the concomitant effects on the structure refinement. It is shown that RDC data sets obtained experimentally from currently available alignment media and measurement methods are of sufficient quality to allow relative configuration determination even when the relative configuration of the analyte is completely unknown.

Concordance of X-ray and AlphaFold2 Models of SARS-CoV-2 Main Protease with Residual Dipolar Couplings Measured in Solution.

The 68-kDa homodimeric 3C-like protease of SARS-CoV-2, Mpro (3CLpro/Nsp5), is a promising antiviral drug target. We evaluate the concordance of models generated by the newly introduced AlphaFold2 structure prediction program with residual dipolar couplings (RDCs) measured in solution for 15N–1HN and 13C′–1HN atom pairs. The latter were measured using a new, highly precise TROSY-AntiTROSY Encoded RDC (TATER) experiment. Three sets of AlphaFold2 models were evaluated: (1) MproAF, generated using the standard AlphaFold2 input structural database; (2) MproAFD, where the AlphaFold2 implementation was modified to exclude all candidate template X-ray structures deposited after Jan 1, 2020; and (3) MproAFS, which excluded all structures homologous to coronaviral Mpro. Close agreement between all three sets of AlphaFold models and experimental RDC data is found for most of the protein. For residues in well-defined secondary structure, the agreement decreases somewhat upon Amber relaxation. For these regions, MproAF agreement exceeds that of most high-resolution X-ray structures. Residues from domain 2 that comprise elements of both the active site and the homo-dimerization interface fit less well across all structures. These results indicate novel opportunities for combining experimentation with molecular dynamics simulations, where solution RDCs provide highly precise input for QM/MM simulations of substrate binding/reaction trajectories.

NMR hawk-eyed view of AlphaFold2 structures.

The prediction of the three‐dimensional (3D) structure of proteins from the amino acid sequence made a stunning breakthrough reaching atomic accuracy. Using the neural network‐based method AlphaFold2, 3D structures of almost the entire human proteome have been predicted and made available (https://www.alphafold.ebi.ac.uk). To gain insight into how well AlphaFold2 structures represent the conformation of proteins in solution, I here compare the AlphaFold2 structures of selected small proteins with their 3D structures that were determined by nuclear magnetic resonance (NMR) spectroscopy. Proteins were selected for which the 3D solution structures were determined on the basis of a very large number of distance restraints and residual dipolar couplings and are thus some of the best‐resolved solution structures of proteins to date. The quality of the backbone conformation of the AlphaFold2 structures is assessed by fitting a large set of experimental residual dipolar couplings (RDCs). The analysis shows that experimental RDCs fit extremely well to the AlphaFold2 structures predicted for GB3, DinI, and ubiquitin. In the case of GB3, the accuracy of the AlphaFold2 structure even surpasses that of a 1.1 Å crystal structure. Fitting of experimental RDCs furthermore allows identification of AlphaFold2 structures that are best representative of the protein's conformation in solution as seen for the EF hands of the N‐terminal domain of Ca2+‐ligated calmodulin. Taken together, the analysis shows that structures predicted by AlphaFold2 can be highly representative of the solution conformation of proteins. The combination of AlphaFold2 structures with RDCs promises to be a powerful approach to study structural changes in proteins.

Determination of Configuration and Conformation of a Reserpine Derivative with Seven Stereogenic Centers Using Molecular Dynamics with RDC-Derived Tensorial Constraints*.

NMR‐based determination of the configuration of complex molecules containing many stereocenters is often not possible using traditional NOE data and coupling patterns. Making use of residual dipolar couplings (RDCs), we were able to determine the relative configuration of a natural product containing seven stereocenters, including a chiral amine lacking direct RDC data. To identify the correct relative configuration out of 32 possible ones, experimental RDCs were used in three different approaches for data interpretation: by fitting experimental data based singular value decomposition (SVD) using a single alignment tensor and either (i) a single conformer or (ii) multiple conformers, or alternatively (iii) using molecular dynamics simulations with tensorial orientational constraints (MDOC). Even though in all three approaches one and the same configuration could be selected and clear discrimination between possible configurations was achieved, the experimental data was not fully satisfied by the methods based on single tensor approaches. While these two approaches are faster, only MDOC is able to fully reproduce experimental results, as the obtained conformational ensemble adequately covers the conformational space necessary to describe the molecule with inherent flexibility.

