Monomeric α-Synuclein Binds Congo Red Micelles in a Disordered Manner

The histological dye Congo Red (CR) previously has been shown to inhibit α-synuclein (aS) fibrillation, but the mode of this inhibition remained unclear. Because of favorable exchange kinetics, interaction between CR and aS lends itself to a detailed nuclear magnetic resonance study, and relaxation dispersion measurements yield the bound fraction and time scales for the interaction of aS with CR. We find that at pH 6, CR exists as a micelle, and at a CR:aS molar ratio of ∼1, only a small fraction of aS (∼2%) is bound to these micelles. Rapid exchange (<i>k</i>_ex ∼ 3000 s^–1) between the free and CR-bound states broadens and strongly attenuates resonances of aS by two processes: a magnetic field-dependent contribution, caused by the chemical shift difference between the two states, and a nearly field-independent contribution caused by slower tumbling of aS bound to the CR micelle. The salt dependence of the interaction suggests a predominantly electrostatic mechanism for the 60 N-terminal residues, while the weaker interaction between residues 61–100 and CR is mostly hydrophobic. Chemical shift and transferred NOE data indicate that aS becomes slightly more helical but remains largely disordered when bound to CR. Results indicate that inhibition of fibril formation does not result from binding of CR to free aS and, therefore, must result from interaction of aS fibrils or protofibrils with CR micelles

Imino Hydrogen Positions in Nucleic Acids from Density Functional Theory Validated by NMR Residual Dipolar Couplings

Hydrogen atom positions of nucleotide bases in RNA structures solved by X-ray crystallography are commonly derived from heavy-atom coordinates by assuming idealized geometries. In particular, N1–H1 vectors in G and N3–H3 vectors in U are commonly positioned to coincide with the bisectors of their respective heavy-atom angles. We demonstrate that quantum-mechanical optimization of the hydrogen positions relative to their heavy-atom frames considerably improves the fit of experimental residual dipolar couplings to structural coordinates. The calculations indicate that deviations of the imino N–H vectors in RNA U and G bases result from H-bonding within the base pair and are dominated by the attractive interaction between the H atom and the electron density surrounding the H-bond-acceptor atom. DFT optimization of H atom positions is impractical in structural biology studies. We therefore have developed an empirical relation that predicts imino N–H vector orientations from the heavy-atom coordinates of the base pair. This relation agrees very closely with the DFT results, permitting its routine application in structural studies

Contrast-Matched Small-Angle X‑ray Scattering from a Heavy-Atom-Labeled Protein in Structure Determination: Application to a Lead-Substituted Calmodulin–Peptide Complex

The information content in 1-D solution X-ray scattering profiles is generally restricted to low-resolution shape and size information that, on its own, cannot lead to unique 3-D structures of biological macromolecules comparable to all-atom models derived from X-ray crystallography or NMR spectroscopy. Here we show that contrast-matched X-ray scattering data collected on a protein incorporating specific heavy-atom labels in 65% aqueous sucrose buffer can dramatically enhance the power of conventional small- and wide-angle X-ray scattering (SAXS/WAXS) measurements. Under contrast-matching conditions the protein is effectively invisible and the main contribution to the X-ray scattering intensity arises from the heavy atoms, allowing direct extraction of pairwise distances between them. In combination with conventional aqueous SAXS/WAXS data, supplemented by NMR-derived residual dipolar couplings (RDCs) measured in a weakly aligning medium, we show that it is possible to position protein domains relative to one another within a precision of 1 Å. We demonstrate this approach with respect to the determination of domain positions in a complex between calmodulin, in which the four Ca²⁺ ions have been substituted by Pb²⁺, and a target peptide. The uniqueness of the resulting solution is established by an exhaustive search over all models compatible with the experimental data, and could not have been achieved using aqueous SAXS and RDC data alone. Moreover, we show that the correct structural solution can be recovered using only contrast-matched SAXS and aqueous SAXS/WAXS data

Side Chain Conformational Distributions of a Small Protein Derived from Model-Free Analysis of a Large Set of Residual Dipolar Couplings

