title: The ADAMTS13 metalloprotease domain: roles of subsites in enzyme activity and specificity.
abstract: ADAMTS13 modulates von Willebrand factor (VWF) platelet-tethering function by proteolysis of the Tyr1605-Met1606 bond in the VWF A2 domain. To examine the role of the metalloprotease domain of ADAMTS13 in scissile bond specificity, we identified 3 variable regions (VR1, -2, and -3) in the ADAMTS family metalloprotease domain that flank the active site, which might be important for specificity. Eight composite sequence swaps (to residues in ADAMTS1 or ADAMTS2) and 18 single-point mutants were generated in these VRs and expressed. Swapping VR1 (E184-R193) of ADAMTS13 with that of ADAMTS1 or ADAMTS2 abolished/severely impaired ADAMTS13 function. Kinetic analysis of VR1 point mutants using VWF115 as a short substrate revealed reduced proteolytic function (k(cat)/K(m) reduced by 2- to 10-fold) as a result of D187A, R190A, and R193A substitutions. Analysis of VR2 (F216-V220) revealed a minor importance of this region. Mutants of VR3 (G236-A261) proteolysed wild-type VWF115 normally. However, using either short or full-length VWF substrates containing the P1' M1606A mutation, we identified residues within VR3 (D252-P256) that influence P1' amino acid specificity, we hypothesize, by shaping the S1' pocket. It is concluded that 2 subsites, D187-R193 and D252-P256, in the metalloprotease domain play an important role in cleavage efficiency and site specificity.
authors.affiliation: Department of Haematology, Imperial College London, Hammersmith Hospital Campus, London, United Kingdom. r.degroot06@imperial.ac.uk
authors.forename: Rens
authors.initials: R
authors.lastname: de Groot
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*; D000071120:ADAMTS13 Protein; D000595:Amino Acid Sequence; D002460:Cell Line; D015870:Gene Expression; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human