The simulation of NMR data of flexible molecules: sagittamide A as an example for MD simulations with orientational constraints.

The recently developed MDOC (Molecular Dynamics with Orientational Constraints) simulation is applied for the first time to a fully flexible molecule. MDOC simulations aim to single out the naturally existing configuration of molecules and to elucidate conformer populations. The performance of the method was first demonstrated on a well-studied test case, the five-membered ring lactone (α-methylene-γ-butyrolactone). In the case of sagittamide A, one-bond 1H-13C residual dipolar couplings (RDC) are used as orientational constraints that reorient the molecule or parts of it. In addition, NOE distances and 3J scalar couplings are used as constraints. Five possible configurations of sagittamide A (labelled a to e) are considered. One experimental RDC value per flexible unit was available and this was not sufficient to single out one valid configuration. The problem could be solved by including NOE distances as well as 3J couplings as complementary constraints into the MDOC simulations. In accordance with former investigations, we confirmed the configuration a for the natural product. A detailed analysis of conformers of the central chain of 6 chiral carbon atoms could be given by inspecting the MDOC trajectory. The relative abundance of these conformers is crucial in fulfilling all three sets of constraints.

Quantitative Conformational Analysis of Functionally Important Electrostatic Interactions in the Intrinsically Disordered Region of Delta Subunit of Bacterial RNA Polymerase.

Electrostatic interactions play important roles in the functional mechanisms exploited by intrinsically disordered proteins (IDPs). The atomic resolution description of long-range and local structural propensities that can both be crucial for the function of highly charged IDPs presents significant experimental challenges. Here, we investigate the conformational behavior of the δ subunit of RNA polymerase from Bacillus subtilis whose unfolded domain is highly charged, with 7 positively charged amino acids followed by 51 acidic amino acids. Using a specifically designed analytical strategy, we identify transient contacts between the two regions using a combination of NMR paramagnetic relaxation enhancements, residual dipolar couplings (RDCs), chemical shifts, and small-angle scattering. This strategy allows the resolution of long-range and local ensemble averaged structural contributions to the experimental RDCs, and reveals that the negatively charged segment folds back onto the positively charged strand, compacting the conformational sampling of the protein while remaining highly flexible in solution. Mutation of the positively charged region abrogates the long-range contact, leaving the disordered domain in an extended conformation, possibly due to local repulsion of like-charges along the chain. Remarkably, in vitro studies show that this mutation also has a significant effect on transcription activity, and results in diminished cell fitness of the mutated bacteria in vivo. This study highlights the importance of accurately describing electrostatic interactions for understanding the functional mechanisms of IDPs.

NMR‐assisted protein structure prediction with MELDxMD

We describe the performance of MELD-accelerated molecular dynamics (MELDxMD) in determining protein structures in the NMR-data-assisted category in CASP13. Seeded from web server predictions, MELDxMD was found best in the NMR category, over 17 targets, outperforming the next-best groups by a factor of ~4 in z-score. MELDxMD gives ensembles, not single structures; succeeds on a 326-mer, near the current upper limit for NMR structures; and predicts structures that match experimental residual dipolar couplings even though the only NMR-derived data used in the simulations was NOE-based ambiguous atom-atom contacts and backbone dihedrals. MELD can use noisy and ambiguous experimental information to reduce the MD search space. We believe MELDxMD is a promising method for determining protein structures from NMR data.

Maximizing accuracy of RNA structure in refinement against residual dipolar couplings.

