title: Essential role of the disintegrin-like domain in ADAMTS13 function.
abstract: ADAMTS13 is a highly specific multidomain plasma metalloprotease that regulates the multimeric size and function of von Willebrand factor (VWF) through cleavage at a single site in the VWF A2 domain. The precise role that the ADAMTS13 disintegrin-like domain plays in its function remains uncertain. Truncated ADAMTS13 variants suggested the importance of the disintegrin-like domain for both enzyme activity and specificity. Targeted mutagenesis of nonconserved regions (among ADAMTS family members) in the disintegrin-like domain identified 3 of 8 ADAMTS13 mutants (R349A, L350G, V352G) with reduced proteolytic activity. Kinetic analyses revealed a 5- to 20-fold reduction in catalytic efficiency of VWF115 (VWF residues 1554-1668) proteolysis by these mutants. These residues form a predicted exposed exosite on the surface of the disintegrin-like domain that lies approximately 26 A from the active site. Kinetic analysis of VWF115 carrying the D1614A mutation suggested that Arg349 in the ADAMTS13 disintegrin-like domain interacts directly with Asp1614 in VWF A2. We hypothesize that this interaction assists in positioning the scissile bond within the active site of ADAMTS13 and therefore plays a major role in determining cleavage parameters (K(m) and k(cat)), as opposed to binding affinity (K(d)) of ADAMTS13 for VWF, the latter being primarily determined by the spacer domain.
authors.affiliation: Department of Haematology, Imperial College London, London, United Kingdom.
authors.forename: Rens
authors.initials: R
authors.lastname: de Groot
authors.forename: Ajoy
authors.initials: A
authors.lastname: Bardhan
authors.forename: Nalisha
authors.initials: N
authors.lastname: Ramroop
authors.forename: David A
authors.initials: DA
authors.lastname: Lane
authors.forename: James T B
authors.initials: JT
authors.lastname: Crawley
mesh_terms: D051722:ADAM Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000071120:ADAMTS13 Protein; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000502:physiology; D020134:Catalytic Domain / Q000235:genetics; D002478:Cells, Cultured; D019483:Disintegrins / Q000737:chemistry; D006801:Humans; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000502:physiology; D050505:Mutant Proteins / Q000737:chemistry / Q000302:isolation & purification; D020641:Polymorphism, Single Nucleotide; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism
chemical_list: D019483:Disintegrins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human