Mechanisms by which von Willebrand Disease Mutations Destabilize the A2 Domain*

Xu, A.J.; Springer, Timothy A.

doi:10.1074/jbc.m112.422618

Cited by 21 publications

(28 citation statements)

References 25 publications

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“…S10), and the remaining mutations exhibited only modest enhancements in VWF73 proteolysis. A number of VWD-2A mutations have been shown to lower the threshold of shear required to unfold the VWF A2 domain, more readily exposing the cryptic scissile bond to ADAMTS13 (32,(34)(35)(36). Taken together, these data strongly suggest that VWF73 requires additional structural features within full-length VWF to effectively model the effect of VWD-2A mutations on ADAMTS13 proteolysis.…”

Section: High-throughput Sequencing Of Uncleaved Phages Defines the Psupporting

confidence: 51%

Massively parallel enzyme kinetics reveals the substrate recognition landscape of the metalloprotease ADAMTS13

Kretz

Dai

Söylemez

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Proteases play important roles in many biologic processes and are key mediators of cancer, inflammation, and thrombosis. However, comprehensive and quantitative techniques to define the substrate specificity profile of proteases are lacking. The metalloprotease ADAMTS13 regulates blood coagulation by cleaving von Willebrand factor (VWF), reducing its procoagulant activity. A mutagenized substrate phage display library based on a 73-amino acid fragment of VWF was constructed, and the ADAMTS13-dependent change in library complexity was evaluated over reaction time points, using high-throughput sequencing. Reaction rate constants (k cat /K M ) were calculated for nearly every possible single amino acid substitution within this fragment. This massively parallel enzyme kinetics analysis detailed the specificity of ADAMTS13 and demonstrated the critical importance of the P1-P1′ substrate residues while defining exosite binding domains. These data provided empirical evidence for the propensity for epistasis within VWF and showed strong correlation to conservation across orthologs, highlighting evolutionary selective pressures for VWF.phage display | protease | high-throughput sequencing | ADAMTS13 | von Willebrand factor P rotease specificity is critical for maintaining diversity and compartmentalization of function, and is tightly controlled. For many proteases, a substrate initially docks to an exosite, which captures and orients the substrate scissile bond toward the active site of the enzyme. At the active site, the Px-Px′ (1) substrate amino acid side chains align with the complementary Sx-Sx′ pockets of the enzyme to optimize recognition by the active site residues that execute the proteolytic reaction (2).Conventional techniques for probing the substrate recognition requirements of a protease are cumbersome and time-consuming and require intimate knowledge of the enzyme/substrate pair. Such methods include engineering deletion mutants (3), use of competitive ligands (4, 5), and site-directed mutagenesis (6, 7). In contrast to these techniques, substrate phage display is a highthroughput, unbiased approach to studying protease substrate specificity (8-10). In this method, a library consisting of 10 6 -10 9 independent phage clones, each expressing a unique potential substrate on its surface, is panned for multiple rounds with a protease, and the cleaved or uncleaved phages after each reaction are removed and amplified for subsequent rounds of selection. In this manner, the library complexity is iteratively reduced and becomes populated by peptide sequences that are most informative. This methodology, although useful, is limited by the number of clones selected for individual Sanger sequencing after the last round of selection, and the selection of phages based on competitive growth advantages unrelated to enzyme specificity. The availability of high-throughput DNA sequencing technology (11) has facilitated detailed analysis of the changing complexity within a phage display library (12-16) without requiring multip...

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Section: High-throughput Sequencing Of Uncleaved Phages Defines the Psupporting

confidence: 51%

Massively parallel enzyme kinetics reveals the substrate recognition landscape of the metalloprotease ADAMTS13

Kretz

Dai

Söylemez

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…It has previously been demonstrated that VWD type 2A mutations in the isolated VWF A2 domain also cause reduction in thermostability. 32 It is likely that the structural defects caused by the naturally occurring VWD type 2A group II mutants reduce the stability of the domain and lower the force threshold at which the VWF A2 domain unfolds, resulting in increased ADAMTS13 proteolysis and reduction in the high-MW VWF multimer species under conditions in which the WT would be resistant to ADAMTS13 proteolysis. For personal use only.…”