Structural information about ribonucleic acid (RNA) is lagging behind that of proteins, in part due to its high charge and conformational variability. Molecular dynamics (MD) has played an important role in describing RNA structure, complementing information from both nuclear magnetic resonance (NMR), or X-ray crystallography. We examine the impact of the choice of the empirical force field for RNA structure refinement using cross-validation against residual dipolar couplings (RDCs) as structural accuracy reporter. Four force fields, representing both the state-of-the art in RNA simulation and the most popular selections in NMR structure determination, are compared for a prototypical A-RNA helix. RNA structural accuracy is also evaluated as a function of both density and nature of input NMR data including RDCs, anisotropic chemical shifts, and distance restraints. Our results show a complex interplay between the experimental restraints and the force fields indicating two best-performing choices: high-fidelity refinement in explicit solvent, and the conformational database-derived potentials. Accuracy of RNA models closely tracks the density of 1-bond C-H RDCs, with other data types having beneficial, but smaller effects. At lower RDC density, or when refining against NOEs only, the two selected force fields are capable of accurately describing RNA helices with little or no experimental RDC data, making them available for the higher order structure assembly or better quantification of the intramolecular dynamics. Unrestrained simulations of simple RNA motifs with state-of-the art MD force fields appear to capture the flexibility inherent in nucleic acids while also maintaining a good agreement with the experimental observables.

Progressive Stereo Locking (PSL): A Residual Dipolar Coupling Based Force Field Method for Determining the Relative Configuration of Natural Products and Other Small Molecules.

Establishing the relative configuration of a bioactive natural product represents the most challenging part in determining its structure. Residual dipolar couplings (RDCs) are sensitive probes of the relative spatial orientation of internuclear vectors. We adapted a force field structure calculation methodology to allow free sampling of both R and S configurations of the stereocenters of interest. The algorithm uses a floating alignment tensor in a simulated annealing protocol to identify the conformations and configurations that best fit experimental RDC and distance restraints (from NOE and J-coupling data). A unique configuration (for rigid molecules) or a very small number of configurations (for less rigid molecules) of the structural models having the lowest chiral angle energies and reasonable magnitudes of the alignment tensor are provided as the best predictions of the unknown configuration. For highly flexible molecules, the progressive locking of their stereocenters into their statistically dominant R or S state dramatically reduces the number of possible relative configurations. The result is verified by checking that the same configuration is obtained by initiating the locking from different regions of the molecule. For all molecules tested having known configurations (with conformations ranging from mostly rigid to highly flexible), the method accurately determined the correct configuration.

Pressure-induced structural transition of mature HIV-1 protease from a combined NMR/MD simulation approach.

We investigate the pressure-induced structural changes in the mature human immunodeficiency virus type 1 protease dimer, using residual dipolar coupling (RDC) measurements in a weakly oriented solution. (1)DNH RDCs were measured under high-pressure conditions for an inhibitor-free PR and an inhibitor-bound complex, as well as for an inhibitor-free multidrug resistant protease bearing 20 mutations (PR20). While PR20 and the inhibitor-bound PR were little affected by pressure, inhibitor-free PR showed significant differences in the RDCs measured at 600 bar compared with 1 bar. The structural basis of such changes was investigated by MD simulations using the experimental RDC restraints, revealing substantial conformational perturbations, specifically a partial opening of the flaps and the penetration of water molecules into the hydrophobic core of the subunits at high pressure. This study highlights the exquisite sensitivity of RDCs to pressure-induced conformational changes and illustrates how RDCs combined with MD simulations can be used to determine the structural properties of metastable intermediate states on the folding energy landscape.

Backbone hydration determines the folding signature of amino acid residues.

