Extensive contacts between ADAMTS13 exosites and von Willebrand factor domain A2 contribute to substrate specificity

Abstract

The metalloprotease ADAMTS13 efficiently cleaves only the Tyr(1605)-Met(1606) bond in the central A2 domain of multimeric von Willebrand factor (VWF), even though VWF constitutes only 0.02% of plasma proteins. This remarkable specificity depends in part on binding of the noncatalytic ADAMTS13 spacer domain to the C-terminal alpha-helix of VWF domain A2. By kinetic analysis of recombinant ADAMTS13 constructs, we show that the first thrombospondin-1, Cys-rich, and spacer domains of ADAMTS13 interact with segments of VWF domain A2 between Gln(1624) and Arg(1668), and together these exosite interactions increase the rate of substrate cleavage by at least approximately 300-fold. Internal deletion of Gln(1624)-Arg(1641) minimally affected the rate of cleavage, indicating that ADAMTS13 does not require a specific distance between the scissile bond and auxiliary substrate binding sites. Smaller deletions of the P2-P9 or the P4'-P18' residues on either side of the Tyr(1605)-Met(1606) bond abolished cleavage, indicating that the metalloprotease domain interacts with additional residues flanking the cleavage site. Thus, specific recognition of VWF depends on cooperative, modular contacts between several ADAMTS13 domains and discrete segments of VWF domain A2.

Figure 1

ADAMTS13 and VWF substrates. (A) Active ADAMTS13 consists of a metalloprotease domain (M), a disintegrin-like domain (D), a thrombospondin type 1 repeat (TSP1, T), a Cys-rich domain (Cys, C), a spacer domain (Spacer, S), 7 additional TSP1 repeats (2-8), and 2 CUB domains. In addition to full-length ADAMTS13 (FL), truncated enzymes were constructed with stop codons after Gln²⁸⁹ (M), Gly³⁸⁵ (MD), Glu⁴³⁹ (MDT), Cys⁵⁵⁵ (MDTC), and Ala⁶⁸⁵ (MDTCS). All constructs contain a C-terminal V5 epitope and 6 × His tag. (B) GST fusion substrates consist of a GST moiety followed by the linker SDLEVLFQGPLGS, a segment of VWF domain A2 (VWFxxx), and 6 His residues (6 × His). Rhinovirus 3C protease cleaves the indicated Gln-Gly bond in the linker sequence to produce substrates for MALDI MS analysis. (C) The amino acid sequence of VWF from Asp¹⁵⁹⁶-Arg¹⁶⁶⁸ is annotated to show the extent of num-bered α-helices and β-strands from a homology model of the A2 domain. The Tyr¹⁶⁰⁵-Met¹⁶⁰⁶ bond cleaved by ADAMTS13 is marked by arrowheads. Segments of VWF contained in GST constructs are indicated. (D) Substrates VWF73nl and VWF106nl represent N-terminal elongations of VWF64 and contain the sequence shown, annotated to indicate the extent of predicted α-helices and β-strands. The asterisk (*) indicates N-glycosylated residue Asn¹⁵⁷⁴.

Figure 2

Cleavage of C-terminal deletion substrates. GST-VWF46, GST-VWF35, and GST-VWF28 (60-100 nM) were incubated with the indicated proteases (1 nM) for 5 hours or 24 hours without (−) or with (+) 10 mM EDTA. Substrates and 28-kDa cleavage products () were detected by gel electrophoresis and Western blotting with anti-GST antibody. The blots for GST-VWF28 were exposed to film for 35 seconds; other blots were exposed to film for 15 seconds. Results are representative of 3 independent experiments.

Figure 3

Cleavage of internal deletion substrates. Substrates (65 nM) were incubated (A) with the indicated recombinant proteases (2 nM) for 1 hour, or (B) with plasma ADAMTS13 (0.3 nM) for 2 hours, without (−) or with (+) 10 mM EDTA. Substrates and 28-kDa cleavage products () were detected by gel electrophoresis and Western blotting with anti-GST antibody. Results are representative of 3 independent experiments.

Figure 4

Cleavage of N-terminal deletion and elongation substrates. (A) GST-VWFd5 (65 nM) was incubated at 37°C for the indicated time with 2 nM recombinant full-length ADAMTS13 (FL), truncated constructs MD or MDT, or 0.3 nM plasma ADAMTS13 (PL). No cleavage product () was detected by gel electrophoresis and Western blotting with anti-GST antibody. (B) GST-VWF64, GST-VWF73nl, and GST-VWF106nl (65 nM) were incubated for 2 hours with 2 nM recombinant ADAMTS13 (FL), or for 6 hours with 0.3 nM plasma ADAMTS13 (PL). Substrates and cleavage products () were detected by gel electrophoresis and Western blotting with anti-GST antibody. Asterisks (*) indicates nonspecific bands observed reproducibly for GST-VWFd5 and GST-VWF106nl. Results are representative of 3 independent experiments. (C) Time course for cleavage of 6.8 nM GST-VWF64 (○), GST-VWF73nl (●), and GST-VWF106nl (□) by 1 nM ADAMTS13 at 37°C. Cleavage products were quantitated by an ELISA method. Data points represent the mean values for 2 independent experiments; the range was 2% to 9% of the mean.

Figure 5

ADAMTS13-VWF interactions. (A) A homology model of the ADAMTS13 metalloprotease (magenta) and disintegrin-like domains (cyan) is shown (MD) to provide a sense of scale, with active site Zn²⁺ ion (green) and 3 structural Ca²⁺ ions (gray) as spheres. The VWF A2 domain is predicted to consist of a 6-stranded β-sheet surrounded by 5 α-helices. Residues Tyr¹⁶⁰⁵-Met¹⁶⁰⁶ (side chains in red) are buried in strand β4. Exposure of this bond to ADAMTS13 requires substantial unfolding of domain A2; more distal segments that interact with specific domains of ADAMTS13 are labeled. The locations of these ADAMTS13 domains relative to the MD moiety are not known. Deletion of strand β5 through helix α4 (dispensable) has a minimal effect on the rate of substrate cleavage. Molecular graphics prepared with PyMOL (DeLano Scientific, Palo Alto, CA). (B) The minimal segment of VWF domain A2 that ADAMTS13 is known to cleave consists of residues Arg¹⁵⁹⁷-Ile¹⁶²³. VWD type 2A mutations that increase cleavage are shown. A VWF polymorphism that may not impair cleavage is shown in parentheses to indicate that cleavability of VWF(1601T) has not been studied directly. Synthetic substrate FRETS-VWF73 is cleaved rapidly with replacement of side chains of Gln¹⁵⁹⁹ by N-methylanthranylate and Asn¹⁶¹⁰ by 2,4-dinitrophenyl. Alanine substitutions at 3 positions reduce the rate of VWF cleavage. Substrates with deletions VWFd4 and VWFd5 (Figure 1C) have the sequences shown, with altered residues in lowercase and preserved residues in uppercase, and neither substrate is cleaved detectably.