Mapping Paratope on Antithrombotic Antibody 6B4 to Epitope on Platelet Glycoprotein Ibalpha via Molecular Dynamic Simulations

<div><p>Binding of platelet receptor glycoprotein Ibα (GPIbα) to the A1 domain of von Willebrand factor (vWF) is a critical step in both physiologic hemostasis and pathologic thrombosis, for initiating platelet adhesion to subendothelium of blood vessels at sites of vascular injury. Gain-of-function mutations in GPIbα contribute to an abnormally high-affinity binding of platelets to vWF and can lead to thrombosis, an accurate complication causing heart attack and stroke. Of various antithrombotic monoclonal antibodies (mAbs) targeting human GPIbα, 6B4 is a potent one to inhibit the interaction between GPIbα and vWF-A1 under static and flow conditions. Mapping paratope to epitope with mutagenesis experiments, a traditional route in researches of these antithrombotic mAbs, is usually expensive and time-consuming. Here, we suggested a novel computational procedure, which combines with homology modeling, rigid body docking, free and steered molecular dynamics (MD) simulations, to identify key paratope residues on 6B4 and their partners on GPIbα, with hypothesis that the stable hydrogen bonds and salt bridges are the important linkers between paratope and epitope residues. Based on a best constructed model of 6B4 bound with GPIbα, the survival ratios and rupture times of all detected hydrogen bonds and salt bridges in binding site were examined via free and steered MD simulations and regarded as indices of thermal and mechanical stabilizations of the bonds, respectively. Five principal paratope residues with their partners were predicted with their high survival ratios and/or long rupture times of involved hydrogen bonds, or with their hydrogen bond stabilization indices ranked in top 5. Exciting, the present results were in good agreement with previous mutagenesis experiment data, meaning a wide application prospect of our novel computational procedure on researches of molecular of basis of ligand-receptor interactions, various antithrombotic mAbs and other antibodies as well as theoretically design of biomolecular drugs.</p> </div

Hydrogen bonds and salt bridges with higher stabilization in Top 8.

*<p>HBSI expresses the index of hydrogen bond stabilization (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042263#s2" target="_blank">Materials and Methods</a>).</p

Variation of interatomic distance versus steered simulation time.

<p>The interatomic distances of the six representative bonds under stretching were plotted against simulation time, where all descriptions for line types, bonds and their lengths are same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042263#pone-0042263-g004" target="_blank">Figure 4</a>. These time courses of interatomic distances showed that, the 5^th and 16^th bonds were very quickly ruptured (A and D), in comparison with others, in which the 9^th and 1^st bonds would maintain more long time (C and F) than 4^th and 10^th bonds (B and E).</p

Time courses of interatomic distances of six representative bonds in binding site of 6B4/GPIbα complex.

<p>The interatomic distances of six representative bonds were plotted against simulation time, where the interatomic distances were from the oxygen atoms of acidic residues and their respective partners, the nitrogen atoms of basic residues, for three salt bridges, 5^th (A), 4^th (B) and 9^th (C) bonds, or from doners to their respective acceptors for three hydrogen bonds, 16^th (D), 10^th (E) and 1^st (F) bonds. The salt bridges and hydrogen bonds were simulated with the initial conformation I (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042263#pone-0042263-g003" target="_blank">Fig. 3 A</a>) and II (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042263#pone-0042263-g003" target="_blank">Fig. 3 B</a>), respectively. The gray dashed line expresses the distance cut-off of 0.35 nm beyond which the bonds breaks, and the blue, green and red lines exhibit the variation of interatomic distances (nm) of a bond against simulation time (ns) for thrice-repeat independent free MD simulations, respectively. The thermal stabilizations of the 4^th and 10^th bonds (B and E) seemed to be higher than those of the 5^th and 16^th bonds but lower than those of the 9^th and 1^st bonds. Remarkable difference in the thrice-repeat independent simulations showed a random behavior of intermolecular interactions.</p

Hydrophobic and polar interactions from docking analysis.

<p>Hydrophobic and polar interactions from docking analysis.</p

Ensemble workflow of computational procedure.

<p>Ensemble workflow of computational procedure.</p

Models of free and bound 6B4.

<p>A, model of 6B4-ScFv via Homology modeling, where the heavy chain (iceblue), light chain (cyan) and (Gly₄Ser)₃ linker (yellow) were shown in transparent newcartoon representation, and the six complementarity determining regions (CDRs) (mauve), i.e. CDR H1, H2, H3, L1, L2 and L3, were marked; B, conformation of the 339^th complex of 6B4 bound to GPIbα subunit (orange); C, structural superposition of 6B4/GPIbα and A1-GPIbα complex (PDB code 1SQ0), where A1 is shown in transparent lime and 6B4 in prunosus; D, the back side view of C.</p

The false discovery rate, sensitivity and specificity of three different positive criterions.

<p>The false discovery rate was evaluated by Eq. 1, and Eq. 3 was used to estimated the sensitivity and specificity, with use of data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042263#pone.0042263.s006" target="_blank">Table S2</a> in Suppl. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042263#s2" target="_blank">Materials</a>.</p

The CDRs and identified paratope residues of 6B4 [16].

*<p>The positions of residues in 6B4-ScFv were expressed with serial numbering from N-terminal of heavy chain to C-terminal of light chain.</p>†<p>The serial numbers, 166, 167, 168, 233 and 106, are corresponding to those in Kabat numbering, such as 27D, 27E, 28, 93 and 100C, respectively. ^†The residue sequences, contributed to their respective CDRs (CDR H1, H2, H3, L1, L2 and L3), follow the serial numbering too.</p

Summary of survival ratios, rupture times and involved residues of hydrogen bonds and salt bridges obtained from free and steered simulations.

*<p>ECC is an abbreviation of equilibrated complex conformation. Letter H and S expresses the two types of bonds, such as hydrogen bond and salt bridge, respectively. The heading I and II denote two different ECCs of of 6B4 bound to GPIbα, the superscript numbers on residues (Column 5 and 3) designate the positions of their respective involved residues in sequences of 6B4 and GPIbα with serial numbering, and, the donor- and acceptor-atoms (Column 6) on paratope residues (Column 5) together with their respectively partners (Column 4) on epitope residues (column 3) contribute to bonds in binding site. All bonds, which were derived from static analyses and/or from thrice independent free and steered MD simulations with equilibrated conformation I and II, respectively, were designated by the symbol “+” and/or nonzero values (mean ± SD) of survival ratios and rupture times of bonds.</p