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High School Poster

HS Poster

Poster 151 - The role of vicinal disulfide bond in mechanical properties of the A2 domain and modulation in A1- GPIbα interaction

Saturday, October 26, 2024
10:00 AM - 12:00 PM EST
Location: Exhibit Hall E, F & G

Introduction: Von Willebrand factor (VWF) is a plasma protein that facilitates platelet clot formation by functioning as a crosslinker between platelets and the injured site of blood vessels. Specifically, the A1 domain of VWF contains the binding site where the protein binds to platelet glycoprotein Ib (GPIb), while the A2 domain is suggested to be involved in the autoinhibition of A1-GPIb interaction. Butera et al. found that a vicinal disulfide bond in the A2 domain modulates the interaction between the A1 domain and GPIb. Mutagenesis of the disulfide bond showed reduced platelet adhesion under fluid shear conditions. Additionally, the removal of this vicinal disulfide bond in the A2 domain and full-length VWF increased their susceptibility to ADMATS13 degradation. However, the exact role of the A2 domain in VWF autoinhibition is not completely understood. In this study, we evaluated the effect of the vicinal disulfide bond on the mechanical unfolding of the A2 domain and A1-A2 domain interaction using optical tweezers and microscale thermophoresis (MST).

Materials and

Methods: Optical tweezer experiments were performed to examine the unfolding behaviors of recombinant wildtype (WT) A2 domain and its mutant lacking the vicinal disulfide bond (CC-AA A2). Streptavidin (SA) modified polystyrene beads were incubated with either SpyCatcher-DNA handle or Spytag-A2 fragment at room temperature in Tris-buffered saline for 15 minutes. One SA bead was fixed by a micropipette, while the other was optically trapped. The beads were allowed to approach each other and to facilitate the formation of an isopeptide covalent bond between Spytag and SpyCatcher, enabling the pulling of A2 fragments at various pulling speeds. MST measurements were conducted to estimate the effect of the disulfide bond on the binding affinities between A1 and A2 fragments. The purified A1 domain was labeled with RED-NHS dye. A2 domain and CC-AA A2 were 2-fold diluted in a 15-step serial dilution in phosphate-buffered saline with 0.01 % Pluronic® F-127, mixed with labeled A1 domain with a final concentration of 40 nM, and loaded into 16 capillaries equally. At least four independent replicates were analyzed to estimate the binding affinity (Kd) using the MO Affinity Analysis software.

Results, Conclusions, and Discussions: For optical tweezer experiments, the unfolding of WT A2 and CC-AA A2 was defined by abrupt extension and force-dropping events (marked by arrows in Figure 1A). Compared to the representative force-extension trace of WT A2 (Figure 1A(a)), which showed a single unfolding with an extension of around 40 nm, CC-AA A2 displayed various unfolding events in each approach-retraction cycle: single unfolding event with long extension (Figure 1A(b)) or short extension (Figure 1A(c)), and multiple unfolding events with one long extension and one short extension (Figure 1A(d-e)). Via MST measurement, the binding affinity of CC-AA A2 to A1 domain was 10.0 ± 2.6 nM, implying that the interaction between CC-AA A2 and A1 has a five-fold higher affinity compared to the 62.8 ± 9.1 nM binding affinity of the interaction between WT A2 and A1.

Using optical tweezers, the unfolding behaviors of WT A2 and CC-AA A2 were evaluated. The removal of the vicinal disulfide bond results in the alteration of the A2 domain’s mechanical unfolding. The enhanced binding affinity of CC-AA A2 to A1 observed in MST measurements supports our hypothesis that the absence of the disulfide bond makes A2 easier to unmask A1, thereby allowing A1 to bind with platelets. Together, the study provides additional direct evidence of how the disulfide bond influences A1-GPIb interaction and suggests the vicinal disulfide bond in the A2 domain modulates A1-GPIb interaction through the alteration of A2’s mechanical unfolding and regulating the autoinhibition of VWF-platelet interaction. Additional research could be conducted using AlphaFold 3 to investigate further how the absence of the vicinal disulfide bond affects the A1-A2 interaction.

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