The relation between the sequence of a protein and its three-dimensional structure remains largely unknown. A lasting dream is to elucidate the side-chain-dependent driving forces that govern the folding process. Different structural data suggest that aromatic amino acids play a particular role in the stabilization of protein structures. To better understand the underlying mechanism, we studied peptides of the sequence EGAAXAASS (X = Gly, Ile, Tyr, Trp) through comparison of molecular dynamics (MD) trajectories and NMR residual dipolar coupling (RDC) measurements. The RDC data for aromatic substitutions provide evidence for a kink in the peptide backbone. Analysis of the MD simulations shows that the formation of internal hydrogen bonds underlying a helical turn is key to reproduce the experimental RDC values. The simulations further reveal that the driving force leading to such helical-turn conformations arises from the lack of hydration of the peptide chain on either side of the bulky aromatic side chain, which can potentially act as a nucleation point initiating the folding process.

Elucidating Molecular Motion through Structural and Dynamic Filters of Energy-Minimized Conformer Ensembles

Complex RNA structures are constructed from helical segments connected by flexible loops that move spontaneously and in response to binding of small molecule ligands and proteins. Understanding the conformational variability of RNA requires the characterization of the coupled time evolution of interconnected flexible domains. To elucidate the collective molecular motions and explore the conformational landscape of the HIV-1 TAR RNA, we describe a new methodology that utilizes energy-minimized structures generated by the program “Fragment Assembly of RNA with Full-Atom Refinement (FARFAR)”. We apply structural filters in the form of experimental residual dipolar couplings (RDCs) to select a subset of discrete energy-minimized conformers and carry out principal component analyses (PCA) to corroborate the choice of the filtered subset. We use this subset of structures to calculate solution T1 and T1ρ relaxation times for 13C spins in multiple residues in different domains of the molecule using two simulation protocols that we previously published. We match the experimental T1 times to within 2% and the T1ρ times to within less than 10% for helical residues. These results introduce a protocol to construct viable dynamic trajectories for RNA molecules that accord well with experimental NMR data and support the notion that the motions of the helical portions of this small RNA can be described by a relatively small number of discrete conformations exchanging over time scales longer than 1 μs.

Aromatic Amino Acids Promote Peptide Folding by Reducing Backbone Hydration

The relation between the sequence of a protein and its tridimensional structure remains largely unknown. To better understand how single residues impact on the local protein structure, we studied peptides of sequence EGAAXAASS (X = Gly, Ile, Tyr, Trp) through comparison of molecular dynamics (MD) trajectories and NMR residual dipolar coupling (RDC) measurements. The RDC patterns of the peptide with X = Gly or Ile are rather flat, suggesting extended, unfolded peptides, while the contrasted patterns for peptides with X = Tyr or Trp suggest compact folded structures. The comparison shows that the formation of internal hydrogen bonds underlying helical-turns is key to reproduce experimental RDC values for the peptides containing aromatic residues. The simulations further reveal that the driving force leading to such helical-turn conformation arises from the lack of hydration of the peptide chain on either side of the bulky aromatic side chain, which can potentially act as a nucleation point initiating the folding process. These results provide a starting point to understand the amino acid code underlying the mechanism of protein folding.

Unfolded protein ensembles, folding trajectories, and refolding rate prediction

Computer simulations can provide critical information on the unfolded ensemble of proteins under physiological conditions, by explicitly characterizing the geometrical properties of the diverse conformations that are sampled in the unfolded state. A general computational analysis across many proteins has not been implemented however. Here, we develop a method for generating a diverse conformational ensemble, to characterize properties of the unfolded states of intrinsically disordered or intrinsically folded proteins. The method allows unfolded proteins to retain disulfide bonds. We examined physical properties of the unfolded ensembles of several proteins, including chemical shifts, clustering properties, and scaling exponents for the radius of gyration with polymer length. A problem relating simulated and experimental residual dipolar couplings is discussed. We apply our generated ensembles to the problem of folding kinetics, by examining whether the ensembles of some proteins are closer geometrically to their folded structures than others. We find that for a randomly selected dataset of 15 non-homologous 2- and 3-state proteins, quantities such as the average root mean squared deviation between the folded structure and unfolded ensemble correlate with folding rates as strongly as absolute contact order. We introduce a new order parameter that measures the distance travelled per residue, which naturally partitions into a smooth "laminar" and subsequent "turbulent" part of the trajectory. This latter conceptually simple measure with no fitting parameters predicts folding rates in 0 M denaturant with remarkable accuracy (r = -0.95, p = 1 × 10(-7)). The high correlation between folding times and sterically modulated, reconfigurational motion supports the rapid collapse of proteins prior to the transition state as a generic feature in the folding of both two-state and multi-state proteins. This method for generating unfolded ensembles provides a powerful approach to address various questions in protein evolution, misfolding and aggregation, transient structures, and molten globule and disordered protein phases.