Accurate quantitative measurement of structural dispersion in proteins remains a prime challenge to both X-ray crystallography and NMR spectroscopy. Here we use a model-free approach based on measurement of many residual dipolar couplings (RDCs) in differentially orienting aqueous liquid crystalline solutions to obtain the side chain χ₁ distribution sampled by each residue in solution. Applied to the small well-ordered model protein GB3, our approach reveals that the RDC data are compatible with a single narrow distribution of side chain χ₁ angles for only about 40% of the residues. For more than half of the residues, populations greater than 10% for a second rotamer are observed, and four residues require sampling of three rotameric states to fit the RDC data. In virtually all cases, sampled χ₁ values are found to center closely around ideal <i>g</i>^–, <i>g</i>⁺ and <i>t</i> rotameric angles, even though no rotamer restraint is used when deriving the sampled angles. The root-mean-square difference between experimental ³J_HαHβ couplings and those predicted by the Haasnoot-parametrized, motion-adjusted Karplus equation reduces from 2.05 to 0.75 Hz when using the new rotamer analysis instead of the 1.1-Å X-ray structure as input for the dihedral angles. A comparison between observed and predicted ³J_HαHβ values suggests that the root-mean-square amplitude of χ₁ angle fluctuations within a given rotamer well is ca. 20°. The quantitatively defined side chain rotamer equilibria obtained from our study set new benchmarks for evaluating improved molecular dynamics force fields, and also will enable further development of quantitative relations between side chain chemical shift and structure

Improved Fitting of Solution X-ray Scattering Data to Macromolecular Structures and Structural Ensembles by Explicit Water Modeling

A new procedure, AXES, is introduced for fitting small-angle X-ray scattering (SAXS) data to macromolecular structures and ensembles of structures. By using explicit water models to account for the effect of solvent, and by restricting the adjustable fitting parameters to those that dominate experimental uncertainties, including sample/buffer rescaling, detector dark current, and, within a narrow range, hydration layer density, superior fits between experimental high resolution structures and SAXS data are obtained. AXES results are found to be more discriminating than standard Crysol fitting of SAXS data when evaluating poorly or incorrectly modeled protein structures. AXES results for ensembles of structures previously generated for ubiquitin show improved fits over fitting of the individual members of these ensembles, indicating these ensembles capture the dynamic behavior of proteins in solution

The Impact of Hydrogen Bonding on Amide ¹H Chemical Shift Anisotropy Studied by Cross-Correlated Relaxation and Liquid Crystal NMR Spectroscopy

Site-specific ¹H chemical shift anisotropy (CSA) tensors have been derived for the well-ordered backbone amide moieties in the B3 domain of protein G (GB3). Experimental input data include residual chemical shift anisotropy (RCSA), measured in six mutants that align differently relative to the static magnetic field when dissolved in a liquid crystalline Pf1 suspension, and cross-correlated relaxation rates between the ¹H^N CSA tensor and either the ¹H−¹⁵N, the ¹H−¹³C′, or the ¹H−¹³C^α dipolar interactions. Analyses with the assumption that the ¹H^N CSA tensor is symmetric with respect to the peptide plane (three-parameter fit) or without this premise (five-parameter fit) yield very similar results, confirming the robustness of the experimental input data, and that, to a good approximation, one of the principal components orients orthogonal to the peptide plane. ¹H^N CSA tensors are found to deviate strongly from axial symmetry, with the most shielded tensor component roughly parallel to the N−H vector, and the least shielded component orthogonal to the peptide plane. DFT calculations on pairs of <i>N</i>-methyl acetamide and acetamide in H-bonded geometries taken from the GB3 X-ray structure correlate with experimental data and indicate that H-bonding effects dominate variations in the ¹H^N CSA. Using experimentally derived ¹H^N CSA tensors, the optimal relaxation interference effect needed for narrowest ¹H^N TROSY line widths is found at ∼1200 MHz

Quantitative Residue-Specific Protein Backbone Torsion Angle Dynamics from Concerted Measurement of ³<i>J</i> Couplings

