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

Author(s): Kretz, Colin A; Dai, Manhong; Soylemez, Onuralp; Yee, Andrew; Desch, Karl C; Siemieniak, David R; Tomberg, Kärt; Kondrashov, Fyodor A; Meng, Fan; Ginsburg, David B
Article Title: Massively parallel enzyme kinetics reveals the substrate recognition landscape of the metalloprotease ADAMTS13
Abstract: 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 (kcat/KM) 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.
Keywords: Protease; ADAMTS13; High-throughput sequencing; Phage display; Von Willebrand factor
Journal Title: PNAS
Volume: 112
Issue 30
ISSN: 1091-6490
Publisher: National Academy of Sciences  
Date Published: 2015-07-28
Start Page: 9328
End Page: 9333
DOI: 10.1073/pnas.1511328112
Notes: We thank Isabel Wang and Vivian Cheung from the Life Sciences Institute, University of Michigan, for assistance with high- throughput sequencing experiments and valuable discussions. We also thank J. Evan Sadler (Washington University) and Sriram Krishnaswamy (Children’s Hospital of Philadelphia) for helpful discussions. We thank Jeff Weitz (McMaster University), Jim Fredenburgh (McMaster University), and Steve Weiss (University of Michigan) for critical review of the manuscript. C.A.K. was awarded the Judith Graham Pool Fellowship from National Hemophilia Foundation. This work was supported by the National Institutes of Health (R01 HL039693), the National Heart, Lung, and Blood Institute (P01- HL057346), Ministerio de Economía y Competitividad Grants BFU2012- 31329 and Sev-2012-0208, and European Research Council Starting Grant 335980_EinME. D.G. is an investigator of the Howard Hughes Medical In- stitute, and F.A.K. is a Howard Hughes Medical Institute International Early Career Scientist.
Open access: no