Shen et al 2006 Supplement - Paper - Lecture 4

SUPPLEMENTARY INFORMATION for Shen et al., Nature Manuscript #2006-03-02695B Supplemental figure 1 E. coli pitrilysin Human IDE Chimpanzee, Pan troglodytes Mouse, Mus musculus Rat, Rattus norvegicus Honey bee, Apis mellifera Fruit fly, Drosophila melanogaster Mosquito, Anopheles gambiae Nematode, Caenorhabditis briggsae Nematode, Caenorhabditis elegans Cress, Arabidopsis thaliana Tomato, Lycopersicon esculentum Rice, Oryza sativa Fungi, Gibberella zeae Fungi, Neurospora crassa Yeast, Candida glabrata Yeast, Saccharomyces cerevisiae Yeast, Kluyveromyces lactis Yeast, Candida albicans Yeast, Yarrowia lipolytica Fungi, Ustilago maydis fungi, Crystococcus neoformans Yeast, Schizosaccharomyces pombe 904 aa 38%/ 24% AAB40468 1019 aa 100%/100% NP004960 1044 aa 97%/ 97% XP507922 1019 aa 96%/ 95% AAH41675 1019 aa 96%/ 95% NP037291 904 aa 57%/ 44% XP396981 990 aa 59%/ 44% P22817 984 aa 58%/ 44% EAA07246 934 aa 54%/ 39% CAE64571 1051 aa 53%/ 39% AF016421 970 aa 50%/ 36% NP181710 971 aa 50%/ 46% CAC67408 988 aa 50%/ 36% XP478770 1023 aa 49%/ 36% EAA76500 1082 aa 48%/ 35% XP956166 1008 aa 49%/ 35% CAG60009 1027 aa 49%/ 35% AAB82351 1004 aa 50%/ 36% XP454175 1107 aa 44%/ 32% XP719241 1027 aa 52%/ 38% XP505854 1292 aa 42%/ 31% XP759404 1162 aa 45%/ 31% EAL18851 969 aa 48%/ 34% CAA20142 Length Similarity/identity Sequence ID Phylogenic tree of insulin degrading enzyme. The amino acid length and protein sequence ID of IDE homologues from mammals, insects, round worms, plants, and fungi are indicated. The sequence similarity and identity of IDE is compared to human IDE. HSQGTFTSDYSKYLDSRRAQDFVQWLMNT HADGTFTSDVSSYLKDQAIKDFVDRLKAGQVRRE Glucagon GLP SLRRSSCFGGRIDRIGAQSGLGCNSFRY DSGCFGRRLDRIGSLSGLGCNVLRRY GLSKGCFGLKLDRIGSMSGLGC ANP BNP CNP FVNQHLCGSHLVEALYLVCGERGFFYTPKT GIVEQCCTSICSLYQLENYCNInsulin IGF-II IGF-I Proinsulin binding degradation IC 50 =~10 nM 1 rapid 2 IC 50 > 1 µM 1 slow 2 n/a slow 2 AYRPSETLCGGELVDTLQFVCGDRGFYFSRPA--SRVSRRSR--GIVEECCFRSCDLALLETYCAT--PAKSE GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA FVNQHLCGSHLVEALYLVCGERGFFYTPKT GIVEQCCTSICSLYQLENYCN RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR IC 50 =5.3 µM 3 rapid 3 none 4 n/a Km=85 nM 5 rapid 5 Supplemental figure 2 Sequence alignment of IDE interacting peptides. ANP, Atrial natriuretic peptide; BNP, Brain natriuretic peptide; CNP, C-type natriuretic peptide; GLP, glucagon-like peptide; IGF, insulin-like growth factor. Glucagon, GLP, insulin, IGF-I, IGF-II and proinsulin are from human. ANP is from rat, and BNP and CNP are from pig. The degradation of Insulin and proinsulin were measured using human IDE while ANP, BNP, CNP, glucagon, GLP, IGF-II and IGF-I were performed using rat IDE. Postively charged residues at the C-terminus of peptides that are not preferred by IDE are colored red. n/a means not available. 