Three-bond ³<i>J</i>_C′C′ and ³<i>J</i>_HNHα couplings in peptides and proteins are functions of the intervening backbone torsion angle ϕ. In well-ordered regions, ³<i>J</i>_HNHα is tightly correlated with ³<i>J</i>_C′C′, but the presence of large ϕ angle fluctuations differentially affects the two types of couplings. Assuming the ϕ angles follow a Gaussian distribution, the width of this distribution can be extracted from ³<i>J</i>_C′C′ and ³<i>J</i>_HNHα, as demonstrated for the folded proteins ubiquitin and GB3. In intrinsically disordered proteins, slow transverse relaxation permits measurement of ³<i>J</i>_C′C′ and ³<i>J</i>_HNH couplings at very high precision, and impact of factors other than the intervening torsion angle on ³<i>J</i> will be minimal, making these couplings exceptionally valuable structural reporters. Analysis of α-synuclein yields rather homogeneous widths of 69 ± 6° for the ϕ angle distributions and ³<i>J</i>_C′C′ values that agree well with those of a recent maximum entropy analysis of chemical shifts, <i>J</i> couplings, and ¹H–¹H NOEs. Data are consistent with a modest (≤30%) population of the polyproline II region

Improved Cross Validation of a Static Ubiquitin Structure Derived from High Precision Residual Dipolar Couplings Measured in a Drug-Based Liquid Crystalline Phase

The antibiotic squalamine forms a lyotropic liquid crystal at very low concentrations in water (0.3-3.5% w/v), which remains stable over a wide range of temperature (1-40 °C) and pH (4-8). Squalamine is positively charged, and comparison of the alignment of ubiquitin relative to 36 previously reported alignment conditions shows that it differs substantially from most of these, but is closest to liquid crystalline cetyl pyridinium bromide. High precision residual dipolar couplings (RDCs) measured for the backbone ¹H-¹⁵N, ¹⁵N-¹³C′, ¹H^α-¹³C^α, and ¹³C′-¹³C^α one-bond interactions in the squalamine medium fit well to the static structural model previously derived from NMR data. Inclusion into the structure refinement procedure of these RDCs, together with ¹H-¹⁵N and ¹H^α-¹³C^α RDCs newly measured in Pf1, results in improved agreement between alignment-induced changes in ¹³C′ chemical shift, ³<i>J</i>_HNHα values, and ¹³C^α-¹³C^β RDCs and corresponding values predicted by the structure, thereby validating the high quality of the single-conformer structural model. This result indicates that fitting of a single model to experimental data provides a better description of the average conformation than does averaging over previously reported NMR-derived ensemble representations. The latter can capture dynamic aspects of a protein, thus making the two representations valuable complements to one another

Crystal Structures of the LsrR Proteins Complexed with Phospho-AI‑2 and Two Signal-Interrupting Analogues Reveal Distinct Mechanisms for Ligand Recognition

Quorum sensing (QS) is a cell-to-cell communication system responsible for a variety of bacterial phenotypes including virulence and biofilm formation. QS is mediated by small molecules, autoinducers (AIs), including AI-2 that is secreted by both Gram-positive and -negative microbes. LsrR is a key transcriptional regulator that governs the varied downstream processes by perceiving AI-2 signal, but its activation via autoinducer-binding remains poorly understood. Here, we provide detailed regulatory mechanism of LsrR from the crystal structures in complexes with the native signal (phospho-AI-2, D5P) and two quorum quenching antagonists (ribose-5-phosphate, R5P; phospho-isobutyl-AI-2, D8P). Interestingly, the bound D5P and D8P molecules are not the diketone forms but rather hydrated, and the hydrated moiety forms important H-bonds with the carboxylate of D243. The D5P-binding flipped out F124 of the binding pocket, and resulted in the disruption of the dimeric interface-1 by unfolding the α7 segment. However, the same movement of F124 by the D8P′-binding did not cause the unfolding of the α7 segment. Although the LsrR-binding affinity of R5P (<i>K</i>_d, ∼1 mM) is much lower than that of D5P and D8P (∼2.0 and ∼0.5 μM), the α-anomeric R5P molecule fits into the binding pocket without any structural perturbation, and thus stabilizes the LsrR tetramer. The binding of D5P, not D8P and R5P, disrupted the tetrameric structure and thus is able to activate LsrR. The detailed structural and mechanistic insights from this study could be useful for facilitating design of new antivirulence and antibiofilm agents based on LsrR