Molecular Dynamics Simulated Unfolding of von Willebrand Factor A Domains by Force

Chen, Wei; Lou, Jizhong; Zhu, Cheng

doi:10.1007/s12195-009-0051-0

Cited by 20 publications

(25 citation statements)

References 35 publications

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“…One mode might be unfolding of the tertiary structure of the A2 domain (far right panel), as l d of approximately 50 nm is consistent with the contour length increase predicted for A2 unfolding. 27 The short length increase (l d ϳ 20 nm) might represent either partial unfolding of the A2 domain or uncoupling of the quaternary structure of the A1A2A3 tridomain by disrupting the domaindomain interactions (2 middle panels). Unfolding of A1 or A3 is improbable because each domain contains a disulfide bond between cystines at the N-and C-termini that makes it strongly resistant to unfolding.…”

mentioning

confidence: 99%

Force-induced cleavage of single VWFA1A2A3 tridomains by ADAMTS-13

Wu¹,

Lin

Crúz

et al. 2010

Blood

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A disintegrin and metalloprotease with a thrombospondin type 1 motifs 13 (ADAMTS-13) regulates hemostasis by cleaving the folded A2 domain of von Willebrand factor (VWF). The cleavage is regulated by forces as it occurs in flowing blood. We tested the hypothesis that force-induced A2 domain unfolding facilitates cleavage using atomic force microscopy to pull single VWF A1A2A3 tridomain polypeptides by platelet glycoprotein Ibalpha or antibodies to measure time, distance, and force. Structural destabilization of A1A2A3 was induced by 5- to 80-pN forces, manifesting as an abrupt molecular length increase distributed around 20 and 50 nm, probably because of uncoupling A1A2A3 (or partially unfolding A2) and fully unfolding A2, respectively. Time required to destabilize A1A2A3 first increased (catch), reaching a maximum of 0.2 seconds at 20pN, then decreased (slip) with increasing force, independent of ADAMTS-13. The time required to rupture A1A2A3 exhibited a similar catch-slip behavior when pulled by glycoprotein Ibalpha but only slip behavior when pulled by antibody, which was progressively shortened by increasing concentration of ADAMTS-13 after (but not before) structural destabilization, indicating that cleavage of A2 requires the force-induced A2 unfolding. Analysis with a model for single-substrate trimolecular enzymatic kinetics estimated a cleavage rate k(cat) of 2.9 (+/- 59) seconds and a K(d) of 5.6 (+/- 3.4) nM for ADAMTS-13/A1A2A3 binding. These findings quantify the mechanical regulation of VWF cleavage by ADAMTS-13 at the level of single A1A2A3 tridomain.

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mentioning

confidence: 99%

Force-induced cleavage of single VWFA1A2A3 tridomains by ADAMTS-13

Wu¹,

Lin

Crúz

et al. 2010

Blood

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101

120

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“…Recent molecular dynamics simulations 17,18 and studies using optical tweezers 19 have suggested that the A2 domain unfolds progressively from its C-terminus; the ␣6-helix is peeled off first, followed by the ␤6 and ␣5-helix. 17,18 We propose that the vicinal disulfide bond primarily influences the initial uncoupling of the ␣6-helix and therefore directly modulates the exposure of the high-affinity ADAMTS13-binding site. Thereafter, a second unfolding step is required for the detachment of the ␤5 and ␣4-less loop and exposure of the cleavage site in the ␤4 strand, as A2-⌬CC is cleaved inefficiently without denaturant.…”

mentioning

confidence: 99%

The importance of vicinal cysteines, C1669 and C1670, for von Willebrand factor A2 domain function

