Compressed sensing (CS), mutual information (MI), and ensemble classifier predictions of HIV-1 Env positions constituting bnMAb epitopes for PGT 123, 123, 125, and 126.

<p>The experimentally identified positions are defined as those at which alanine point mutations were observed to increase the measured IC₅₀ of the mutant by more than 30-fold relative to that of the wild type JR-CSF. Alanine scans were performed as part of the present work for PGT 143 and 145; data for PGT 121–135 were taken from Ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080562#pone.0080562-Walker2" target="_blank">[51]</a>.</p><p><i>Footnote</i>: For each of the ten HIV-1 broadly neutralizing monoclonal antibodies (bnMAb) considered in this study, we report the residues identified by the compressed sensing (CS) classifier, positions identified by the mutual information (MI) classifier, and positions identified by the ensemble classifier (formed by combining the CS and MI predictions) predicted to lie within the bnMAb epitope. The number of residues identified by the CS classifier, <i>n_CS</i>, number of positions identified by the MI classifier, <i>n_MI</i>, number of positions predicted by the ensemble classifier, <i>n_ENS</i>, and number of positions identified by alanine scans, <i>n_EXPT</i>, may differ between bnMAbs.</p

Mutual information (MI) selection of PGT-123 epitope positions.

<p>The redundancy spectrum produced by application of the mutual information classification algorithm to the neutralization activity of bnMAb PGT-123 against a panel of 141 HIV-1 pseudoviruses (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080562#pone.0080562.s001" target="_blank">Table S1</a>) using an IC₅₀ cutoff of 10 µg/ml. The ordinate records the computed redundancy of the residue identity in each position with the observed neutralization activity. The abscissa lists the positions of the protein in decreasing order of redundancy. The dashed line indicates the cutoff computed by the shuffling procedure described in Materials and Methods, <i>R_cutoff</i> = 0.15, above which redundancy values should be considered statistically significant. These results suggest that the three top ranked positions – respectively, 332, 334 and 330– be retained in the model (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080562#pone-0080562-t001" target="_blank">Table 1</a>). For clarity of viewing, plots are terminated at the 100-component model.</p

Supplemental Material for Walker et al., 2023

Supplemental Materials for the manuscript,   Parallel evolution of the G protein-coupled receptor GrlG and the loss of fruiting body formation in the social amoeba Dictyostelium discoideum evolved under low relatedness.   Abstract:  Aggregative multicellularity relies on cooperation among formerly independent cells to form a multicellular body. Previous work with Dictyostelium discoideum showed that experimental evolution under low relatedness profoundly decreased cooperation, as indicated by the loss of fruiting body formation in many clones and an increase of cheaters that contribute proportionally more to spores than to the dead stalk. Using whole-genome sequencing and variant analysis of these lines we identified 38 single nucleotide polymorphisms (SNPs) in 29 genes. Each gene had one variant except for grlG (encoding a G protein-coupled receptor), which had ten unique SNPs and five structural variants. Variants in the 5' half of grlG — the region encoding the signal peptide and the extracellular binding domain — were significantly associated with the loss of fruiting body formation; the association was not significant in the 3' half of the gene. These results suggest that the loss of grlG was adaptive under low relatedness and that at least the 5' half of the gene is important for cooperation and multicellular development. This is surprising given some previous evidence that grlG encodes a folate receptor involved in predation, which occurs only during the single-celled stage. However, non-fruiting mutants showed little increase in a parallel evolution experiment where the multicellular stage was prevented from happening. This shows that non-fruiting mutants are not generally selected by any predation advantage, but rather by something – likely cheating – during the multicellular stage. Contents: FileS1 contains all supporting information and supplemental figures S1 - S3.  FileS2 contains supplemental tables S1 - S7. FileS3 is a compressed folder containing three VCF files, each containing all variant calls that survived hard filtration (before manual review) from each of the three variant callers (GATK, Freebayes and Delly). FileS4 is a .txt file that contains the Dictyostelium discoideum gene annotations downloaded from NCBI on 10-25-19. </p

Compressed sensing (CS), mutual information (MI), and ensemble classifier predictions of HIV-1 Env positions constituting bnMAb epitopes for PGT 135, 143, and 145.

<p>The experimentally identified positions are defined as those at which alanine point mutations were observed to increase the measured IC₅₀ of the mutant by more than 30-fold relative to that of the wild type JR-CSF. Alanine scans were performed as part of the present work for PGT 143 and 145; data for PGT 121–135 were taken from Ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080562#pone.0080562-Walker2" target="_blank">[51]</a>.</p><p><i>Footnote</i>: See footnote to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080562#pone-0080562-t001" target="_blank">Table 1</a>.</p

Compressed sensing (CS), mutual information (MI), and ensemble classifier predictions of HIV-1 Env positions constituting bnMAb epitopes for PGT 127, 128, and 130.

