Structure prediction and automated modeling of HL4E10.

<p>Cartoon representation of the superimposition of the experimentally determined HL4E10 heavy and light chain variable domain structures and the three top scoring heavy (1W72, 2G75, 1ADQ) and light chain (1A7P, 1GIG, and 1DL7) computational models. Heavy and light chains are shown in dark and light gray, respectively. The CDR loops of HL4E10 are color coded as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019828#pone-0019828-g001" target="_blank">Fig. 1</a>&<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019828#pone-0019828-g002" target="_blank">2</a>: CDR L1 yellow, CDR L2 cyan, CDR L3 orange, CDR H1 blue, CDR H2 pink, CDR H3 green. The Cα atoms of the experimentally determined HL4E10 structure and the computational models superimpose well, with an average r.m.s.d. of 0.68 Å for the heavy chains and 0.77 Å for the light chains, respectively. The largest deviations are observed, as expected, in the CDR loops, namely L1, L3 and H3.</p

Hierarchical clustering of the HL4E10 protein sequence with known immunoglobulins.

<p>Distances were calculated between protein sequences of (<b>A</b>) heavy chains and (<b>B</b>) light chains as the number of amino acid substitutions per site. Scale bar indicates distances. GenBank accession numbers of sequences included in the analysis are indicated within branch labels. Hierarchical clustering was performed on distance matrices generated from protein sequences with removed signal peptides.</p

Amino-acid sequence alignment of the HL4E10 hamster IgG light chain with those of other hamster antibodies.

<p>The HL4E10 lambda light chain is aligned with H28.710 (Genbank accession no. U17165 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019828#pone.0019828-Whitters1" target="_blank">[32]</a>), 145.2c11 (U17870 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019828#pone.0019828-Alegre1" target="_blank">[31]</a>), and 1F4 (S80615 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019828#pone.0019828-Mallender1" target="_blank">[30]</a>). Identical residues are in red and homologous exchanges are in green. Signal peptides are underlined, CDR loops are shaded: CDR L1 is in yellow, CDR L2 is in cyan, CDR L3 is in orange-red, and residues which are rarely observed in antibodies at particular locations are shaded gray. Kabat numbering is used throughout, as well as the definition of CDRs.</p

Amino-acid sequence alignment of the HL4E10 hamster IgG heavy chain with those of other hamster antibodies.

<p>The HL4E10 heavy chain is aligned with H28.710 (U17166 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019828#pone.0019828-Collins1" target="_blank">[33]</a>), 145.2c11 (U17871 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019828#pone.0019828-Alegre1" target="_blank">[31]</a>), and 3A5-1 (S80616 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019828#pone.0019828-Mallender1" target="_blank">[30]</a>). Color coding and shading is used as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019828#pone-0019828-g001" target="_blank">Fig. 1</a>, CDR H1 is in blue, CDR H2 in pink, CDR H3 in green, and the glycine-, proline-, cysteine-rich hinge region between V_HC_H1 and C_H2C_H3 is shaded gray.</p

Crystal structure of the HL4E10 Fab.

<p>(<b>A</b>) Cartoon representation of the superimposition of the two HL4E10 Fab structures in the asymmetric unit. The two HL4E10 Fabs (LH and AB) are shown in dark and light gray, respectively. The CDR loops are color coded as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019828#pone-0019828-g001" target="_blank">Fig. 1</a>&<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019828#pone-0019828-g002" target="_blank">2</a>: CDR L1 yellow, CDR L2 cyan, CDR L3 orange, CDR H1 blue, CDR H2 pink, CDR H3 green. The Cα atoms of Fab LH and Fab AB superimpose with an r.m.s.d. of 0.63 Å. (<b>B</b>) Superimposition of the combining sites of HL4E10 Fab LH (CDR loops colored) and Fab AB (CDR loops gray) (in a similar orientation to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019828#pone-0019828-g004" target="_blank">Fig. 4C</a>) reveals a rigid assembly without significant conformational differences. (<b>C</b>) Wall-eyed stereo representation of the molecular interactions which rigidify the HL4E10 CDR loops and lock the side chains in conformations predefined for high affinity ligand binding. For example, hydrogen bonds (black), CH-π interactions (grey), and hydrophobic stacking interactions occur at the interface of CDR L3 with CDRs H3, H2, and H1.</p