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.
authors.affiliation: Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109;
authors.forename: Colin A
authors.initials: CA
authors.lastname: Kretz
authors.affiliation: Department of Psychiatry and Molecular and Behavioral Neuroscience Institute, University of Michigan, Ann Arbor, MI 48109;
authors.forename: Manhong
authors.initials: M
authors.lastname: Dai
authors.affiliation: Bioinformatics and Genomics Programme, Centre for Genomic Regulation, Barcelona, Spain 08003; Universitat Pompeu Fabra, Barcelona, Spain 08003;
authors.forename: Onuralp
authors.initials: O
authors.lastname: Soylemez
authors.affiliation: Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109;
authors.forename: Andrew
authors.initials: A
authors.lastname: Yee
authors.affiliation: Department of Pediatrics, University of Michigan Medical School, Ann Arbor, MI 48109;
authors.forename: Karl C
authors.initials: KC
authors.lastname: Desch
authors.affiliation: Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109; Howard Hughes Medical Institute, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109;
authors.forename: David
authors.initials: D
authors.lastname: Siemieniak
authors.affiliation: Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI 48109;
authors.forename: Kärt
authors.initials: K
authors.lastname: Tomberg
authors.affiliation: Bioinformatics and Genomics Programme, Centre for Genomic Regulation, Barcelona, Spain 08003; Universitat Pompeu Fabra, Barcelona, Spain 08003; Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain 08003;
authors.forename: Fyodor A
authors.initials: FA
authors.lastname: Kondrashov
authors.affiliation: Department of Psychiatry and Molecular and Behavioral Neuroscience Institute, University of Michigan, Ann Arbor, MI 48109;
authors.forename: Fan
authors.initials: F
authors.lastname: Meng
authors.affiliation: Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109; Department of Pediatrics, University of Michigan Medical School, Ann Arbor, MI 48109; Howard Hughes Medical Institute, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109; Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI 48109; Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109 ginsburg@umich.edu.
authors.forename: David
authors.initials: D
authors.lastname: Ginsburg
mesh_terms: D051722:ADAM Proteins / Q000737:chemistry*; D000071120:ADAMTS13 Protein; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D001777:Blood Coagulation; D003001:Cloning, Molecular; D004843:Epistasis, Genetic; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D019151:Peptide Library; D011485:Protein Binding / Q000235:genetics; D059748:Proteolysis; D013379:Substrate Specificity; D014841:von Willebrand Factor / Q000737:chemistry
chemical_list: D019151:Peptide Library; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human
keywords: ADAMTS13; high-throughput sequencing; phage display; protease; von Willebrand factor