1 Muller, D., et al., Atrial natriuretic peptide (ANP) is a high-affinity substrate for rat insulin-degrading enzyme. Eur. J. Biochem., 202:285-292, 1991. 2 Muller, D., et al., Rat Insulin-degrading enzyme: cleavage pattern of the natriuretic peptide hormones ANP, BNP and CNP revealed by HPLC and mass spectrometry. Biochemistry, 31:11138-11143, 1992. 3 Duckworth, W. C. & Kitabchi, A. E., Insulin and glucagon degradation by the same enzyme. Diabetes, 23:536-543, 1974. 4 Authier, F., et al., Degradation of the cleaved leader peptide of thiolase by a peroxisomal proteinase. Proc. Nat. Acad. Sci. USA, 92:3859-3863, 1995. 5 Chesneau V. & Rosner, M. R., Functional human insulin-degrading enzyme can be expressed in bacteria. Protein Expr. Purif., 19:91-98, 2000. 6 Misbin, R. I., et al., Inhibition of insulin degradation by insulin-like growth factors. Endocrinology, 113:1525-1527, 1983. 7 Kitabchi, A. E., et al., Direct measurement of proinsulin in human plasma by the use of an insulin-degrading enzyme. J. Clin. Invest., 50: 1792-1799, 1971. IC 50 = 50 nM 6 rapid 6 IC 50 > 150 nM 6 slow 6 n/a slow 7 N C ?1 ?2 ?3 ?4 ?1 ?5 ?2 ?6?7 ?3 ?8 ?4 ?5 ?6?7 ?8 ?9 ?10 ?11 ?10 ?11 ?12?13 ?12 ?13 ?14 ?15 ?16 ?17 ?14 ?15 ?16 ?18 ?19 ?17 ?18 ?19 ?20 ?21 ?20?21 ?22 ?23 ?24 ?25 ?26 ?22 ?27 ?23 ?24 ?25 ?28 ?29 ?26 ?30 ?31 ?32 ?33 ?34 ?28 ?35 ?29 ?36 ?30 ?9 ?27 Supplemental figure 3 Domain analysis of human insulin-degrading enzyme (IDE). (A) Secondary structure representation of IDE. The four IDE domains are colored green, blue, yellow and red. The location of the zinc metal binding, catalytic site of IDE is marked by a red star. (B) Pairwise sequence comparison of the four IDE domains. Similarity(%)/ Identity(%) Domain 1 Domain 2 Domain 3 Domain 2 21/11 Domain 3 21/12 24/12 Domain 4 20/7 24/15 15/7 B A Domain 1 Domain 2 Domain 3 Domain 4 Sequence alignment of IDE domain 1 (A), domain 2 (B), domain 3 (C), and domain 4 (D). Residues below asterisks indicate the conserved residues for zinc binding and catalysis. Residues above "S" are those that are involved in substrate binding. Residues above "I" are those that are located at the interface between IDE-N and IDE-C and are involved in the interaction between these two domains (see the list in Supplemental figure 4E). Residues in red with yellow shade are residues that are identical within six members of IDE. Residues in blue are mostly identical among IDE family. Residue in green shade are homologous residues among IDE family. (E) List of atoms from IDE-N and IDE-C that are in close contact and the distance measurement. Supplemental figure 4A ------------------MRYRLAWLLHPALPSTFRSVLGARLPPPERLCGFQKKHuman -----------------------MTIAESSQKSATRKPDSMEP------------Fruitfly (1) ------------------------MILSTVFGRSIRRVSTLSIR-----------Zebrafish_1 (1) --------------------------------MAVEKSNTTVG------------Cress (1) MGVSLLASSSAFVTKPLLTQLVHLSPISLNFTVRRFKPFTCLSRYYTTN------Yeast_Ste23P (1) MSLSLLSKFSRGVPSPALAHIAKRTFSSVNFSQILRRPQNSSLRLRLVRNISNSNNematode (1) -TYSKMNNPAIKRIGNHITKSPEDKREYRGLELANGIKVLL SDPTTDKSSAAHuman (38) -------------ILRLNNIEKSLQDTRDYRGLQLENGLKVLL SDPNTDVSAAAFruitfly (21) -------MSDPAVKRVVSDIIRSPEDKREYRGLEFTNGLKAILISDPTTDKSSAAZebrafish_1 (21) ----------------GVEILKPRTDNREYRMIVLKNLLQVLLISDPDTDKCAASCress (12) -----PYNMTSNFKTFNLDFLKPDLDERSYRFIELPNKLKALLIQDPKADKAAASYeast_Ste23P (50) PLPKMTEAGKNIVLKRHDLIVKGAQDAREYRGLELTNGIRVLLVSDPTTDKSAAANematode (56) LDVHIGSLSDPPNIAGLSHFCEHMLFLGTKKYPKENEYSQFLSEHAGSSNAFTSGHuman (90) LSVQVGHMSDPTNLPGLAHFCEHMLFLGTEKYPHENGYTTYLSQSGGSSNAATYPFruitfly (63) LDVHMGSLSDPENISGLAHFCEHMLFLGTEKYPKENEYSQFLSEHAGSSNAFTSGZebrafish_1 (69) MSVSVGSFSDPQGLEGLAHFLEHMLFYASEKYPEEDSYSKYITEHGGSTNAYTASCress (51) LDVNIGAFEDPKNLPGLAHFCEHLLFMGSEKFPDENEYSSYLSKHGGSSNAYTASYeast_Ste23P (100) LDVKVGHLMDPWELPGLAHFCEHMLFLGTAKYPSENEYSKFLAAHAGSSNAYTSSNematode (111) EHTNYYFDVSHEHLEGALDRFAQFFLCPLFDESCKDREVNAVDSEHEKNVMNDAWHuman (145) LMTKYHFHVAPDKLDGALDRFAQFFIAPLFTPSATEREINAVNSEHEKNLPSDLWFruitfly (118) EHTNYYFDVSHEHLQGALDRFAQFFLCPLFDESCKDREVNAVDSEHEKNLMNDAWZebrafish_1 (124) EETNYHFDVNADCFDEALDRFAQFFIKPLMSADATMREIKAVDSENQKNLLSDGWCress (106) QNTNYFFEVNHQHLFGALDRFSGFFSCPLFNKDSTDKEINAVNSENKKNLQNDIWYeast_Ste23P (155) DHTNYHFDVKPDQLPGALDRFVQFFLSPQFTESATEREVCAVDSEHSNNLNNDLWNematode (166) RLFQLEKATGNPKHPFSKFGTGNKYTLETRPNQEGIDVRQELLKFHSAYYSSNLMHuman (200) RIKQVNRHLAKPDHAYSKFGSGNKTTLSEIPKSKNIDVRDELLKFHKQWYSANIMFruitfly (173) RLFQLEKATGNPKHPFSKFGTGNKLTLETRPSQQGIDIREELLKFHSTYYSSNLMZebrafish_1 (179) RIRQLQKHLSKEDHPYHKFSTGNMDTLHVRPQAKGVDTRSELIKFYEEHYSANIMCress (161) RIYQLDKSLTNTKHPYHKFSTGNIETLGTLPKENGLNVRDELLKFHKNFYSANLMYeast_Ste23P (210) RFLQVDRSRSKPGHDYGKFGTGNKQTLLEDARKKGIEPRDALLQFHKKWYSSDIMNematode (221) AVCVLGRESLDDLTNLVVKLFSEVE-NKNVPHuman (255) CLAVIGKESLDELEGMVLEKFSEIE-NKNVKFruitfly (228) GLCVLGRETLDELTSMVVKLFGEVE-NKNVPZebrafish_1 (234) HLVVYGKESLDKIQDLVERMFQEIQ-NTNKVCress (216) KLCILGREDLDTLSDWTYDLFKDVA-NNGREYeast_Ste23P (265) TCCIVGKEPLNVLESYLGTLEFDAIENKKVENematode (276) ?1 ?2 ?3 ?4 ?5 ?6 ?7 ?8 ?9 ?1 ?2 ?3 ?4 ?5 ?6 ?7 ?8 *** * ssss sss sss ss II III I I I I I LPEFPEHPFQEEHLKQLYKIVPIK Human (285) VPGWPRHPYAEERYGQKVKIVPIK Fruitfly (258) VPEFPTHPFQEEHLRQFYKVVPIK Zebrafish_1 (264) VPRFPGQPCTADHLQILVKAIPIK Cress (246) VPLYAEPIMQPEHLQKIIQVRPVK Yeast_Ste23P (295) RKVWEEFPYGPDQLAKRIDVVPIK Nematode (307) DIRNLYVTFPIPDLQKYYKSNPGHYLGHLIGHEGPGSLLSELKSKGWVNTLVGGQHuman (309) DIRSLTISFTTDDLTQFYKSGPDNYLTHLIGHEGKGSILSELRRLGWCNDLMAGHFruitfly (282) DIRNLYVTFPIPDLQKYYKSNPGHYLGHLIGHEGPGSLLSELKSKGWVNTLVGGQZebrafish_1 (288) QGHKLGVSWPVTPSIHHYDEAPSQYLGHLIGHEGEGSLFHALKTLGWATGLSAGECress (270) DLKKLEISFTVPDMEEHWESKPPRILSHLIGHEGSGSLLAHLKKLGWANELSAGGYeast_Ste23P (319) DTRLVSISFPFPDLNGEFLSQPGHYISHLIGHEGPGSLLSELKRLGWVSSLQSDSNematode (331) KEGARGFMFFIINVDLTEEGLLHVEDIILHMFQYIQKLRAEGPQEWVFQECKDLNHuman (364) QNTQNGFGFFDIVVDLTQEGLEHVDDIVKIVFQYLEMLRKEGPKKWIFDECVKLNFruitfly (337) KEGARGFMFFIINVDLTEEGLLHVEDIIFHMFQYIQKLRTEGPQEWVFQECKDLNZebrafish_1 (343) GEWTLDYSFFKVSIDLTDAGHEHMQEILGLLFNYIQLLQQTGVCQWIFDELSAICCress (325) HTVSKGNAFFAVDIDLTDNGLTHYRDVIVLIFQYIEMLKNSLPQKWIFNELQDISYeast_Ste23P (374) HTQAAGFGVYNVTMDLSTEGLEHVDEIIQLMFNYIGMLQSAGPKQWVHDELAELSNematode (386) AVAFRFKDKERPR-GYTSKIAGILHYYPLEEVLTAEYLLEEFRPDLIEMVLDKLRHuman (419) EMRFRFKEKEQPE-NLVTHAVSSMQIFPLEEVLIAPYLSNEWRPDLIKGLLDELVFruitfly (392) TVAFRFKDKERPR-GYTSKVAGLLHYYPLEEILAAEYLLEEFRPDLIEMVLDKLRZebrafish_1 (398) ETKFHYQDKIPPM-SYIVDIASNMQIYPTKDWLVGSSLPTKFNPAIVQKVVDELSCress (380) NATFKFKQAGSPSSTVSSLAKCLEKDYIPVSRILAMGLLTKYEPDLLTQYTDALVYeast_Ste23P (429) AVKFRFKDKEQPM-TMAINVAASLQYIPFEHILSSRYLLTKYEPERIKELLSMLSNematode (441) PENVRVAIVSKSFEG-KTDRTEEWYGTQYKQEAIPDEVIKKWQNADLNGKFKLPTHuman (473) PSKSRIVIVSQSFEP-DCDLAEPYYKTKYGITRVAKDTVQSWENCELNENLKLALFruitfly (446) PENVRVAVVSKSFEG-QTDRTEEWYGTQYKQEAITDEAIKKWDNADLNGKFKLPMZebrafish_1 (452) PSNFRIFWESQKFEG-QTDKAEPWYNTAYSLEKITSSTIQEWVQSAPDVHLHLPACress (434) PENSRVTLISRSLET---DSAEKWYGTAYKVVDYPADLIKNMKSPGLNPALTLPRYeast_Ste23P (484) PANMQVRVVSQKFKGQEGNTNEPVYGTEMKVTDISPETMKKYENALKTSHHALHLNematode (495) Supplemental figure 4B ?9 ?10 ?11 ?12 ?13 ?14 ?15 ?16 ?17 ?18 ?10 ?11 ?12 ?13 ?14 ?15 ?16 ss sss I II I II I II IDE domain 2 alignment. Residues above "S" are those that are involved in substrate binding. Residues above "I" are those that are located at the interface between IDE-N and IDE-C and are involved in the interaction between these two domains. Residues in red with yellow shade are residues that are identical within six members of IDE. Residues in blue are mostly identical residues and those in green shade are homologous residues. IDE domain 3 alignment. Residues above "S" are those that are involved in substrate binding. Residues above "I" are those that are located at the interface between IDE-N and IDE-C and are involved in the interaction between these two domains. Residues in red with yellow shade are residues that are identical within six members of IDE. Residues in blue are mostly identical residues and those in green shade are homologous Supplemental figure 4C K--NEFIPTNFEILPLE-KEATPYPALIKDTAMSKLWFKQDDK F PKACLNFEFHuman (527) P--NSFIPTNFDISDVP-ADAPKHPTIILDTPILRVWHKQDNQFNKPKACMTFDMFruitfly (500) K--NEFIPTNFEIYPLE-KDSPSAPTLIKDTAMSKVWFKQDDKFFLPKACLNFEFZebrafish_1 (506) P--NVFIPTDLSLKDAD--DKETVPVLLRKTPFSRLWYKPDTMFSKPKAYVKMDFCress (488) P--NEFVSTNFKVDKIDGIKPLDEPVLLLSDDVSKLWYKKDDRFWQPRGYIYLSFYeast_Ste23P (536) PEKNEYIATNFDQKPRE-SVKNEHPRLISDDGWSRVWFKQDDEYNMPKQETKLALNematode (550) FSPFAYVDPLHCNMAYLYLELLKDSLNEYAYAAELAGLSYDLQNTIYGMYLSVKGHuman (579) SNPIAYLDPLNCNLNHMMVMLLKDQLNEYLYDAELASLKLSVMGKSCGIDFTIRGFruitfly (552) FSPFAYVDPLHCNMAYLYLELLKDSLNEYAYAAELAGLSYDLQNTVYGMYLSVKGZebrafish_1 (558) NCPLAVSSPDAAVLTDIFTRLLMDYLNEYAYYAQVAGLYYGVSLSDNGFELTLLGCress (539) KLPHTHASIINSMLSTLYTQLANDALKDVQYDAACADLRISFNKTNQGLAITASGYeast_Ste23P (589) TTPMVAQNPRMSLLSSLWLWCLSDTLAEETYNADLAGLKCQLESSPFGVQMRVYGNematode (604) YNDKQPILLKKIIEKMATFEIDEKRFEIIKEAYMRSLNNFRAEQPHQHAMYYLRLHuman (634) FSDKQVVLLEKLLDHLFDFSIDEKRFDILKEEYVRSLKNFKAEQPYQHSIYYLALFruitfly (607) YNDKQHILLKKIIEKMATFEIDEKRFDIIKEAYMRSLNNFRAEQPHQHAMYYLRLZebrafish_1 (613) YNHKLRILLETVVGKIANFEVKPDRFAVIKETVTKEYQNYKFRQPYHQAMYYCSLCress (594) FNEKLIILLTRFLQGVNSFEPKKDRFEILKDKTIRHLKNLLYEVPYSQMSNYYNAYeast_Ste23P (644) YDEKQALFAKHLANRMTNFKIDKTRFDVLFESLKRALTNHAFSQPYLLTQHYNQLNematode (659) LMTEVAWTKDELKEALDDVTLPRLKAFIPQLLSRLHIEALLHGNITKQAALGIMQHuman (689) LLTENAWANMELLDAMELVTYDRVLNFAKEFFQRLHTECFIFGNVTKQQATDIAGFruitfly (662) LMTEVAWTKDELRDALDDVTLPRLKAFIPQLLSRLHIEALLHGNITKQSALEMMQZebrafish_1 (668) ILQDQTWPWTEELDVLSHLEAEDVAKFVPMLLSRTFIECYIAGNVENNEAESMVKCress (649) IINERSWSTAEKLQVFEKLTFEQLINFIPTIYEGVYFETLIHGNIKHEEALEVDSYeast_Ste23P (699) LIVDKVWSKEQLLAVCDSVTLEDVQGFAKEMLQAFHMELFVHGNSTEKEAIQLSKNematode (714) ?19 ?17 ?20 ?21 ?22 ?23 ?24 ?25 ?26 ?27 ?18 ?19 ?20 ?21 ?22 ?23 s III I IIII I IIII F L MVEDTLIEH--AHTKPLLPSQLVRYREHuman (744) RVNTRLEATN-ASKLPILARQMLKKREFruitfly (717) MLEDTLIEH--AHTKPLLPSQLIRYREZebrafish_1 (723) HIEDVLFNDPKPICRPLFPSQHLTNRVCress (704) LIKSLIPNN-----IHNLQVSNNRLRSYeast_Ste23P (754) ELMDVLKSAA-PNSRPLYRNEHNPRRENematode (769) Supplemental figure 4D IDE domain 4 alignment. Residues above "S" are those that are involved in substrate binding. Residues above "I" are those that are located at the interface between IDE-N and IDE-C and are involved in the interaction between these two domains. Residues in red with yellow shade are residues that are identical within six members of IDE. Residues in blue are mostly identical residues and those in green shade are homologous residues. YQTDMQSTSENMFLELFCQIISEPCFNTLRTKEQLGYIVFSGPRRANGIQ LRFIHuman (795) LQCGAQTDHTNIMVNLVSQVLSEPCYDCLRTKEQLGYIVFSGVRKVNGAN IRIIFruitfly (769) YQTDMQNTHENMLLELFCQIISEPCFNTLRTKEQLGYIVFSGPRRANGVQGLRFIZebrafish_1 (774) IQVHRDDFSMNIKLQLFGLVAKQATFHQLRTVEQLGYITALAQRNDSGIYGVQFICress (759) TQLDVYSEDLSALSGLFAQLIHEPCFDTLRTKEQLGYVVFSSSLNNHGTANIRILYeast_Ste23P (804) YQIGVQNTYDNAVVGLIDQLIREPAFNTLRTNEALGYIVWTGSRLNCGTVALNVINematode (821) IQSEKP-PHYLESRVEAFLITMEKSIEDMTEEAFQKHIQALAIRRLDKPKKLSAEHuman (850) VQSAKH-PSYVEDRIENFLQTYLQVIEDMPLDEFERHKEALAVKKLEKPKTIFQQFruitfly (824) IQSEKA-PHYLESRVEAFLKTMEKSVEEMGDEAFQKHIQALAIRRLDKPKKLAAEZebrafish_1 (829) IQSSVKGPGHIDSRVESLLKNFESKLYEMSNEDFKSNVTALIDMKLEKHKNLKEECress (814) IQSEHT-TPYLEWRINNFYETFGQVLRDMPEEDFEKHKEALCNSLLQKFKNMAEEYeast_Ste23P (859) VQGPKS-VDHVLERIEVFLESVRKEIAEMPQEEFDNQVSGMIARLEEKPKTLSSRNematode (876) CAKYWGEIISQQYNFDRDNTEVAYLKTLTKEDIIKFYKEMLAVDAPRRHKVSVHVHuman (904) FSQFYGEIAMQTYHFEREEAEVAILRKISKADFVDYFKKFIAKDGEERRVLSVHIFruitfly (878) CAKYWGEIISQQYNFDRDNIEVAYLKTLTKEHIMQFYRDLLAIDAPRRHKVSVHVZebrafish_1 (883) SRFYWREIQSGTLKFNRKEAEVSALKQLQKQELIDFFDEYIKVGAARKKSLSIRVCress (869) SARYTAAIYLGDYNFTHRQKKAKLVANITKQQMIDFYENYIMSENASKLILHLKSYeast_Ste23P (913) FRRFWNEIECRQYNFARREEEVALLKTIKKDDVLELFDKKIRKDAAERRKLAVFVNematode (930) LAREMDSCPVVGEFPCQNDINLSQAPALPQPEVIQNMTEFKRGLPLFPLVKPHINHuman (959) VSQQTDEN-------ATSEAEPVEITNMERHKPISDIVTFKSCKELYPIALPFLDFruitfly (933) LSREMDSCPLVGEFPAQNDVNLAPAPSLPQPSLVQDMTEFKRSLPLFPLTKPHINZebrafish_1 (938) YGSQHLKEMAS-------DKDEVP--SP--SVEIEDIVGFRKSQPLHGSFRGCGQCress (924) QVENKELN----------E-NELDTAKYPTGQLIEDVGAFKSTLFVAPVRQPMKDYeast_Ste23P (968) HGKNEDQEAVNTIIKKNAESGKKEKEVLYSDQLRQFLPLYGRPIAAIDLKPIGVDNematode (985) VQLPDRGWFVYQQR--NEVHNNCGIEIYHuman (769) YKLLAGDSYLFEKE--NEFHKSSCAQLYFruitfly (743) VQVPDGGWYVYQQR--NEVHNNCGIEIYZebrafish_1 (748) VKLGEGMKYFYHQDGSNPSDENSALVHYCress (731) YLLPKGKTFRYETALKDSQNVNSCIQHVYeast_Ste23P (776) LQLNNGDEYVYRHL--QKTHDVGCVEVTNematode (795) FMAAKL----------Human (1014) IKAKGARSKL------Fruitfly (981) FMAAKL----------Zebrafish_1 (993) PKL-------------Cress (968) FEISAPPKLNNSSESEYeast_Ste23P (1012) PLEHQETTKSKY----Nematode (1040) ?24 ?25 ?26 ?27?28 ?29 ?30 ?31 ?32 ?33 ?34 ?35 ?36 ?28 ?29 ?30 ss II I I I I III G G Supplemental figure 4E Atoms from IDE-N Atoms from IDE-C distance (Angstrom) ==================================================== [ ASP 84 OD2 ] [ LYS 896 N ] 3.1 [ LYS 85 NZ ] [ ASP 895 OD2 ] 3.3 [ ASN 125 OD1 ] [ GLU 817 OE1 ] 3.1 [ SER 132 O ] [ GLN 813 NE2 ] 3.2 [ SER 132 O ] [ ARG 892 NH2 ] 3.3 [ GLY 136 O ] [ ARG 892 NH1 ] 2.6 [ ARG 181 NH1 ] [ THR 825 O ] 2.8 [ GLU 182 OE1 ] [ ARG 824 NH2 ] 3.2 [ GLU 182 OE2 ] [ GLN 828 OE1 ] 3.3 [ ALA 185 N ] [ GLN 828 NE2 ] 3.1 [ SER 188 OG ] [ TYR 831 N ] 2.7 [ LYS 308 NZ ] [ GLU 676 OE1 ] 2.8 [ ASP 309 O ] [ ARG 668 NH1 ] 2.8 [ ASP 309 N ] [ ASN 672 ND2 ] 2.8 [ ARG 311 NH2 ] [ GLU 664 OE2 ] 2.5 [ ARG 311 NH2 ] [ ARG 668 NE ] 3.1 [ GLU 341 OE2 ] [ ASN 605 OD1 ] 2.9 [ SER 348 OG ] [ GLU 606 OE2 ] 2.7 [ LYS 351 NZ ] [ ASP 602 OD2 ] 2.9 [ LYS 351 O ] [ LYS 657 NZ ] 2.9 [ ASN 357 OD1 ] [ ARG 658 NH2 ] 3.0 [ PHE 424 O ] [ LYS 571 NZ ] 2.8 [ ASP 426 OD1 ] [ LYS 571 NZ ] 2.9 [ LYS 527 O ] [ GLU 529 N ] 3.1 [ ASN 528 OD1 ] [ PHE 530 N ] 2.9 [ ASN 528 ND2 ] [ ALA 610 O ] 2.9 ==================================================== Supplemental figure 5 9090 o IDE-N IDE-C Zn 2+ 80 o N C IDE-N IDE-C 36Å 30Å 35Å 34Å A B C Analysis of the substrate binding chamber of IDE. (A) Surface representation of the IDE substrate binding chamber. Surface of IDE is shown in light yellow and the substrate binding chamber is colored brown Zinc ion and insulin are colored magenta and orange, respectively. (B) Electrostatic surface representation of IDE-N and IDE-C. Triangles colored cyan indicate the shape of the substrate binding chamber. The GRASP program (Nicholls et al., Proteins 11:281, 1991) was used to calculate the surface potential. Negative surface is colored red, positive surface blue, and neutral surface white. (C) Models to depict how the enclosed substrate binding chamber is just large enough to accommodate intact insulin at its R state (left) or T-state (right). The substrate binding cavity is represented by the gray mesh. The space-filling and ribbon models of insulin (pdb code: 1G7A) are colored yellow and red, respectively. A B Asp1 Ala2 Glu3 Lys16 Leu17 Val18 Phe19 Phe20 Ala21 Glu22 Asp23 Stereo view of simulated annealing omit map contour at 3.5? of IDE-bound insulin B chain (A) and IDE-bound A? (1-40) (B). The map is colored cyan and the backbone is colored green. Atoms oxygen, nitrogen and carbon are colored red, blue, and green, respectively. Supplemental figure 6 Phe1 Val2 Asn3 Gln4 Ala5 Glu13 Ala14 Leu15 Tyr16 Leu17 Val18 Cys19 Gly20 Supplemental figure 6 (continued) C D Lys1Cys2 Asn3 Thr4 Ala11 Leu12 Ala13 Asn14 Phe15 Leu16 Val17 His18 Ser19 Ser20 Asn21 Asn22 Stereo view of simulated annealing omit map contour at 3.5? of IDE-bound amylin (C) and IDE-bound glucagon (D). The map is colored cyan and the backbone is colored green. Atoms oxygen, nitrogen and carbon are colored red, blue, and green, respectively. Ala23 His1 Ser2 Gln3 Gly4 Phe22 Gln24 Trp25 Leu26 Met27 Asn28 Val23 Supplemental figure 6 (continued) Stereo view of simulated annealing omit map contour at 3.5? of IDE-bound insulin in the presence of 5 mM EDTA (E). The map is colored cyan and the backbone is colored green. Atoms oxygen, nitrogen and carbon are colored red, blue, and green, respectively. Phe1 Val2 Asn3 Gln4 Ala12 Ala25 E Supplemental figure 7 A B Mass (m/z) 4333 Mass (m/z) 1999 3405 4811 6217 7623 9029 Relativ e int ensit y (%) 0 50 100 3908 1499 2804 4109 5415 6720 8026 0 50 100 3485 1499 2603 3708 4813 5918 7023 0 50 100 3432 2163 3127 4091 5055 60191199 Relativ e int ensit y (%) 0 50 100 IDE-E111Q/insulin (+ EDTA) 3432 999 2805 4611 6417 8223 10030 0 50 100 IDE-E111Q/insulin (- EDTA) IDE-E111Q/A?