Structures of VWF tubules before and after concatemerization reveal a mechanism of disulfide bond exchange

Anderson, Jacob R; Li, Jing; Springer, T.A.; Brown, Alan

doi:10.1182/blood.2022016467

Cited by 17 publications

(18 citation statements)

References 47 publications

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“…34 While this work was under revision, another structure of A1-containing tubules was reported, accompanied by a well-reasoned hypothesis for the polypeptide connectivity between the A1 domain and other regions of VWF. 35 Structures are available of the A1 domain in complex with various binding partners, allowing comparison between the interfaces used to incorporate A1 into the tubule and those used for downstream function. Particularly interesting is a comparison with the binding mode of A1 to GPIbα.…”

Section: Resultsmentioning

confidence: 99%

Assembly of von Willebrand factor tubules with in vivo helical parameters requires A1 domain insertion

Javitt

Yeshaya

Khmelnitsky

et al. 2022

Blood

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The von Willebrand factor (VWF) glycoprotein is stored in tubular form in Weibel-Palade bodies (WPBs) prior to secretion from endothelial cells into the bloodstream. The organization of VWF in the tubules promotes formation of covalently linked VWF polymers and enables orderly secretion without polymer tangling. Recent studies have described the high-resolution structure of helical tubular cores formed in vitro by the D1D2 and D´D3 amino-terminal protein segments of VWF. Here we show that formation of tubules with the helical geometry observed for VWF in intracellular WPBs requires also the VWA1 (A1) domain. We reconstituted VWF tubules from segments containing the A1 domain and discovered it to be inserted between helical turns of the tubule, altering helical parameters and explaining the increased robustness of tubule formation when A1 is present. The conclusion from this observation is that the A1 domain has a direct role in VWF assembly, along with its known activity in hemostasis post-secretion.

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Section: Resultsmentioning

confidence: 99%

Assembly of von Willebrand factor tubules with in vivo helical parameters requires A1 domain insertion

Javitt

Yeshaya

Khmelnitsky

et al. 2022

Blood

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“…3A). Therefore, their polymerization mechanisms can be described interchangeably, and progress in understanding each of these molecules has informed research on the other [40,[66][67][68][69][70].…”

Section: Disulfide-mediated Complex Formation In the Golgimentioning

confidence: 99%

“…It can be explained, however, by an indirect role for low pH in bringing together the proper protein domains to position cysteines for disulfide bonding. High-resolution analysis of low-pH assembly intermediates of mucins and VWF amino-terminal regions [66,69,70,77], together with extensive lowresolution studies of the domain organization of VWF [78], led to a model in which the amino termini of the tail-linked dimers interact non-covalently in such a way that they are prevented from disulfide bonding with one another. Instead, the low pH of the Golgi lumen induces filamentation of the amino-terminal regions, which juxtaposes the relevant cysteines from separate dimers.…”

Section: Disulfide-mediated Complex Formation In the Golgimentioning

confidence: 99%

Disulfide bond formation and redox regulation in the Golgi apparatus

Reznik

Fass

2022

FEBS Letters

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Formation of disulfide bonds in secreted and cell‐surface proteins involves numerous enzymes and chaperones abundant in the endoplasmic reticulum (ER), the first and main site for disulfide bonding in the secretory pathway. Although the Golgi apparatus is the major station after the ER, little is known about thiol‐based redox activity in this compartment. QSOX1 and its paralog QSOX2 are the only known Golgi‐resident enzymes catalyzing disulfide bonding. The localization of disulfide catalysts in an organelle downstream of the ER in the secretory pathway has long been puzzling. Recently, it has emerged that QSOX1 regulates particular glycosyltransferases, thereby influencing a central activity of the Golgi. Surprisingly, a few important disulfide‐mediated multimerization events occurring in the Golgi were found to be independent of QSOX1. These multimerization events depend, however, on the low pH of the Golgi lumen and secretory granules. We compare and contrast disulfide‐mediated multimerization in the ER vs. the Golgi to illustrate the variety of mechanisms controlling covalent supramolecular assembly of secreted proteins.

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“…In case of vWF, while the importance of cysteine redox states and disulfide connections has been recognized, structural data on the major disulfide bonded domains C and D have only very recently become available [14,[20][21][22]. Hence, a mechanistic and quantitative view on redox regulation and the role of force therein is currently lacking.…”

Section: Introductionmentioning

confidence: 99%

“…Beyond conferring structural integrity, disulfide bonds have been recently discovered to directly participate in hemostatic functions. Thiol disulfide shuffling has been suggested to mediate vWF multimer size [12], oligomerization [13, 14], and platelet binding [15]. Moreover, auto-inhibition of vWF for the binding of platelets, initiated by A1-A2 inter-domain interactions, is controlled by a disulfide bond switch in the vWF A2 domain [16].…”

Section: Introductionmentioning

confidence: 99%

Dynamic Disulfide Bond Topologies in von-Willebrand-Factor’s C4-Domain Undermine Platelet Binding

Kutzki¹,

Butera

Lay

et al. 2022

Preprint

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Background: The von Willebrand Factor (vWF) is a key player in regulating hemostasis through adhesion of platelets to sites of vascular injury. It is a large multi-domain mechanosensitive protein stabilized by a net of disulfide bridges. Binding to platelet integrin is achieved by the vWF-C4 domain which exhibits a fixed fold, even under conditions of severe mechanical stress, but only if critical internal disulfide bonds are closed. Objective: To quantitatively determine C4's disulfide topologies and their implication in vWF's platelet-binding function via integrin. Methods: We employed a combination of classical Molecular Dynamics and quantum mechanical simulations, mass spectrometry, site-directed mutagenesis, and platelet binding assays. Results: We quantitatively show that two disulfide bonds in the vWF-C4 domain, namely the two major force-bearing ones, are partially reduced in human blood. Reduction leads to pronounced conformational changes within C4 that considerably affect the accessibility of the RGD-integrin binding motif, and thereby impair integrin-mediated platelet binding. Our combined approach also reveals that reduced species in the C4 domain undergo specific thiol/disulfide exchanges with the remaining disulfide bridges, in a process in which mechanical force may increase the proximity of specific reactant cysteines, further trapping C4 in a state of low integrin-binding propensity. We identify a multitude of redox states in all six vWF-C domains, suggesting disulfide bond reduction and swapping to be a general theme. Conclusion: Overall, our data put forward a mechanism in which disulfide bonds dynamically swap cysteine partners and control the interaction of vWF with integrin and potentially other partners, thereby critically influencing its hemostatic function.

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Structures of VWF tubules before and after concatemerization reveal a mechanism of disulfide bond exchange

Cited by 17 publications

References 47 publications

Assembly of von Willebrand factor tubules with in vivo helical parameters requires A1 domain insertion

Assembly of von Willebrand factor tubules with in vivo helical parameters requires A1 domain insertion

Disulfide bond formation and redox regulation in the Golgi apparatus

Dynamic Disulfide Bond Topologies in von-Willebrand-Factor’s C4-Domain Undermine Platelet Binding

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