IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ ADAMTS13 substrate recognition of von Willebrand factor A2 domain. ADAMTS13 substrate recognition of von Willebrand factor A2 domain. J. Biol. Chem. (0021-9258) 2006 imex curation Zanardelli S., Crawley JT., Chion CK., Lam JK., Preston RJ., Lane DA. BioCreative - Critical Assessment of Information Extraction systems in Biology HPLC and SDS-PAGE were used to follow the proteolytic reaction. Mass spectrometry or N-terminal sequencing was used to identify the polypeptide in peaks eluted using HPLC so as to establish the site of proteolysis. j.crawley@imperial.ac.uk. 14-DEC-2006: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro protease assay protease assay predetermined predetermined participant predetermined Zanardelli S., Crawley JT., Chion CK., Lam JK., Preston RJ., Lane DA. BioCreative - Critical Assessment of Information Extraction systems in Biology HPLC and SDS-PAGE were used to follow the proteolytic reaction. Mass spectrometry or N-terminal sequencing was used to identify the polypeptide in peaks eluted using HPLC so as to establish the site of proteolysis. j.crawley@imperial.ac.uk. Accepted 2006-SEP-15 by ORCHARD. 14-DEC-2006: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation ADAMTS13 substrate recognition of von Willebrand factor A2 domain. ADAMTS13 substrate recognition of von Willebrand factor A2 domain. J. Biol. Chem. (0021-9258) 2006 imex curation Zanardelli S., Crawley JT., Chion CK., Lam JK., Preston RJ., Lane DA. BioCreative - Critical Assessment of Information Extraction systems in Biology HPLC and SDS-PAGE were used to follow the proteolytic reaction. Mass spectrometry or N-terminal sequencing was used to identify the polypeptide in peaks eluted using HPLC so as to establish the site of proteolysis. j.crawley@imperial.ac.uk. 14-DEC-2006: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro spr surface plasmon resonance Optical biosensor BIAcore(r) BIAcore(r) Optical biosensor spr predetermined predetermined participant predetermined Zanardelli S., Crawley JT., Chion CK., Lam JK., Preston RJ., Lane DA. BioCreative - Critical Assessment of Information Extraction systems in Biology HPLC and SDS-PAGE were used to follow the proteolytic reaction. Mass spectrometry or N-terminal sequencing was used to identify the polypeptide in peaks eluted using HPLC so as to establish the site of proteolysis. j.crawley@imperial.ac.uk. Accepted 2006-SEP-15 by ORCHARD. 14-DEC-2006: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation vwf_human von Willebrand factor VWF F8VWF protein protein human Homo sapiens Human MIPARFAGVLLALALILPGTLCAEGTRGRSSTARCSLFGSDFVNTFDGSMYSFAGYCSYLLAGGCQKRSFSIIGDFQNGKRVSLSVYLGEFFDIHLFVNGTVTQGDQRVSMPYASKGLYLETEAGYYKLSGEAYGFVARIDGSGNFQVLLSDRYFNKTCGLCGNFNIFAEDDFMTQEGTLTSDPYDFANSWALSSGEQWCERASPPSSSCNISSGEMQKGLWEQCQLLKSTSVFARCHPLVDPEPFVALCEKTLCECAGGLECACPALLEYARTCAQEGMVLYGWTDHSACSPVCPAGMEYRQCVSPCARTCQSLHINEMCQERCVDGCSCPEGQLLDEGLCVESTECPCVHSGKRYPPGTSLSRDCNTCICRNSQWICSNEECPGECLVTGQSHFKSFDNRYFTFSGICQYLLARDCQDHSFSIVIETVQCADDRDAVCTRSVTVRLPGLHNSLVKLKHGAGVAMDGQDVQLPLLKGDLRIQHTVTASVRLSYGEDLQMDWDGRGRLLVKLSPVYAGKTCGLCGNYNGNQGDDFLTPSGLAEPRVEDFGNAWKLHGDCQDLQKQHSDPCALNPRMTRFSEEACAVLTSPTFEACHRAVSPLPYLRNCRYDVCSCSDGRECLCGALASYAAACAGRGVRVAWREPGRCELNCPKGQVYLQCGTPCNLTCRSLSYPDEECNEACLEGCFCPPGLYMDERGDCVPKAQCPCYYDGEIFQPEDIFSDHHTMCYCEDGFMHCTMSGVPGSLLPDAVLSSPLSHRSKRSLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVALERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKKRVTILVEGGEIELFDGEVNVKRPMKDETHFEVVESGRYIILLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQNNDLTSSNLQVEEDPVDFGNSWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRILTSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTATLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKILDELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQICHCDVVNLTCEACQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHDFYCSRLLDLVFLLDGSSRLSEAEFEVLKAFVVDMMERLRISQKWVRVAVVEYHDGSHAYIGLKDRKRPSELRRIASQVKYAGSQVASTSEVLKYTLFQIFSKIDRPEASRITLLLMASQEPQRMSRNFVRYVQGLKKKKVIVIPVGIGPHANLKQIRLIEKQAPENKAFVLSSVDELEQQRDEIVSYLCDLAPEAPPPTLPPDMAQVTVGPGLLGVSTLGPKRNSMVLDVAFVLEGSDKIGEADFNRSKEFMEEVIQRMDVGQDSIHVTVLQYSYMVTVEYPFSEAQSKGDILQRVREIRYQGGNRTNTGLALRYLSDHSFLVSQGDREQAPNLVYMVTGNPASDEIKRLPGDIQVVPIGVGPNANVQELERIGWPNAPILIQDFETLPREAPDLVLQRCCSGEGLQIPTLSPAPDCSQPLDVILLLDGSSSFPASYFDEMKSFAKAFISKANIGPRLTQVSVLQYGSITTIDVPWNVVPEKAHLLSLVDVMQREGGPSQIGDALGFAVRYLTSEMHGARPGASKAVVILVTDVSVDSVDAAADAARSNRVTVFPIGIGDRYDAAQLRILAGPAGDSNVVKLQRIEDLPTMVTLGNSFLHKLCSGFVRICMDEDGNEKRPGDVWTLPDQCHTVTCQPDGQTLLKSHRVNCDRGLRPSCPNSQSPVKVEETCGCRWTCPCVCTGSSTRHIVTFDGQNFKLTGSCSYVLFQNKEQDLEVILHNGACSPGARQGCMKSIEVKHSALSVELHSDMEVTVNGRLVSVPYVGGNMEVNVYGAIMHEVRFNHLGHIFTFTPQNNEFQLQLSPKTFASKTYGLCGICDENGANDFMLRDGTVTTDWKTLVQEWTVQRPGQTCQPILEEQCLVPDSSHCQVLLLPLFAECHKVLAPATFYAICQQDSCHQEQVCEVIASYAHLCRTNGVCVDWRTPDFCAMSCPPSLVYNHCEHGCPRHCDGNVSSCGDHPSEGCFCPPDKVMLEGSCVPEEACTQCIGEDGVQHQFLEAWVPDHQPCQICTCLSGRKVNCTTQPCPTAKAPTCGLCEVARLRQNADQCCPEYECVCDPVSCDLPPVPHCERGLQPTLTNPGECRPNFTCACRKEECKRVSPPSCPPHRLPTLRKTQCCDEYECACNCVNSTVSCPLGYLASTATNDCGCTTTTCLPDKVCVHRSTIYPVGQFWEEGCDVCTCTDMEDAVMGLRVAQCSQKPCEDSCRSGFTYVLHEGECCGRCLPSACEVVTGSPRGDSQSSWKSVGSQWASPENPCLINECVRVKEEVFIQQRNVSCPQLEVPVCPSGFQLSCKTSACCPSCRCERMEACMLNGTVIGPGKTVMIDVCTTCRCMVQVGVISGFKLECRKTTCNPCPLGYKEENNTGECCGRCLPTACTIQLRGGQIMTLKRDETLQDGCDTHFCKVNERGEYFWEKRVTGCPPFDEHKCLAEGGKIMKIPGTCCDTCEEPECNDITARLQYVKVGSCKSEVEVDIHYCQGKCASKAMYSIDINDVQDQCSCCSPTRTEPMQVALHCTNGSVVYHEVLNAMECKCSPRKCSK DIP protein P04275 original sequence version: 134 DIP protein P04275 original sequence version: 127 DIP protein P04275 original sequence version: 133 D5C1C78360917C29 ats13_human A disintegrin and metalloproteinase with thrombospondin motifs 13 von Willebrand factor-cleaving protease ADAMTS13 C9orf8 UNQ6102/PRO20085 protein protein human Homo sapiens Human MHQRHPRARCPPLCVAGILACGFLLGCWGPSHFQQSCLQALEPQAVSSYLSPGAPLKGRPPSPGFQRQRQRQRRAAGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTGFDLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGLAWSPCSRRQLLSLLSAGRARCVWDPPRPQPGSAGHPPDAQPGLYYSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDPLDQSSCSRLLVPLLDGTECGVEKWCSKGRCRSLVELTPIAAVHGRWSSWGPRSPCSRSCGGGVVTRRRQCNNPRPAFGGRACVGADLQAEMCNTQACEKTQLEFMSQQCARTDGQPLRSSPGGASFYHWGAAVPHSQGDALCRHMCRAIGESFIMKRGDSFLDGTRCMPSGPREDGTLSLCVSGSCRTFGCDGRMDSQQVWDRCQVCGGDNSTCSPRKGSFTAGRAREYVTFLTVTPNLTSVYIANHRPLFTHLAVRIGGRYVVAGKMSISPNTTYPSLLEDGRVEYRVALTEDRLPRLEEIRIWGPLQEDADIQVYRRYGEEYGNLTRPDITFTYFQPKPRQAWVWAAVRGPCSVSCGAGLRWVNYSCLDQARKELVETVQCQGSQQPPAWPEACVLEPCPPYWAVGDFGPCSASCGGGLRERPVRCVEAQGSLLKTLPPARCRAGAQQPAVALETCNPQPCPARWEVSEPSSCTSAGGAGLALENETCVPGADGLEAPVTEGPGSVDEKLPAPEPCVGMSCPPGWGHLDATSAGEKAPSPWGSIRTGAQAAHVWTPAAGSCSVSCGRGLMELRFLCMDSALRVPVQEELCGLASKPGSRREVCQAVPCPARWQYKLAACSVSCGRGVVRRILYCARAHGEDDGEEILLDTQCQGLPRPEPQEACSLEPCPPRWKVMSLGPCSASCGLGTARRSVACVQLDQGQDVEVDEAACAALVRPEASVPCLIADCTYRWHVGTWMECSVSCGDGIQRRRDTCLGPQAQAPVPADFCQHLPKPVTVRGCWAGPCVGQGTPSLVPHEEAAAPGRTTATPAGASLEWSQARGLLFSPAPQPRRLLPGPQENSVQSSACGRQHLEPTGTIDMRGPGQADCAVAIGRPLGEVVTLRVLESSLNCSAGDMLLLWGRLTWRKMCRKLLDMTFSSKTNTLVVRQRCGRPGGGVLLRYGSQLAPETFYRECDMQLFGPWGEIVSPSLSPATSNAGGCRLFINVAPHARIAIHALATNMGAGTEGANASYILIRDTHSLRTTAFHGQQVLYWESESSQAEMEFSEGFLKAQASLRGQYWTLQSWVPEMQDPQSWKGKEGT DIP protein Q76LX8 original sequence version: 50 A2103AFABC1A4445 adamts13-vwf 1 3 enzyme target enzyme target substrate substrate unspecified