Luken

Winn

Emsley

et al. 2010

Blood

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The von Willebrand factor (VWF) A2 crystal structure has revealed the presence of a rare vicinal disulfide bond between C1669 and C1670, predicted to influence domain unfolding required for proteolysis by ADAMTS13. We prepared VWF A2 domain fragments with (A2-VicCC, resi- Introductionvon Willebrand factor (VWF) forms disulfide-linked multimers, the largest of which are most potent in binding collagen and the platelet receptor glycoprotein Ib␣. [1][2][3][4] Mechanical shear forces in the bloodstream induce conformational changes in VWF 5 and modulate the exposure of both platelet and ADAMTS13-binding sites. VWF multimer size and function are regulated through proteolysis by ADAMTS13. 6,7 Unfolding of the central VWF A2 domain is required to expose the Y1605-M1606 cleavage site, which is normally buried within the central ␤-sheet of the domain. 8 The VWF A2 domain is highly homologous to the VWF A1 and A3 domains (supplemental Figure 1A, available on the Blood Web site; see the Supplemental Materials link at the top of the online article) but lacks an intradomain disulfide bond that connects the ␤1-sheet with the ␣6-helix. However, the recent VWF A2 domain crystal structure revealed a disulfide bond between adjacent C1669 and C1670 at the C-terminus of the ␣6-helix. 8 Such a vicinal disulfide bond is rare, as an 8-membered ring is formed that bends the protein backbone in an unusual constrained conformation. 9 In the crystal structure, the disulfide bond directly interacts to the hydrophobic core of the domain, suggesting that it stabilizes the domain conformation. 8 We therefore examined the influence of this unusual vicinal disulfide bond on VWF A2 domain function. Methods Recombinant proteinsVWF A2-⌬CC (amino acids 1473-1668), A2-VicCC (1473A2-VicCC ( -1670, and both N1493C/C1669G (A2-CC1) and N1493C/C1670G (A2-CC2) mutants (supplemental Figure 1B) were cloned in the pCEP4 vector (Invitrogen) containing a C-terminal myc/His tag. A2 domain fragments were expressed in HEK293EBNA cells, purified by Ni 2ϩ chromatography, and quantified by the BCA protein assay kit (Thermo Scientific).Mutations N1493C/C1669G (VWF-CC1), N1493C/C1670G (VWF-CC2), and C1669G/C1670G (VWF-VicGG) were introduced into pcDNA3.1-VWF 10 and full-length VWF variants expressed in HEK293EBNA cells. Conditioned media were dialyzed and VWF concentrations determined by enzyme-linked immunosorbent assay. 10 AD-AMTS13 was expressed and purified as previously described. 11 Maleimide-PEG 2 -biotin labeling of free thiolsFree thiols were detected both before and after reduction of VWF A2 variants by labeling with maleimide-PEG 2 -biotin (Thermo Scientific). Labeled thiols were detected by Western blotting using streptavidin-peroxidase. Reduction and carboxymethylation of VWF A2 variantsTo permanently disrupt disulfide bonds, VWF A2 variants were reduced with dithiothreitol (DTT) and carboxymethylated with iodoacetic acid before use in the proteolysis and binding assays. Proteolysis by ADAMTS13Proteolysis of VWF A2 variants was performed as previously de...

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“…This is similar to the first level exposure observed in the unfolding of A2 by force. 5 Then the SASA of the sidechains was elevated up to~150 Å 2 while the SASA of the backbones increased up to~25 Å 2 . This is similar to the second level exposure observed in the unfolding of A2 by force.…”

Section: Exposure Of the Proteolytic Site Of A2mentioning

confidence: 99%

“…12,25 A suggested mechanism for the enhancing effect is that shear flow or denaturants unfold the A2 domain and expose the proteolytic site for cleavage by ADAMTS-13. 1 In our previous atomic force microscopy (AFM) experiments 29 and molecular dynamics (MD) simulations, 5 we have demonstrated that tensile force can unfold the VWF A2 domain and expose its cryptic proteolytic site. Here we studied the thermal unfolding of the VWF A domains with MD simulations, which were performed at high temperature to accelerate conformational sampling.…”

Section: Introductionmentioning

confidence: 95%

Simulated Thermal Unfolding of the von Willebrand Factor A Domains

2010

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The A1 and A2 domains of von Willebrand factor (VWF) have important functions: A1 contains a binding site for platelet glycoprotein Ib (GPIb) while A2 contains a cryptic proteolytic site for the VWF-cleavage enzyme, A Disintegrin And Metalloprotease with a ThromboSpondin type 1 motifs 13 (ADAMTS-13). Because the proteolytic site is fully buried in the native A2 structure, A2 needs to be unfolded to expose its proteolytic site for ADAMTS-13 cleavage. To study the unfolding mechanism of the VWF A domains, we used molecular dynamics (MD) to simulate in atomic details the thermal unfolding of A1 and A2 at high temperatures. The thermal unfolding of A1 and A2 appears very different from their unfolding by tensile forces. At 500 K, unfolding of the central b sheet of A2 starts from the two edges and propagates into the center. b4 and b5 in the center are structurally the most stable and unfolded the latest. However, A2 could be unfolded along different pathways and the unfolded A2 structure is highly flexible. By comparison, A1 is unfolded slower than A2 at 500 K. In even longer time, the unfolding of A1 is limited to the edges of the central b sheet, suggesting a protective role of the N-C terminal disulfide bond.

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Molecular Dynamics Simulated Unfolding of von Willebrand Factor A Domains by Force

Cited by 20 publications

References 35 publications

Force-induced cleavage of single VWFA1A2A3 tridomains by ADAMTS-13

Force-induced cleavage of single VWFA1A2A3 tridomains by ADAMTS-13

The importance of vicinal cysteines, C1669 and C1670, for von Willebrand factor A2 domain function

Simulated Thermal Unfolding of the von Willebrand Factor A Domains

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