PhIP-Seq results for 1C6 Fv.

<p>PhIP-Seq results for 1C6 Fv.</p

SPR sensorgrams of the interactions between 4E10 and the indicated ESs are shown.

<p>Time (in seconds) is plotted on the x-axis and SPR response (in RUs) is plotted on the y-axis. Double-referenced binding data are shown in black with corresponding kinetics fits to the data shown in red. Details of the experiments are given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004403#ppat-1004403-t001" target="_blank">Table 1</a>.</p

Epitope contacts for 4E10 and GEPs determined from complex crystal structures; electrostatic contacts are <b>bolded</b>; otherwise, the contacts are mediated by van der Waals interactions.

<p>Asterisks indicate residues that differ between GEPs and 4E10.</p><p>Epitope contacts for 4E10 and GEPs determined from complex crystal structures; electrostatic contacts are <b>bolded</b>; otherwise, the contacts are mediated by van der Waals interactions.</p

Effects of 4E10/gp140₃ pre-binding on 447-52D and b12 binding.

<p>Double-corrected sensorgrams of the SPR response of 10 nM analytes of Fab b12 (red and green traces) or Fab 447-52D (blue and black traces) to chip-coupled SF162 gp140₃ in the absence (red and black traces) or presence (green and blue traces) of pre-bound Fv 4E10 are shown. Fv 4E10, at a concentration of 3 µM, was flowed over the chip starting 5 minutes before the 447 or b12 injections and continued throughout the experiment. The black and blue 447-52D traces were completely superimposed, showing no effect of 4E10 pre-binding on 447-52D binding. While the dissociation phases of the b12 traces are nearly parallel, showing no minimal effect of 4E10 pre-binding on the dissociation of b12 from the bound state, the association phases are clearly not parallel, resulting in a lower peak response in the presence of 4E10 pre-binding, showing a modest, but important, reduction in the association rate. This result suggests that the CD4 binding site adopted a distinct conformational state prior to b12 binding as a consequence of 4E10 binding at the MPER.</p

PhIP-Seq results for GEP 4 Fv.

<p>PhIP-Seq results for GEP 4 Fv.</p

Interdomain movements within Fv cassettes are limited.

<p>(<b>A</b>) Superpositions of the V_H domains from two 4E10 ligand-bound structures (2FX7.pdb, 3LH2.pdb), unbound 4E10 (4LLV.pdb), ligand-bound GEP 1 (4M8Q.pdb), unbound GEP 1 (4LRN.pdb), ligand-bound GEP 2 (4M62.pdb), ligand-bound GEP 7 (4ODX.pdb), and unbound GEP 7 (4OB5.pdb) are shown in Cα backbone representations, colored as indicated. Residue P14H in each Fv, chosen as a reference point to illustrate interdomain movement upon binding between 4E10 and GEPs, is shown as a sphere and colored to match the corresponding backbone. When isolated V_H domains are superimposed, the P14H spheres are nearly coincident, indicating that the V_H domain structure is highly conserved among these Abs. (<b>B</b>) Fv cassettes of 4E10 and GEPs, colored as in (<b>A</b>), superimposed only on V_L domains (oriented on the right side of the panel), are shown as represented in (<b>A</b>). In this view, interdomain movements can be visualized as the relative movement of V_H domains, particularly at the P14H reference point. (<b>C</b>) Orthogonal view of the P14H spheres excerpted from (<b>B</b>) illustrating the pattern and degree of interdomain movements.</p

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Peptidome binding results.

<p>(<b>A</b>) PhIP-Seq results are plotted as −Log₁₀<i>P</i>-values, one replicate on the abscissa, the other on the ordinate, colored by Ab as indicated; note the discontinuity in axis scales. The top scoring 4E10 peptide derived from IP₃R is highlighted with a red arrow; one peptide, derived from the zinc finger Ran-binding domain-containing protein 3, which bound to both GEP 2 and GEP 4, is highlighted with purple arrows. Proximity to the diagonal indicates good replicate concordance; peptides with highly discordant replicate values, falling along the axes, were discarded from the analysis. Overall library scoring statistics are: 4E10, average = 0.32, σ = 0.35; GEP 2, average = 0.22, σ = 0.44; GEP 4, average = 0.25, σ = 0.52; 1C6, average = 0.27, σ = 0.50. (<b>B</b>) The molecular surfaces of the Fv domains of 1C6 (4LCI.pdb), unbound 4E10 (4LLV.pdb) and unbound GEP 7 (4OB5.pdb) are shown, oriented with V_H domains at left and the V_L domains at right. The surface is colored to show hydrophobic patches, defined by the program HotPatch <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004403#ppat.1004403-Pettit1" target="_blank">[80]</a>; patches are colored in descending order of total area (red, orange, yellow, …). The total surface area for red and orange hydrophobic patches is shown. The crystal structure of GEP 7 is partially disordered in HCDR1 and 3 and so patch area is underrepresented in the calculation.</p

SPR methods and results.

<p>SPR methods and results.</p

CDR restructuring.

<p>Views from the side (A) and below (B) are shown of superpositions of isolated V_H domains, in cartoon representations, with FRW regions colored grey and β-strands indicated as arrows. HCDRs are shown in a B-factor putty representation (tube width correlates with crystallographic Debye-Waller, or B, factor), colored by Ab. On the left in both frames is shown the superposition of V_H domains from ligand-bound 4E10 (3LH2.pdb, with green HCDRs) vs. unbound 4E10 (4LLV.pdb, with blue HCDRs), and on the right in both frames is shown the superposition of ligand-bound structures of GEP 1 (4M8Q.pdb), GEP 2 (4M62.pdb), and GEP 7 (4ODX.pdb), all with pink HCDRs, vs. unbound GEP 1 (4LRN.pdb) and GEP 7 (4OB5.pdb), both with orange HCDRs. (<b>C</b>) RMSD values for V_H, V_L, or Fv superpositions, calculated with or without CDR residues, are plotted for Cα atoms only (<i>left</i>) or all atoms (<i>right</i>).</p