role unspecified role region VWF76 binding region binding-associated region binding region deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range xpress epitope n-ter Xpress epitope tag tag tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecoli Escherichia coli (strain K12) 4 enzyme enzyme unspecified role unspecified role n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. protein cleavage protein cleavage kcat/Km 6.39 (plus or minus 1.18) E4 per M per s ADAMTS13 was incubated at 37 �C with either VWF76, VWF115, or VWF115-A3 in the presence of physiological concentrations of Ca2 and NaCl. Sub-samples were stopped between 0 and 6 h and analyzed by SDS-PAGE and Coomassie staining to give a qualitative assessment of each reaction (Fig. 2, A�C). In each case, only the full-length substrate band was visible at 0 h. Thereafter, the intensity of this band diminished and two smaller cleavage product bands appeared. In each case, these bands corresponded to the predicted molecular masses of fragments generated following cleavage at the Tyr1605�Met1606 bond. N-terminal sequencing was performed upon the smallest cleavage product and confirmed that proteolysis had occurred between the Tyr1605�Met1606 bond. Visually, VWF76, VWF115, and VWF115-A3 appeared to be proteolyzed by ADAMTS13 at similar rates. Parallel samples were also analyzed by HPLC. The resolution of the full-length substrate from the two cleavage fragments (peaks 1 and 2) arising from proteolysis (Fig. 2) of each different substrate allowed precise quantification of the extent of cleavage at each time point All of the residue positions given map onto the Uniprot entry P04275 for VWF except Glu 1874 which the Uniprot entry states is a Gly. 2A, 4A, 4C, Table 1 Cleavage product fractions were collected and analyzed by mass spectrometry. For all substrates, peak 1 corresponded to the tagged N-terminal cleavage product up to Tyr1605 and peak 2 to the C-terminal fragment, in each case starting at Met1606. This confirmed that the Tyr1605-Met1606 was cleaved. adamts13-vwf-8 1 4 enzyme enzyme unspecified role unspecified role n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. 3 enzyme target enzyme target substrate substrate unspecified role unspecified role n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range xpress epitope n-ter Xpress epitope tag tag tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range P04275:p.Arg1583Ala alters kinetics of interaction mutation mutation alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain R A Wed May 02 17:06:58 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'arg1583ala' Tue Jun 30 20:04:40 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Arg1583Ala' P04275:p.[Asp1614Ala;Glu1615Ala;Lys1617Ala] asp1614ala, glu1615ala, lys1617ala alters kinetics of interaction mutation mutation alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain E A certain certain sequence position certain certain certain sequence position certain K A certain certain sequence position certain certain certain sequence position certain D A Fri Jul 22 11:12:16 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'glu1615ala,lys1617ala,asp1614ala' Wed May 02 15:05:55 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'asp1614ala,glu1615ala,lys1617ala' Tue Jun 30 18:11:45 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Asp1614Ala;Glu1615Ala;Lys1617Ala]' P04275:p.Asp1587Ala alters kinetics of interaction mutation mutation alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain D A Wed May 02 17:16:51 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'asp1587ala' Tue Jun 30 18:41:01 