A Mechanism for Localized Dynamics-driven Affinity Regulation of the Binding of von Willebrand Factor to Platelet Glycoprotein Ibα

Journal of Molecular Biology

Machha

Frontroth

et al. 2017

Mutation of the cysteines forming the disulfide loop of the platelet GPIbα adhesive A1 domain of von Willebrand factor causes quantitative VWF deficiencies in the blood and von Willebrand disease. We report two cases of transient severe thrombocytopenia induced by DDAVP-treatment. Cys1272Trp and Cys1458Tyr mutations identified by genetic sequencing implicate an abnormal gain-of-function phenotype, evidenced by thrombocytopenia, that quickly relapses back to normal platelet counts and deficient plasma VWF. Using surface plasmon resonance, analytical rheology, and hydrogen-deuterium exchange mass spectrometry (HXMS), we decipher mechanisms of A1-GPIbα mediated platelet adhesion and resolve dynamic secondary structure elements that regulate the binding pathway. Constrained by the disulfide, conformational selection between weak and tight binding states of A1 takes precedence and drives normal platelet adhesion to VWF. Less restrained through mutation, loss of the disulfide preferentially diverts binding through an induced-fit disease pathway enabling high-affinity GPIbα binding and firm platelet adhesion to a partially disordered A1 domain. HXMS reveals a dynamic asymmetry of flexible and ordered regions common to both variants indicating that the partially disordered A1 lacking the disulfide retains native-like structural dynamics. Both binding mechanisms share common structural and thermodynamic properties, but the enhanced local disorder in the disease state perpetuates high-affinity platelet agglutination, characteristic of type 2B VWD, upon DDAVP-stimulated secretion of VWF leading to transient thrombocytopenia and a subsequent deficiency of plasma VWF, characteristic of type 2A VWD.

Section: Resultsmentioning

confidence: 99%

Enhanced Local Disorder in a Clinically Elusive von Willebrand Factor Provokes High-Affinity Platelet Clumping

Journal of Molecular Biology

Machha

Frontroth

et al. 2017

“…has not yet been completely tested, but other computational methods have also implicated a localized dynamic role of the a2 helix (23). Taking into account the fact that GPIba has been cocrystallized with a helical peptide (OS1) that inhibits platelet interactions with VWF, it is possible that the a2-loop-a3 helices could play a direct role in the binding under shear stress (24).…”

Section: Discussionmentioning

confidence: 98%

Structural Origins of Misfolding Propensity in the Platelet Adhesive Von Willebrand Factor A1 Domain

Zimmermann

Whitten

et al. 2015

Biophysical Journal

The von Willebrand factor (VWF) A1 and A3 domains are structurally isomorphic yet exhibit distinct mechanisms of unfolding. The A1 domain, responsible for platelet adhesion to VWF in hemostasis, unfolds through a molten globule intermediate in an apparent three-state mechanism, while A3 unfolds by a classical two-state mechanism. Inspection of the sequences or structures alone does not elucidate the source of this thermodynamic conundrum; however, the three-state character of the A1 domain suggests that it has more than one cooperative substructure yielding two separate unfolding transitions not present in A3. We investigate the extent to which structural elements contributing to intermediate conformations can be identified using a residue-specific implementation of the structure-energy-equivalence-of-domains algorithm (SEED), which parses proteins of known structure into their constituent thermodynamically cooperative components using protein-group-specific, transfer free energies. The structural elements computed to contribute to the non-two-state character coincide with regions where Von Willebrand disease mutations induce misfolded molten globule conformations of the A1 domain. This suggests a mechanism for the regulation of rheological platelet adhesion to A1 based on cooperative flexibility of the α2 and α3 helices flanking the platelet GPIbα receptor binding interface.

“…The structure of A1 is a αβ‐Rossmann type fold with a central β‐sheet flanked by six α‐helices, three on each side, linked by a single disulfide bond at the N‐ and C‐terminus. All structures of A1 are highly similar in the conformation of the backbone chain, but detailed structural analyses of the complex crystal structure suggest that the rearrangement of intermolecular electrostatics between residue side chains alter the dynamics of the α1β2 loop causing a pivot of the α2 helix . Such a conformational propensity is supported by recent hydrogen‐deuterium exchange experiments and is thought to be “on—pathway” toward high—affinity …”

Section: Introductionmentioning

confidence: 94%

“…All structures of A1 are highly similar in the conformation of the backbone chain, 12 but detailed structural analyses of the complex crystal structure suggest that the rearrangement of intermolecular electrostatics between residue side chains alter the dynamics of the α1β2 loop 8,13,19 causing a pivot of the α2 helix. 20 Such a conformational propensity is supported by recent hydrogen-deuterium exchange experiments 21 and is thought to be "on-pathway" toward high-affinity. 14 Likewise, 13 structures have been determined of WT and PT-VWD variants of free GPIbα, [22][23][24] GPIbα in complex with the A1 domain, 10,13,14 thrombin, 23,25,26 and a peptide inhibitor.…”

mentioning

confidence: 92%

Platelet‐type von Willebrand disease: Local disorder of the platelet GPIbα β‐switch drives high‐affinity binding to von Willebrand factor

Journal of Thrombosis and Haemostasis

Machha

Moon‐Tasson

et al. 2019

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.