Abstract
BACKGROUND: Mutations in the β-switch of GPIbα cause gain-of-function in the platelet-type von Willebrand disease. Structures of free and A1-bound GPIbα suggest that the β-switch undergoes a conformational change from a coil to a β-hairpin. OBJECTIVES: Platelet-type von Willebrand disease (VWD) mutations have been proposed to stabilize the β-switch by shifting the equilibrium in favor of the β-hairpin, a hypothesis predicated on the assumption that the complex crystal structure between A1 and GPIbα is the high-affinity state. METHODS: Hydrogen-deuterium exchange mass spectrometry is employed to test this hypothesis using G233V, M239V, G233V/M239V, W230L, and D235Y disease variants of GPIbα. If true, the expectation is a decrease in hydrogen-deuterium exchange within the β-switch as a result of newly formed hydrogen bonds between the β-strands of the β-hairpin. RESULTS: Hydrogen-exchange is enhanced, indicating that the β-switch favors the disordered loop conformation. Hydrogen-exchange is corroborated by differential scanning calorimetry, which confirms that these mutations destabilize GPIbα by allowing the β-switch to dissociate from the leucine-rich-repeat (LRR) domain. The stability of GPIbα and its A1 binding affinity, determined by surface plasmon resonance, are correlated to the extent of hydrogen exchange in the β-switch. CONCLUSION: These studies demonstrate that GPIbα with a disordered loop is binding-competent and support a mechanism in which local disorder in the β-switch exposes the LRR-domain of GPIbα enabling high-affinity interactions with the A1 domain.