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asp1587Ala' P04275:p.Asp1614Ala alters kinetics of interaction mutation mutation alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain D A Wed May 02 15:45:17 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'asp1614ala' Tue Jun 30 18:42:19 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asp1614Ala' P04275:p.Glu1615Ala alters kinetics of interaction mutation mutation alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain E A Wed May 02 14:42:36 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'glu1615ala' Tue Jun 30 17:09:57 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Glu1615Ala' P04275:p.Lys1617Ala alters kinetics of interaction mutation mutation alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain K A Wed May 02 16:20:53 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys1617ala' Tue Jun 30 16:19:07 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys1617Ala' P04275:p.Glu1638Ala alters kinetics of interaction mutation mutation alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain E A Wed May 02 17:15:08 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'glu1638ala' Tue Jun 30 20:01:11 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Glu1638Ala' P04275:p.Glu1640Ala alters kinetics of interaction mutation mutation alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain E A Wed May 02 17:35:00 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'glu1640ala' Tue Jun 30 15:36:04 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Glu1640Ala' region VWF115 sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) protein cleavage protein cleavage The scissile bond in VWF is predicted from a homology model to be located in the center of the folded A2 domain. For the ADAMTS13-dependent proteolysis of VWF115 (n equals 4), they derived a kcat of 0.14 plus or minus 0.06 per s. The Km for this reaction was 1.61 plus or minus 4.7 E-7 M and the specificity constant (catalytic efficiency, kcat/Km) was 8.70 E4 per M per s. From the gels in Fig. 4A, it seemed as if all of the mutants exhibited reduced rates of proteolysis. All of the residue positions given map onto the Uniprot entry P04275 for VWF except Glu 1874 which the Uniprot entry states is a Gly. 2B, 3D, 4, Table 1 For separate determination of the catalytic efficiency of VWF115, repeated time course experiments were performed using low substrate concentrations (250 �500 nM). Repeated time course experiments (n 8) gave a mean value of 7.83 104 M 1 s 1 for kcat/Km (Table 1). Confirmation of the pseudo-first order conditions was obtained by deriving the same kcat/Km value for VWF115 proteolysis at both 250 and 500 nM. Determination of kcat/Km for VWF76 and VWF115-A3 gave values of 6.39 104 M 1 s 1 and 6.70 104 M 1 s 1, respectively (Table 1). These kinetic data confirmed that the VWFA3 domain does not significantly contribute to the rate of A2 domain proteolysis, despite containing an ADAMTS13 binding site (18). The contribution of each selected charged amino acid to VWF proteolysis was assessed by determination of the catalytic efficiency of all the mutant VWF115 substrates at 500 nM(Fig. 4Cand Table 1). The rate of proteolysis of the R1583A mutant was not significantly different from that of VWF115, suggesting that this residue is not important for cleavage. Mutation of Glu1638 and Glu1640 induced a mild (2�3-fold) reduction in catalytic efficiency. The most affected ( 12-fold reduction) single mutant was D1587A, the kcat/Km of which was 0.64 0.10 104M 1 s 1. The kcat/Km for K1617A, D1614A, and E1615A, which are predicted to lie on the same surface-exposed loop, were 1.90, 