Force-Induced Cooperative Unfolding of Two Distinctive Domains in a Single Gpibalpha Molecule

GPIbα, a major member of the GPIb-IX-V complex, initiates a mechano-signaling pathway that leads to platelet intracellular calcium flux when binding to VWF at the A1 domain¹. Exactly how this signal is transduced across the membrane is unknown. A recent work identifying the unfolding of a jextamembrane mechanosensitive domain (MSD) (Fig.1 A,C) suggested that this unfolding might play a role in the signal transduction of GPIbα². Using molecular dynamics (MD) simulations to pull the GPIbα leucine-rich repeat domain (LRRD) from the VWF A1 domain, we observed unfolding of the LRRD (Fig.1 A) (manuscript under review).

       Here we used a biomembrane force probe (BFP) to study single GPIbα unfolding (Fig. 1E,F). Platelet GPIbα was pulled by A1 coated on the probe through a ramping phase (pink, Fig. 1G) to a clamping phase to wait for bond dissociation under a 25-pN force (red, Fig. 1G). Two unfolding signatures were identified: i) ramped unfolding, featured by a sudden force kink in the ramping phase (dashed circle, Fig. 1G); ii) clamped unfolding, featured by an abrupt force drop in the clamping phase (dashed ellipse, Fig. 1G). Unfolding lengths from both signatures were measured from the force vs. time data (Fig. 1G), based on which the unfolding events of MSD (7-25 nm), LRRD (25-56 nm) or both together (56-80 nm) were differentiated. Intriguingly, LRRD unfolding was only observed in the ramping phase, while MSD could unfold in both ramping and clamping phases.

The frequency of observing both LRRD and MSD unfolding in the same cycle was much higher than the product of the frequencies for LRRD and MSD to unfold (the joint probability for both to unfold if they were independent), suggesting that the two GPIbα domains unfolded cooperatively (Fig 2A). Chi-square tests also rejected the hypothesis that MSD and LRRD unfolded independently (p=5.24×10^-5). Separating the ramped and clamped unfolding of MSD and evaluating their respective cooperativity with LRRD unfolding demonstrated similar results (Fig 2A). Agreeing with these, both the ramped and clamped unfolding of MSD occurred much more frequently in the presence of LRRD unfolding (Fig 2B, left), and vice versa for the unfolding of LRRD (Fig 2B, right). Cooperative unfolding of LRRD and MSD may play a key role in transducing signals across platelet membrane.

 

References

1              Nesbitt, W. S. et al. The Journal of biological chemistry 277, 2965-2972, doi:10.1074/jbc.M110070200 (2002).

2              Zhang, W. et al. Blood 125, 562-569, doi:10.1182/blood-2014-07-589507 (2015).