p.Arg127Gln allosterically enhances the conformational dynamics of the C-terminal disulfide loop of GPIbα. (A) Equilibrium binding of VWF A1 to WT and p.Arg127Gln GPIbα obtained from the maximum SPR response as a function of A1 concentration. Data for p.Trp246Leu, p.Gly249Val and p.Met255Val were taken from Tischer et al 2019.¹⁶  Maximum SPR response (R_max) is 639 ± 4 response units. Panels on the right show representative SPR sensorgrams as a function of A1 concentration. The SPR data indicate that p.Arg127Gln is a mild GOF mutation of GPIbα, as its affinity is slightly enhanced relative to WT. (B) Excess heat capacity curves for thermal unfolding of WT and p.Arg127Gln GPIbα obtained by DSC at a scan rate of 2°C/min. Thermal unfolding is irreversible and dependent on the thermal scan rate. Slower thermal scan rates are shown in the supporting information (supplemental Figure 3). Fitting parameters are reported in supplemental Table 3 . Panels on the right show the scan rate independent thermal transition temperatures (T* in °C) and enthalpies (ΔH* in kJ/mol) where the rate of unfolding, k_unf = 1. Asterisks (*) on the p.Arg127Gln bars indicate a P < 0.05 relative to WT. DSC demonstrates that p.Arg127Gln destabilizes GPIbα, as evident by a diminished T* and ΔH* relative to WT GPIbα. (C) Hydrogen-deuterium Exchange fraction of WT (blue) and p.Arg127Gln GPIbα (gray) as a function of residue number after 1 hour of incubation at 25°C in 80% (vol/vol) deuterium oxide. Additional timepoints ranging 1 minute to 18 hours overnight incubation are included in supplemental Figure 4. Parameters for the exchange experiments are summarized in supplemental Table 8. The opposable thumb region of GPIbα is highlighted in orange. The position of the p.Arg127Gln mutation is indicated by a black vertical line. (D) Exchange fraction mapped onto the crystal structure of GPIbα (pdb ID = 1GWB).⁴⁸  Colors are as follows: black = not resolved, blue = 0, white = 0.25, and red ≥0.5. Structures were rendered using UCSF chimera.⁴⁹  The arrow in p.Arg127Gln indicates the position of the mutation. (E) Peptide envelopes (normalized intensity as a function of the deuterium-induced mass shift relative to all H peaks in absence of deuterium) of 8 peptides spanning the protein after 1 hour of exchange indicate an increased deuterium uptake for p.Arg127Gln relative to WT. Envelopes represent HXMS raw data and were analyzed with EXMS2³⁷  prior to deconvolution of the HX fraction with HDsite. The peptides shown in Figure 3E represent a small portion of the HX raw data that were extracted from the MS data using EXMS2.³⁷  A multitude of such peptide envelopes was used for the deconvoluted exchange fraction (Figure 3C,D). The HXMS data shown in panels (C)-(E) agree with the stability data shown in (B) as p.Arg127Gln destabilizes GPIbα. The primary impact of the mutation, however, can be observed in the opposable thumb region where the exchange is significantly enhanced. This indicates that p.Arg127Gln increases the conformational dynamics of this region. The difference between p.Arg127Gln and other platelet-type VWD mutations occurring within the opposable thumb sequence¹⁶  is that p.Arg127Gln acts allosterically through the LRR of GPIbα.