Analysis of the effects of salt concentration, mutation and overall cassette charge on 4E10/liposome interactions.

<p>Corrected SPR responses are shown for Annexin V or 4E10 IgG analytes (300 nM; duplicate runs) to liposomes incorporating biotinylated lipids captured on streptavidin-coated biosensor chips; liposome compositions are indicated above each frame. (<b>A</b>) SPR responses of wild-type 4E10 IgG analytes at different salt concentrations are plotted. (<b>B</b>) SPR responses of Annexin V (300 nM), wild-type 4E10 IgG (300 nM) or the 4E10 [G(L50)E] mutant IgG (400 nM) are plotted. A higher concentration of 4E10 mutant IgG was used with the expectation that binding might be significantly reduced. Since this did not occur, mutant IgG responses appear elevated due to the concentration differential. (<b>C</b>) The net charge at neutral pH of the Fv cassettes of the anti-HIV Abs with structures currently available through the PDB <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003639#ppat.1003639-Berman1" target="_blank">[103]</a> was calculated with the structure-based algorithm PDB2PQ <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003639#ppat.1003639-Dolinsky1" target="_blank">[51]</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003639#ppat.1003639-Unni1" target="_blank">[54]</a>. Two Abs, PG16 and NIH45-46, were excluded because their structures included modified amino acids that could not be accommodated by PDB2PQ. Fvs are plotted with their names, with assigned PDB accession codes in parentheses. Ab labels are colored by the locale of their epitopes on Env, as indicated; 4E10 is also bolded.</p

Structure of 4E10 free of bound antigen.

<p>(<b>A</b>) Superposition of the three HCDRs from bound and unbound b12 (3RU8.pdb <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003639#ppat.1003639-Azoitei1" target="_blank">[104]</a>, 1HZH.pdb <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003639#ppat.1003639-Saphire1" target="_blank">[110]</a> and 4E10 (2FX7.pdb <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003639#ppat.1003639-Cardoso1" target="_blank">[9]</a>, 4LLV.pdb) are shown in a B-factor putty representation (b12, blue: bound; cyan: unbound. 4E10, pink: bound, yellow: unbound). (<b>B</b>) Superposition of residues from the 4E10 epitope binding site and HCDR1 and 3 from bound (semi-transparent molecular surface in pink; 2FX7.pdb <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003639#ppat.1003639-Cardoso1" target="_blank">[9]</a>) and unbound (mesh molecular surface in yellow; 4LLV.pdb) 4E10 Fv are shown; HCDR1 and 3 are shown in cartoon representations with side-chains of key residues shown in licorice-stick representations and labeled. The molecule is oriented so that the V_L domains are to the left and the V_H domains to the right. Molecular images were generated with MacPyMOL <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003639#ppat.1003639-DeLano1" target="_blank">[109]</a>.</p

Analysis of 4E10H knock-in mice.

<p>(<b>A</b>) The percentages of IgM⁺IgD⁻ pre-B and IgM⁺IgD⁺ immature B cells within the B220⁺ cell population in the bone marrow of B6 WT, B6-KL25H and B6-4E10H mice are shown. Marrow cells were isolated and stained for cell surface markers as described in the methods section. FACS plots were previously gated B220⁺ and on live cell size based on forward and side scatter. Numbers represent the percentages of B220⁺ bone marrow cells falling within each box gate. (<b>B</b>) The proliferation of B220⁺ splenocytes from B6 WT, B6-KL25H, and B6-4E10H mice in response to overnight culture in the presence of B cell stimuli are shown. Cells were loaded with cell proliferation dye before overnight incubation, and proliferation was assessed by FACS analysis after cell surface staining to identify B220⁺ B cells. Histograms were previously gated B220⁺ and on live cell size based on forward and side scatter. Numbers represent the percentage of B220⁺ cells falling within the bar gate on each plot, indicating dilution of the proliferation dye as a result of cell division.</p

Design and characterization of HCDR3-grafted Ubq fusion proteins.

