HLA-F and MHC-I Open Conformers Bind Natural Killer Cell Ig-Like Receptor KIR3DS1 - Fig 3

<p>Surface staining with KIR3DS1-His and HLA-F (A) PBMC from three individuals were stained with NK markers (CD3-, CD56+) and with mAb Z27 and DX9 to detect KIR3DL1 (DX9+, Z27+) and KIR3DS1 (DX9-, Z27+), and stained with HLA-F tetramer as indicated. Three individuals were chosen based on their KIR haplotypes, containing homozygous KIR3DL1, heterozygous KIR3DL1, KIR3DS1, and homozygous KIR3DS1 as indicated. (B) Cells described in A were activated and subjected to the same staining protocols 11 days post activation. (C) Isolated T cells from two individuals with the indicated KIR3DS1L1 genotypes were stained with mAb 3D11 and with KIR3DS1-His as marked beneath each pair of before (<i>left</i>) and after (<i>right</i>) activation profiles.</p

Functional measurement of HLA-F and KIR3DS1 interaction.

<p>PBMC from three donors were activated and stimulated with refolded HLA-F, HLA-control tetramers, or no protein as described in <i>Materials and Methods</i>. (<b>A)</b> A representative FACS staining for the analysis of CD107a expression on cell populations gated in the top panel indicated as KIR3DS1+ and KIR3DS1-. Expression of CD107a was detected with percentages of the total cell population analyzed in bold in the lower right quadrant of each profile. Experiments were performed in the presence of HLA-F and D^b-TRP bound to streptavidin beads and no-protein as indicated above the panels. The percentages CD107a positive cells within the KIR3DS1+ populations (<b>B</b>) and in the KIR3DS1- population (<b>C</b>) for each donor are graphed for comparison. <b>(B and C)</b> Five experiments were performed for each of four KIR3DS1+ donors (Donors CW and EB: KIR3DL1/KIR3DS1, Donors TG and DG KIR3DS1/KIR3DS1). The graphs show the percentage of total cells using the formula showing the mean of 5 replicates for each individual, the error bars represent the standard error of the mean (SEM) of the replicates, and the p-values calculated by performing a paired t-test comparing all values F and D^b across replicates and individuals.</p

Western analysis using the indicated mAbs for detection of gel fractionated protein after pull-down with KIR3DS1-D0-D2stem-His (<i>above</i>).

<p>Four cell lines were incubated with KIR3DS1-His and pull-downs performed followed by Western blot analysis with HCA2 and 3D11. Heterodimerization of KIR-His and HLA-F without tag followed by Ni column purification (<i>below</i>). After gel fractionation, gels were stained with Coomassie blue and proteins visualized. Heterodimerization assays were carried out with KIR alone or with KIR + HLA-F as indicated above each lane. Recombinant HLA-F is included alone for comparison. MW markers are indicated for both gels.</p

Steric clashes with presented peptides introduced by KIR substitutions.

<p>(<i>Left</i>) View of the KIR3DL1/HLA-B*5701 interface (PDB accession code 3VH8; <a href="http://www.rcsb.org/" target="_blank">www.rcsb.org</a>). (<i>Right</i>) Model of KIR3DS1 in complex with HLA-B*5701, shown as at left. The steric clash between Arg166 with p8 is highlighted in red. One rotomer of Arg166 is depicted; however, all accessible Arg166 rotomers sterically clash with residue p8 of the bound peptide. HLA-B*5701 is shown in cartoon representation, colored teal; the bound peptide (LSSPVTKSF) is shown as a ribbon with side-chains in licorice stick representation, colored yellow. KIR3D molecules are shown in semi-transparent surface representation, colored grey. Residue 166 from the KIR3D molecules is shown in licorice stick representation, colored grey (KIR3DL1) or grey and red (KIR3DS1).</p

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

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

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

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

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

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