1.36, and 1.13 104 M 1 s 1, respectively. These results implied that this loop might play an important role in the cleavage reaction. Consequently, we generated a triple mutant DEK1614/5/7AAA) containing all 3 residues substituted for alanine. Whereas individually these mutations caused a 4�7-fold reduction in kcat/Km, in combination the catalytic efficiency was 13- fold less than wild-type VWF115, implying that the amino acids in this charged loop may act in synergy The kcat_Km (E4 per M per s) are as follows: VWF115 7.83 plus or minus 1.18, VWF115-A3 6.70 plus or minus 2.78, VWF76 6.39 plus or minus 1.18, for VWF115 variants: arg1583ala 6.19, asp1640ala, 2.74, glu1638ala 2.74, lys1617ala 1.90, asp1614ala 1.36 plus or minus 0.26, glu1615ala 1.13 plus or minus 0.30, asp1587ala 0.64 plus or minus 0.10, asp1614ala_glu1615ala_lys1617ala 0.59. Mutation of Glu1638 and Glu1640 induced a mild (2�3-fold) reduction in catalytic efficiency. The most affected (over 12-fold reduction) single mutant was D1587A, the kcat/Km of which was 0.64 plus or minus 0.10x 10E4 per M per s. The kcat/Km for K1617A, D1614A, and E1615A, which are predicted to lie on the same surface-exposed loop, were 1.90, 1.36, and 1.13 E4 per M per s, respectively. The triple mutant DEK1614/5/7AAA containing all 3 residues substituted for alanine was generated. Whereas individually these mutations caused a 4�7-fold reduction in kcat/Km, in combination the catalytic efficiency was over 13-fold less than wild-type VWF115, implying that the amino acids in this charged loop may act in synergy. adamts13-vwf-9 1 4 enzyme enzyme unspecified role unspecified role n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. 3 enzyme target enzyme target substrate substrate unspecified role unspecified role n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range xpress epitope n-ter Xpress epitope tag tag tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range region VWFA2 sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain P04275:p.Asp1614Ala alters kinetics of interaction mutation mutation alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain D A Wed May 02 17:42:20 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'asp1614ala' Tue Jun 30 19:58:42 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asp1614Ala' P04275:p.Glu1615Ala alters kinetics of interaction mutation mutation alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain E A Wed May 02 17:31:44 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'glu1615ala' Tue Jun 30 18:48:08 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Glu1615Ala' P04275:p.Asp1587Ala alters kinetics of interaction mutation mutation alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain D A Wed May 02 15:51:34 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'asp1587ala' Tue Jun 30 15:59:18 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asp1587Ala' ecoli Escherichia coli (strain K12) protein cleavage protein cleavage 5 this approach also served to ascertain whether the most affected charged residue mutants (D1587A, D1614A, E1615A) induced gross structural changes. Despite using increased concentrations of ADAMTS13 (16.5 nM) in the absence of a denaturant, the intact wildtype A2 domain was proteolyzed very slowly, as visualized by gel staining (Fig. 5A), when compared with the A2 domain fragments VWF115, VWF76, and VWF115-A3. When samples containing 500 nM VWFA2 were analyzed by HPLC (Fig. 5C), it was clear that the cleavage reaction did not proceed toward completion ( 28% after 5 h), as previously reported (13). The D1587A, D1614A, and E1615A VWFA2 mutants were proteolyzed either at a