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Published on: August 14, 2017
ADAMTS13 substrate recognition of von Willebrand factor A2 domain
Sara Zanardelli1, James T B Crawley, Chan K N Chan Kwo Chion
1Department of Haematology, Imperial College London, London W12 ONN, United Kingdom.
Abstract:
ADAMTS13 controls the multimeric size of circulating von Willebrand factor (VWF) by cleaving the Tyr1605-Met1606 bond in theA2 domain. To examine substrate recognition, we expressed in bacteria and purified three A2 (VWF76-(1593-1668), VWF115-(1554-1668), VWFA2-(1473-1668)) and one A2-A3 (VWF115-A3-(1554-1874)) domain fragments. Using high pressure liquid chromatography analysis, the initial rates of VWF115 cleavage by ADAMTS13 at different substrate concentrations were determined, and from this the kinetic constants were derived (Km 1.61 microM; kcat 0.14 s(-1)), from which the specificity constant kcat/Km was calculated, 8.70 x 10(4) m(-1) s(-1). Similar values of the specificity constant were obtained for VWF76 and VWF115-A3. To identify residues important for recognition and proteolysis of VWF115, we introduced certain type 2A von Willebrand disease mutations by site-directed mutagenesis. Although most were cleaved normally, one (D1614G) was cleaved approximately 8-fold slower. Mutagenesis of additional charged residues predicted to be in close proximity to Asp1614 on the surface of the A2 domain (R1583A, D1587A, D1614A, E1615A, K1617A, E1638A, E1640A) revealed up to 13-fold reduction in kcat/Km for D1587A, D1614A, E1615A, and K1617A mutants. When introduced into the intact VWFA2 domain, proteolysis of the D1587A, D1614A, and E1615A mutants was also slowed, particularly in the presence of urea. Surface plasmon resonance demonstrated appreciable reduction in binding affinity between ADAMTS13 and VWF115 mutants (KD up to approximately 1.3 microM), compared with VWF115 (KD 20 nM). These results demonstrate an important role for Asp1614 and surrounding charged residues in the binding and cleavage of the VWFA2 domain by ADAMTS13.
Insights
ADAMTS13 enzyme activity is crucial for regulating von Willebrand factor size. Key charged residues, particularly Asp1614, are vital for ADAMTS13 binding and cleaving von Willebrand factor A2 domain.
Area of Science:
- Biochemistry
- Molecular Biology
- Hematology
Background:
- ADAMTS13 enzyme regulates von Willebrand factor (VWF) multimeric size by cleaving the VWF A2 domain.
- Understanding VWF-ADAMTS13 interaction is critical for VWF-related disorders.
Purpose of the Study:
- To investigate the substrate recognition and binding mechanisms of ADAMTS13 on the VWF A2 domain.
- To identify key residues involved in VWF cleavage by ADAMTS13.
Main Methods:
- Bacterial expression and purification of VWF A2 and A2-A3 domain fragments.
- High-performance liquid chromatography (HPLC) for kinetic analysis of VWF cleavage.
- Site-directed mutagenesis to create VWF mutants and assess their cleavage kinetics.
- Surface plasmon resonance (SPR) to evaluate binding affinity between ADAMTS13 and VWF mutants.
Main Results:
- Kinetic constants (Km, kcat) and specificity constant (kcat/Km) for VWF A2 domain cleavage by ADAMTS13 were determined.
- Mutagenesis studies identified Asp1614 and surrounding charged residues as critical for efficient VWF cleavage.
- Mutant VWF proteins showed reduced binding affinity to ADAMTS13, impacting proteolysis rates.
Conclusions:
- Asp1614 and adjacent charged residues play a significant role in ADAMTS13's recognition and catalytic activity on the VWF A2 domain.
- These findings provide insights into the molecular basis of ADAMTS13-VWF interaction and type 2A von Willebrand disease mechanisms.