<p>(<b>A</b>) A ribbon representation of Ubq (1UBQ.pdb <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003639#ppat.1003639-VijayKumar1" target="_blank">[106]</a>) is shown at bottom right, colored prismatically from N- (blue) to C-terminus (red); the targeted loop for HCDR3 engraftment is highlighted in gray with red bounding residues. The structures of the three targeted HCDR3 segments in context of their parent Abs are shown above in a licorice-stick representation, colored by atom type (oxygen: red; nitrogen: blue; carbons of buried residues: marine blue; and carbons of exposed residues: gray). Residue exposure was determined with the program GetArea <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003639#ppat.1003639-Fraczkiewicz1" target="_blank">[107]</a>, with greater than 30% solvent accessibility considered “exposed”, based on the structural context in the corresponding crystal structures (2F5: 3IDG.pdb <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003639#ppat.1003639-Bryson1" target="_blank">[108]</a>; b12: 3RU8.pdb <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003639#ppat.1003639-Azoitei1" target="_blank">[104]</a>; 4E10: 2FX7.pdb <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003639#ppat.1003639-Cardoso1" target="_blank">[9]</a>). The three transferred sequences (2F5: AHRRGP<b>T</b>T<b>LFGVPIARG</b>PVNAMDVW; b12: ARVG<b>PYSWDDSP</b>QYNYYMDVW; 4E10: AREGTT<b>GWGWLGKP</b>IGAFAHW; exposed residues <b>bolded</b>), were characterized as “hydrophobic” with the Sigma-Aldrich PEPscreen Library Design Tool (Φ_2F5 = 0.52; Φ_b12 = 0.51; Φ_4E10 = 0.56). Key residues and Cα-Cα distances (Å) are indicated. The inset shows a schematic representation of the design process. Molecular images were generated with MacPyMOL <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003639#ppat.1003639-DeLano1" target="_blank">[109]</a>. (<b>B</b>) SEC analyses of the solution properties of the purified recombinant HCDR3-grafted Ubq fusion proteins are shown, confirming monodispersivity. (<b>C</b>) Corrected SPR responses (duplicate runs) of HCDR3-grafted Ubq fusion proteins to HIV SF162 gp140 protein amine-coupled to biosensor chips; analyte concentrations are indicated. While detectable, these responses are consistent with <i>K</i>_D values >>10 µM and are thus too weak to quantify reliably. (<b>D</b>) SPR responses (duplicate runs) of Annexin V and HCDR3-grafted Ubq fusion protein analytes to liposomes incorporating biotinylated lipids captured on streptavidin-coated biosensor chips are shown, with liposome compositions indicated above each frame. HCDR3-grafted Ubq fusion protein responses are colored as indicated, but none showed detectable binding on any liposome composition.</p

PhIP-Seq analyses of 4E10 and b12.

<p>PhIP-Seq results are plotted as −Log₁₀<i>P</i> values, one replicate on the abscissa, the other on the ordinate; note the discontinuity in axis scales. The top five scoring 4E10 peptides are highlighted with arrows; the three peptides derived from IP₃R isoform sequences are highlighted with green arrows, with the consensus core sequence shown as a MEME logo <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003639#ppat.1003639-Bailey1" target="_blank">[72]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003639#ppat.1003639-Schneider1" target="_blank">[111]</a> in the inset. The orange arrow corresponds to a peptide derived from cytoplasmic dynein 1 and the blue arrow to a peptide derived from complement receptor type 1. The gray arc corresponds to the −Log₁₀<i>P</i> values observed for the highest-scoring b12 peptide, so defines a threshold for potential autoreactive binding events. Proximity to the diagonal indicates good replicate concordance; peptides with highly discordant replicate values, falling along the axes, outside the bounding dashed red lines were discarded from the analysis.</p

Structures of 4E10, b12, 2F5, the CL-binding yeast cytochrome bc1 complex and Annexin V.