Giant Residual Dipolar 13C–1H Couplings in High‐Spin Organoiron Complexes: Elucidation of Their Structures in Solution by 13C NMR Spectroscopy

High-spin Fe(II)-alkyl complexes with bis(pyridylimino)isoindolato ligands were synthesized and their paramagnetic (1)H and (13)C NMR spectra were analyzed comprehensively. The experimental (13)C-(1)H coupling values are temperature (T(-1))- as well as magnetic-field (B(2))-dependent and deviate considerably from typical scalar (1)J(CH) couplings constants. This deviation is attributed to residual dipolar couplings (RDCs), which arise from partial alignment of the complexes in the presence of a strong magnetic field. The analysis of the experimental RDCs allows an unambiguous assignment of all (13)C NMR resonances and, additionally, a structural refinement of the conformation of the complexes in solution. Moreover the RDCs can be used for the analysis of the alignment tensor and hence the tensor of the anisotropy of the magnetic susceptibility.

Configuration verification via RDCs on the example of a tetra‐substituted pyrrolidine ring

The configurational analysis of organic compounds is an important application for high resolution NMR spectroscopy. In the present study, a tetra-substituted pyrrolidine with four chiral carbon atoms is analyzed using classical methods based on (3) J and NOE data in solution and compared and verified with recently introduced alternative approaches via residual dipolar couplings (RDCs) in two weak anisotropic alignment media. The molecule shows sufficient rigidity in the five-membered ring for the configurational characterization with the various techniques. However, the flexibility caused by the many freely rotating bonds potentially poses problems for the interpretation of data. It is shown that RDCs measured in poly-γ-benzyl-l-glutamate and a stretched polydimethylsiloxane gel provide useful information for the distinction of diastereomers, but the success varies with the data interpretation strategy used. Although a general improvement of corresponding correlation factors is observed when limiting data to a subset of dipolar couplings directly connected to the central ring, the distinction power is reduced because of the smaller number of RDCs available for potential model falsification. Singular value decomposition for fitting experimental RDCs is able to distinguish in most cases the correct from incorrect configurations, but the differences in correlation factors can be relatively small. Surprisingly, predicting RDCs using the rod model as implemented in PALES gives best results in distinguishing the eight possible diastereomers. It is also found that the use of proton-phosphorus and carbon-phosphorus RDCs helps with the configurational analysis of the model compound.

Influence of solvent and salt concentration on the alignment properties of acrylamide copolymer gels for the measurement of RDC

The dependence of molecular alignment with solvent nature and salt concentration has been investigated for mechanically stretched polyacrylamide copolymer gels. Residual dipolar couplings (RDCs) were recorded for D(2)O, DMSO-d(6), and DMSO-d(6)/D(2)O solutions containing different proportions of the solvents and different sodium chloride concentrations. Alignment tensors were determined by fitting the experimental RDCs to the DFT-computed structure of N-methylcodeinium ion. Analysis of the tensors shows that the degree of alignment decreases with the proportion of DMSO-d(6) as well as with the concentration of sodium chloride, most likely due to enhanced ion-pair aggregation. Furthermore, rotation of the alignment tensor is observed when increasing the salt concentration.