Section: Discussionmentioning

confidence: 99%

N-linked glycan stabilization of the VWF A2 domain

Lynch

Lane

2016

Blood

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Key Points• Glycosylation at N1574 stabilizes the VWF A2 domain against unfolding and proteolysis by ADAMTS13, and its first GlcNAc is the critical element.• Y1544 is a likely interacting residue with N1574-GlcNAc, and its mutation to aspartic acid stabilizes the domain in the absence of the glycan.Shear forces in the blood trigger a conformational transition in the von Willebrand factor (VWF) A2 domain, from its native folded to an unfolded state, in which the cryptic scissile bond (Y1605-M1606) is exposed and can then be proteolysed by ADAMTS13. The conformational transition depends upon a Ca 21 binding site and a vicinal cysteine disulfide bond. Glycosylation at N1574 has previously been suggested to modulate VWF A2 domain interaction with ADAMTS13 through steric hindrance by the bulky carbohydrate structure. We investigated how the N-linked glycans of the VWF A2 domain affect thermostability and regulate both the exposure of the ADAMTS13 binding sites and the scissile bond. We show by differential scanning fluorimetry that the N-linked glycans thermodynamically stabilize the VWF A2 domain. The essential component of the glycan structure is the first sugar residue (GlcNAc) at the N1574 attachment site. From its crystal structures, N1574-GlcNAc is predicted to form stabilizing intradomain interactions with Y1544 and nearby residues. Substitution of the surface-exposed Y1544 to aspartic acid is able to stabilize the domain in the absence of glycosylation and protect against ADAMTS13 proteolysis in both the VWF A2 domain and FLVWF. Glycan stabilization of the VWF A2 domain acts together with the Ca 21 binding site and vicinal cysteine disulfide bond to control unfolding and ADAMTS13proteolysis. (Blood. 2016;127(13):1711-1718

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“…43,[49][50][51][52][53] Unfolding of A2 is required for cleavage by ADAMTS13 ( Figure 7D), 43 consistent with burial of the specific cleavage site between Tyr 1605 and Met 1606 in the central b-sheet in the folded domain 51,54,55 (Figure 5B). A2 unfolding kinetics increase exponentially with force.…”

Section: A Domains and Von Willebrand Disease (Vwd)mentioning

confidence: 99%

“…A2 unfolding kinetics increase exponentially with force. 43,52,53 Single molecule measurements on A2, and the calculated peak force and rate of force application on VWF tumbling in vivo at 100 dyn/cm 2 , correctly predict the maximal length of VWF to be ;100 monomers. 43 Patients with the bleeding diathesis VWD type 2A have overly short VWF concatemers (Figure 8).…”

Section: A Domains and Von Willebrand Disease (Vwd)mentioning

confidence: 99%

von Willebrand factor, Jedi knight of the bloodstream

Springer

2014

Blood

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383

454

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When blood vessels are cut, the forces in the bloodstream increase and change character. The dark side of these forces causes hemorrhage and death. However, von Willebrand factor (VWF), with help from our circulatory system and platelets, harnesses the same forces to form a hemostatic plug. Force and VWF function are so closely intertwined that, like members of the Jedi Order in the movie Star Wars who learn to use “the Force” to do good, VWF may be considered the Jedi knight of the bloodstream. The long length of VWF enables responsiveness to flow. The shape of VWF is predicted to alter from irregularly coiled to extended thread-like in the transition from shear to elongational flow at sites of hemostasis and thrombosis. Elongational force propagated through the length of VWF in its thread-like shape exposes its monomers for multimeric binding to platelets and subendothelium and likely also increases affinity of the A1 domain for platelets. Specialized domains concatenate and compact VWF during biosynthesis. A2 domain unfolding by hydrodynamic force enables postsecretion regulation of VWF length. Mutations in VWF in von Willebrand disease contribute to and are illuminated by VWF biology. I attempt to integrate classic studies on the physiology of hemostatic plug formation into modern molecular understanding, and point out what remains to be learned.

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Mechanisms by which von Willebrand Disease Mutations Destabilize the A2 Domain*

Cited by 21 publications

References 25 publications

Massively parallel enzyme kinetics reveals the substrate recognition landscape of the metalloprotease ADAMTS13

Massively parallel enzyme kinetics reveals the substrate recognition landscape of the metalloprotease ADAMTS13

N-linked glycan stabilization of the VWF A2 domain

von Willebrand factor, Jedi knight of the bloodstream

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