<p>(<b>A</b>) The structure of the Fv domain of 4E10 (from 2FX7.pdb <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003639#ppat.1003639-Cardoso1" target="_blank">[9]</a>) is shown in a semi-transparent molecular surface representation colored by electrostatic potential (blue: positive; red: negative). Side-chains of key residues are shown in licorice-stick representations and labeled. In this orientation, the V_L domain is in the upper left and the V_H domain is in the lower right. The backbone of the epitope peptide co-crystallized in this structure is shown in a cartoon representation, with side-chains shown in licorice-stick representations colored by atom-type (carbon: gray; oxygen: red; nitrogen: blue). (<b>B</b>) The molecular surfaces of the Fv domains of 4E10 (top) and 2F5 (bottom) are shown, oriented with V_L domains at left and the V_H 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.1003639#ppat.1003639-Pettit1" target="_blank">[113]</a>; patches are colored in descending order of total area (red, orange, yellow …). Key residues and hydrophobic patch areas are indicated. (<b>C</b>) The structure of the Fv domain of b12 (from 3RU8.pdb <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003639#ppat.1003639-Azoitei1" target="_blank">[104]</a>) is shown as in (<b>A</b>). (<b>D</b>) Annexin V (1A8A.pdb <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003639#ppat.1003639-Swairjo1" target="_blank">[114]</a>) is shown as a semi-transparent molecular surface colored by underlying atom type, highlighting the exposed tryptophan side-chain (shown in licorice-stick representation). Calcium ions, shown as green spheres, coordinate the phosphate moiety from PS (shown in a licorice-stick representation, colored by atom-type as in (<b>A</b>) plus phosphorus in orange). (<b>E</b>) The structure of the unbound form of 4E10 (4LLV.pdb) is shown as a semi-transparent molecular surface representation colored as in (<b>A</b>). Side-chains of key residues are shown in licorice-stick representations and labeled. The view of the molecule has been rotated roughly 45° from the orientation of 4E10 shown in (<b>A</b>). The coordinated SO₄ ion is shown in a licorice-stick representation colored by atom type as in (<b>A</b>) plus sulfur in yellow. (<b>F</b>) CL is shown docked onto the unbound structure of 4E10 oriented and colored as in (<b>E</b>). Molecular images were generated with MacPyMOL <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003639#ppat.1003639-DeLano1" target="_blank">[109]</a>.</p

SPR analyses of 4E10/liposome interactions.

<p>Corrected SPR responses are shown (response units: RU; duplicate runs) of Annexin V (black), αCL polyclonal Ab (orange), 4E10 IgG (red), 2F5 IgG (blue), and b12 IgG (green) analytes binding to liposomes incorporating biotinylated lipids captured on streptavidin-coated biosensor chips. All analyte concentrations were 300 nM. The Annexin V analyte buffer included 2.5 mM CaCl₂. Liposome compositions are indicated above each frame.</p

Top scoring 4E10 and b12 PhIP-Seq 36-mer peptides.

<p>The top fifteen scoring 36-mer peptides from the b12 IgG PhIP-Seq analysis (above) and the seventeen top scoring 36-mer peptides from the 4E10 IgG analysis exceeding the threshold set by the highest-scoring b12 peptide (below) are shown, compiled by their relative rank order, replicate-averaged PhIP-Seq score, sequence, identity of the parent protein, hydrophobicity (Φ) and predicted charge at neutral pH. Hydrophobicities were determined with the Sigma-Aldrich PEPscreen Library Design Tool (green: hydrophilic; orange: moderately hydrophobic; red: hydrophobic; no peptides were predicted to be “very hydrophobic”); overall charge was determined with the Innovagen Peptide Property Calculator (red: negative; green: neutral; blue: positive). Segments of low sequence complexity (defined by the program SEG <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003639#ppat.1003639-Wootton1" target="_blank">[112]</a>) are highlighted in red; the core motif in the IP₃R isoform peptides (labeled in bold), identified by MEME <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003639#ppat.1003639-Bailey1" target="_blank">[72]</a>, is underlined. Asterisks indicate peptide sequences selected for expression as fusion constructs for subsequent validation.</p

IHC of serial mouse cerebellar sections.

<p>(<b>A</b>) Immunohistochemical staining with 4E10 IgG in mouse cerebellum sections reveals perinuclear, nodular immunoreactivity in Purkinje neurons; inset shows a magnified view. (<b>B</b>) Human IgG Isotype control staining of mouse cerebellum is shown. (<b>C</b>) Staining for IP₃R1 with an anti-IP₃R1 IgG shows a perinuclear, nodular and diffuse cytoplasmic staining pattern in Purkinje neurons, mirroring that seen in (<b>A</b>); inset shows a magnified view. (<b>D</b>) Rabbit IgG isotype control staining of mouse cerebellum is shown.</p

SPR analyses of Scn-peptide fusions.

<p>(<b>A</b>) The generalized siderocalin fusion construct used for PhIP-Seq peptide expression is shown. (<b>B</b>) Corrected and normalized SPR responses for 4E10 IgG binding to the top five peptides immobilized on a CM5 biosensor chip are plotted. (<b>C</b>) Corrected and normalized SPR responses of 4E10 IgG (300 nM; duplicate runs) binding to peptides (colored as in (<b>B</b>)) at 500 mM NaCl are plotted; inset shows magnified view. Normalized SPR responses are plotted in arbitrary units.</p