Essential role of the disintegrin-like domain in ADAMTS13 function.
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.
de Groot
Rens
R
Department of Haematology, Imperial College London, London, United Kingdom.
Bardhan
Ajoy
A
Ramroop
Nalisha
N
Lane
David A
DA
Crawley
James T B
JT
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
D019483:Disintegrins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human