similar or slower rate (Fig. 5, A and C), suggesting that these mutations do not alter the A2 domain structure markedly. Under denaturing conditions (1 M urea), the wild-type VWFA2 was proteolyzed faster, although it still did not reach completion ( 50% after 5 h) (Fig. 5, B and D). Although all the mutants were cleaved at a higher rate under denaturing conditions, they were cleaved appreciably slower than VWFA2, corroborating the direct influence of these mutations upon VWF115 proteolysis. When samples containing 500 nM VWFA2 were analyzed by HPLC (Fig. 5C), it was clear that the cleavage reaction did not proceed toward completion (approx. 28% after 5 h). The D1587A, D1614A, and E1615A VWFA2 mutants were proteolyzed either at a similar or slower rate (Fig. 5, A and C), suggesting that these mutations do not alter the A2 domain structure markedly. Under denaturing conditions (1 M urea), the wild-type VWFA2 was proteolyzed faster, although it still did not reach completion (approx. 50% after 5 h) (Fig. 5, B and D). Although all the mutants were cleaved at a higher rate under denaturing conditions, they were cleaved appreciably slower than VWFA2, corroborating the direct influence of these mutations upon VWF115 proteolysis. All of the residue positions given map onto the Uniprot entry P04275 for VWF except Glu 1874 which the Uniprot entry states is a Gly. adamts13-vwf-7 2 3 unspecified role unspecified role bait bait xpress epitope n-ter Xpress epitope tag tag tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range region VWF115 contains part of the A2 domain binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range P04275:p.Asp1587Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain D A Wed May 02 15:09:45 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'asp1587ala' Tue Jun 30 16:54:12 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asp1587Ala' P04275:p.Asp1614Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain D A Wed May 02 20:15:55 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'asp1614ala' Tue Jun 30 19:28:07 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asp1614Ala' P04275:p.Glu1615Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain E A Wed May 02 15:50:27 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'glu1615ala' Tue Jun 30 16:09:48 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Glu1615Ala' P04275:p.[Asp1614Ala;Glu1615Ala;Lys1617Ala] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain D A certain certain sequence position certain certain certain sequence position certain E A certain certain sequence position certain certain certain sequence position certain K A Wed May 02 14:52:03 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'asp1614ala,glu1615ala,lys1617ala' Tue Jun 30 18:05:30 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Asp1614Ala;Glu1615Ala;Lys1617Ala]' ecoli Escherichia coli (strain K12) 4 unspecified role unspecified role prey prey n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. direct interaction direct interaction All of the residue positions given map onto the Uniprot entry P04275 for VWF except Glu 1874 which the Uniprot entry states is a Gly. 6 we performed SPR analysis. Monoclonal antibody-mediated orientation-specific coupling of either VWF115 or ADAMTS13 to the sensor chip was not possible due to the relatively fast off-rates of these molecules with the available antibodies to each ligand (not shown). Consequently, we covalently bound either VWF115 or ADAMTS13 to the sensor chip by amine coupling. With VWF115 bound to the chip and ADAMTS13 injected over the sensor, we monitored a concentration-dependent