<p>The experimentally identified positions are defined as those at which alanine point mutations were observed to increase the measured IC₅₀ of the mutant by more than 30-fold relative to that of the wild type JR-CSF. Alanine scans were performed as part of the present work for PGT 143 and 145; data for PGT 121–135 were taken from Ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080562#pone.0080562-Walker2" target="_blank">[51]</a>.</p><p><i>Footnote</i>: See footnote to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080562#pone-0080562-t001" target="_blank">Table 1</a>.</p

Compressed sensing (CS) selection of PGT-123 epitope residues.

<p>Results of the application of the compressed sensing classification algorithm to the neutralization activity of bnMAb PGT-123 against a panel of 141 HIV-1 pseudoviruses (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080562#pone.0080562.s001" target="_blank">Table S1</a>). In each panel, the abscissa indicates the number of non-zero elements in the signal vector computed by the LASSO algorithm, and therefore the number of residues incorporated into the regularized least squares fit of the neutralization data (Eqn. 3). For clarity of viewing, plots are terminated at the 100-component model. As indicated by the arrows, knees in the (a) mean squared error (MSE) over the complete data set and (b) leave-one-out cross-validation mean squared error (LOOCV-MSE) curves were identified using the L method at 11 and 9 residues, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080562#pone.0080562-Salvador1" target="_blank">[77]</a>. The mean of these values motivated the selection of the ten residues constituting this model: I323, H330, N332, N334, S334, S612, N671, Q740, V815, and V843 (c.f. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080562#pone-0080562-t001" target="_blank">Table 1</a>).</p

Logo plot of the variability of selected positions in HIV-1 Env within the 141-strain pseudovirus panel.

<p>We present data for all positions identified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080562#pone-0080562-t001" target="_blank">Tables 1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080562#pone-0080562-t003" target="_blank">3</a> as significant determinants of bnMAb neutralization activity by either the ensemble classifier or experimental alanine scan data.</p

Broadly Neutralizing Antibody PGT121 Allosterically Modulates CD4 Binding via Recognition of the HIV-1 gp120 V3 Base and Multiple Surrounding Glycans

<div><p>New broad and potent neutralizing HIV-1 antibodies have recently been described that are largely dependent on the gp120 N332 glycan for Env recognition. Members of the PGT121 family of antibodies, isolated from an African donor, neutralize ∼70% of circulating isolates with a median IC₅₀ less than 0.05 µg ml⁻¹. Here, we show that three family members, PGT121, PGT122 and PGT123, have very similar crystal structures. A long 24-residue HCDR3 divides the antibody binding site into two functional surfaces, consisting of an open face, formed by the heavy chain CDRs, and an elongated face, formed by LCDR1, LCDR3 and the tip of the HCDR3. Alanine scanning mutagenesis of the antibody paratope reveals a crucial role in neutralization for residues on the elongated face, whereas the open face, which accommodates a complex biantennary glycan in the PGT121 structure, appears to play a more secondary role. Negative-stain EM reconstructions of an engineered recombinant Env gp140 trimer (SOSIP.664) reveal that PGT122 interacts with the gp120 outer domain at a more vertical angle with respect to the top surface of the spike than the previously characterized antibody PGT128, which is also dependent on the N332 glycan. We then used ITC and FACS to demonstrate that the PGT121 antibodies inhibit CD4 binding to gp120 despite the epitope being distal from the CD4 binding site. Together, these structural, functional and biophysical results suggest that the PGT121 antibodies may interfere with Env receptor engagement by an allosteric mechanism in which key structural elements, such as the V3 base, the N332 oligomannose glycan and surrounding glycans, including a putative V1/V2 complex biantennary glycan, are conformationally constrained.</p> </div

Thermodynamic parameters of PGT 121 antibodies and CD4 binding to gp120 and SOSIP.664 trimers measured by isothermal titration calorimetry.

%<p>Reported values are averages from at least two independent measurements and associated errors are approximately 10% of the average. Representative isotherms can be found in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003342#ppat.1003342.s006" target="_blank">Figs. S6</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003342#ppat.1003342.s007" target="_blank">S7</a>.</p>*<p>The binding isotherms do not allow to accurately determining these binding parameters.</p>#<p>The change in Gibbs free energy (ΔG) was determined using the relationship: ΔG_binding = RTlnK_d<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003342#ppat.1003342-deAzevedo1" target="_blank">[75]</a>.</p>&<p>The stoichiometry of binding (N) is directly affected by errors in protein concentration measurements, sample impurity and glycan heterogeneity on gp120.</p

X-ray crystallography statistics.

*<p>Values in parentheses are for the highest resolution shell.</p>†<p>R_sym = Σ|I-<i>|/Σ<i>, where I is the observed intensity, and <i> is the average intensity of multiple observations of symmetry related reflections.</i></i></i></p><i><i><i>‡<p>R = Σhkl∥Fobs|−|Fcalc∥/Σhkl|Fobs|.</p>§<p>R_free calculated from 5% of the reflections excluded from refinement.</p></i></i></i