The difference in angle at the most disrupted site between helices in homologous helix pairs (|<i>θ</i>_max − <i>θ</i>_min|) plotted against the sequence identity between A) the homologous helix sequences, B) the residues in spatial proximity to the homologous helices (neighbouring residues) and C) the homologous chain sequences.

<p>Data from the redundant membrane protein set is shown so that the full range of sequence identity can be seen.</p

The number of aligned helix pairs in each class, and occurrence of proline within that class.

<p>The number of aligned helix pairs in each class, and occurrence of proline within that class.</p

GPCR kink angle variation shown on the PDB structure 1F88, chain A.

<p>Colouring is by a) mean and c) standard deviation of angles at each site in the GPCR family, on a spectrum from the lowest values in blue to the highest in red. Grey residues have no angles measured as they are loop regions or within 6 residues of the end of the consensus helix (the minimum for a cylinder fit). b) is coloured by rainbow from N-terminus (blue) to C-terminus (red). The conserved kink angles in TMH 6 and TMH 7 and the correlated kink angles at sites in TMH 3 and TMH 5 are labelled.</p

Flowchart showing the classification of homologous helix pairs and families.

<p>Pair classification uses the angles of the two helices at the most disrupted site (<i>θ</i>_max, <i>θ</i>_min), angle difference (Δ<i>θ</i>) and the error on each angle (<i>ϵ</i>). Family classification uses analogous statistics to obtain the same classes: the median angle (<i>θ</i>_median), standard deviation (<i>σ</i>_<i>θ</i>), and mean error (<i>μ</i>_<i>ϵ</i>) of the angles in the family.</p

The number of helix families greater than or equal to each group size for the soluble and membrane protein sets.

<p>The number of helix families greater than or equal to each group size for the soluble and membrane protein sets.</p

Two examples of helix pairs, which are (A) not significantly different and (B) significantly different.

<p>PDB code, chain identifier and residue numbers are given for each helix. The black residues are at the most disrupted site (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157553#sec002" target="_blank">Methods</a>) in each helix pair. <i>r</i>_<i>n</i> and <i>r</i>_<i>c</i> give the quality of the cylinder fit (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157553#pone.0157553.e001" target="_blank">Eq (1)</a>) to the backbone atoms on the N- (red) and C- (blue) terminal sides of the kink site. <i>θ</i> is the angle measured between the two cylinders. <i>ε</i> is the estimated error of the angle measurement, calculated from <i>r</i>_<i>n</i> + <i>r</i>_<i>c</i> using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157553#pone.0157553.e006" target="_blank">Eq (4)</a>. If <i>θ</i>_max − <i>θ</i>_min > <i>ε</i>₁ + <i>ε</i>₂, the confidence intervals do not overlap therefore we consider the angles to be significantly different.</p

Distributions of angles measured at each site of the seven transmembrane helices in the GPCR family, after smoothing.

<p>The label at each site shown on the <i>x</i>-axis is the Class A numbering used in the GPCRDB [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157553#pone.0157553.ref024" target="_blank">24</a>]. The broken grey line at 20° is the threshold for the definition of a kink. The most disrupted site in each helix (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157553#sec002" target="_blank">Methods</a>) is shown in grey. In the top left of each graph, the classification, <i>σ</i>_<i>θ</i> (standard deviation of angles), and <i>μ</i>_<i>ε</i> (mean error) of the most disrupted site is given.</p

data_sheet_1_Structurally Mapping Antibody Repertoires.PDF

<p>Every human possesses millions of distinct antibodies. It is now possible to analyze this diversity via next-generation sequencing of immunoglobulin genes (Ig-seq). This technique produces large volume sequence snapshots of B-cell receptors that are indicative of the antibody repertoire. In this paper, we enrich these large-scale sequence datasets with structural information. Enriching a sequence with its structural data allows better approximation of many vital features, such as its binding site and specificity. Here, we describe the structural annotation of antibodies pipeline that maps the outputs of large Ig-seq experiments to known antibody structures. We demonstrate the viability of our protocol on five separate Ig-seq datasets covering ca. 35 m unique amino acid sequences from ca. 600 individuals. Despite the great theoretical diversity of antibodies, we find that the majority of sequences coming from such studies can be reliably mapped to an existing structure.</p

Insight into small molecule binding to the neonatal Fc receptor by X-ray crystallography and 100 kHz magic-angle-spinning NMR.

Aiming at the design of an allosteric modulator of the neonatal Fc receptor (FcRn)-Immunoglobulin G (IgG) interaction, we developed a new methodology including NMR fragment screening, X-ray crystallography, and magic-angle-spinning (MAS) NMR at 100 kHz after sedimentation, exploiting very fast spinning of the nondeuterated soluble 42 kDa receptor construct to obtain resolved proton-detected 2D and 3D NMR spectra. FcRn plays a crucial role in regulation of IgG and serum albumin catabolism. It is a clinically validated drug target for the treatment of autoimmune diseases caused by pathogenic antibodies via the inhibition of its interaction with IgG. We herein present the discovery of a small molecule that binds into a conserved cavity of the heterodimeric, extracellular domain composed of an α-chain and β2-microglobulin (β2m) (FcRnECD, 373 residues). X-ray crystallography was used alongside NMR at 100 kHz MAS with sedimented soluble protein to explore possibilities for refining the compound as an allosteric modulator. Proton-detected MAS NMR experiments on fully protonated [13C,15N]-labeled FcRnECD yielded ligand-induced chemical-shift perturbations (CSPs) for residues in the binding pocket and allosteric changes close to the interface of the two receptor heterodimers present in the asymmetric unit as well as potentially in the albumin interaction site. X-ray structures with and without ligand suggest the need for an optimized ligand to displace the α-chain with respect to β2m, both of which participate in the FcRnECD-IgG interaction site. Our investigation establishes a method to characterize structurally small molecule binding to nondeuterated large proteins by NMR, even in their glycosylated form, which may prove highly valuable for structure-based drug discovery campaigns

Crystal structure of the compound UCB-FcRn-303 (R enantiomer) bound to FcRn_ECD.

<p><b>(A)</b> The protein crystallized as a dimer composed of two β2m (dark grey and green) and two α-chain (light grey and blue) molecules. <b>(B)</b> At the interface of β2m and the α-chain, UCB-FcRn-303 (grey) occupies a binding pocket with Glycine, Cysteine, hydrophobic (Leucine), charged (Histidine, Aspartate), and polar uncharged (Serine, Glutamine) residues. β2m, β2-microglobulin; FcRn, neonatal Fc receptor; FcRn_ECD, extracellular domain of the neonatal Fc receptor.</p