binding response indicative of a high affinity interaction (KD, 20 nM) for this interaction (Fig. 6, inset). Determination of both the association and dissociation rates for the ADAMTS13-VWF115 interaction (n 3) demonstrated a slow off-rate (1.72 10 3 s 1), which would appear to be the major determinant for the high affinity binding. A second approach, in which ADAMTS13 was coupled to the sensor chip and varying concentrations of VWF115 injected, gave a similar high affinity binding component (KD 15 nM, not shown). These data demonstrate that the affinity of ADAMTS13 for VWF115 is significantly higher than is suggested by the Km for VWF115 proteolysis. SPR analysis of VWF115 mutants (D1587A, D1614A, E1615A, and DEK1614/5/7AAA) using both binding approaches revealed a markedly reduced affinity of ADAMTS13 for each of the mutants when compared with wild-type VWF115 (Fig. 6). Because of the low binding response measured, an accurate KD could not be derived from the VWF115 mutant data. Despite the poor fitting the estimate for the KD for the DEK1614/5/7AAA mutant was 1.3 M. It was clear that for each variant the binding with ADAMTS13 was appreciably compromised and thus the likely cause of their reduced specificity constants. The KD was 2.0 E-8 M for VWF115. The mutants has a reduced affinity. The estimate for the KD for the triple mutant was approx. 1.3 E-6 M but due to low binding for mutants KD values could not be derived. The results for the experiment using ADAMTS13 as the bait were not shown but the authors state that the results were similar. adamts13-vwf-1 1 3 enzyme target enzyme target substrate substrate unspecified role unspecified role n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range xpress epitope n-ter Xpress epitope tag tag tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range region VWF115-A3 sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 4 enzyme enzyme unspecified role unspecified role n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. protein cleavage protein cleavage kcat/Km 6.70 (plus or minus 2.78) E4 per M per s ADAMTS13 was incubated at 37 �C with either VWF76, VWF115, or VWF115-A3 in the presence of physiological concentrations of Ca2 and NaCl. Sub-samples were stopped between 0 and 6 h and analyzed by SDS-PAGE and Coomassie staining to give a qualitative assessment of each reaction (Fig. 2, A�C). In each case, only the full-length substrate band was visible at 0 h. Thereafter, the intensity of this band diminished and two smaller cleavage product bands appeared. In each case, these bands corresponded to the predicted molecular masses of fragments generated following cleavage at the Tyr1605�Met1606 bond. N-terminal sequencing was performed upon the smallest cleavage product and confirmed that proteolysis had occurred between the Tyr1605�Met1606 bond. Visually, VWF76, VWF115, and VWF115-A3 appeared to be proteolyzed by ADAMTS13 at similar rates. Parallel samples were also analyzed by HPLC. The resolution of the full-length substrate from the two cleavage fragments (peaks 1 and 2) arising from proteolysis (Fig. 2) of each different substrate allowed precise quantification of the extent of cleavage at each time point All of the residue positions given map onto the Uniprot entry P04275 for VWF except Glu 1874 which the Uniprot entry states is a Gly. 2 C, 4A and C, Table 1 Cleavage product fractions were collected and analyzed by mass spectrometry. For all substrates, peak 1 corresponded to the tagged N-terminal cleavage product up to Tyr1605 and peak 2 to the C-terminal fragment, in each case starting at Met1606. This confirmed that the Tyr1605-Met1606 was cleaved.