(1-40) IDE-E111Q/amylin IDE-E111Q/glucagon MALDI-TOF mass spectrometry analysis of crystals of IDE-substrate complexes. Mass-spec analysis of peptides eluted from IDE-insulin B chain crystals in the presence and absence of EDTA (A) and those from crystals of IDE in complex with A?1-40, amylin, and glucagon in the presence of EDTA (B). The expected molecular weight of these IDE substrates are as following: insulin B chain, 3431 dalton (Da); A? 1-40, 4331 Da, amylin, 3907 Da; and glucagon, 3484 Da. EDTA is used to chelate zinc ion to further reduce the catalysis of the IDE-E111Q mutant. All of the crystals are grown in the presence of 5 mM Tris(2-carboxyethyl) phosphine (TCEP, reducing agent). The disulfide bond of insulin is presumably reduced in the crystal of IDE-E111Q in complex with insulin. For MALDI-ToF experiments, 5 µl of samples were mixed with equal volume of 0.1% trifluoroacetic acid (TFA) and passed through a C-18 ZipTip (Millipore). 1 µl treated samples were then mixed with matrix (?-cyano-4-hydroxycinn) and spotted on the metal plate (ABI). The experiment was done using ABI 4700 Maldi TOF/TOF MS. Supplemental figure 8 Detailed interactions of residues at the IDE catalytic cleft with the cleavage sites of insulin B chain (A), A?1-40 (B), amylin (C), and glucagon (D). The cut-off distance is 4.0Å. E13 A14 L15 Y16 L17 V18 T142 W199 H108 Q111 A140 F141 T142 E189 W199 G219 T220 N139 A140 F141 Y150 E189 Y831 Q111 H112 N139 A140 R824 Y831 K16 L17 V18 F19 F20 A21 T142 W199 H108 Q111 F141 T142 E189 W199 T220 H108 Q111 H112 N139 A140 F141 Y150 E189 Y831 Q111 H112 F115 N139 A140 E182 R824 Y831 AB P3 P2 P1 P1' S3 S2 S1 S1' S3 S2 S1 S1' P3 P2 P1 P1' V23 Q24 W25 L26 M27 T142 W199 Q111 A140 F141 T142 E189 W199 T220 H108 H112 N139 A140 F141 Y150 E189 Y831 Q111 H112 F115 N139 A140 R824 Y831 L12 A13 N14 F15 L16 V17 F141 T142 W199 H108 Q111 A140 F141 T142 E189 W199 T220 A140 F141 Y150 E189 Y831 Q111 H112 F115 N139 A140 R824 Y831 CD P3 P2 P1 P1' P3 P2 P1 P1' S3 S2 S1 S1' S3 S2 S1 S1' F22 Supplemental figure 9 kDa 97 66 45 31 22 14 WT Y831A Y831F R824A R824K E111Q D426C/K899C N184C/Q828C S132C/E817C AB CD Enzymatic activities of IDE mutants. (A) Coomassie blue staining of purified IDE proteins. 1.5 µg protein was loaded on 13% SDS-PAGE gel. (B) Activities of IDE mutants which had a single mutation in domain 4 or the catalytic base in domain 1. The indicated quantities of IDE proteins were used and the fluorescence intensity was measured after the reaction was performed for one hour. (C) Activities of IDE double cysteine mutants in the absence (filled symbols) and presence (open symbols) of TCEP. The assays were done after one hour incubation using indicated quantities of IDE. (D) Comparison of IDE wild type activities in the presence and absence of 1mM reducing agent, TCEP or 1 mM oxidizing agent, K 3 Fe(CN) 6 . The reaction was performed for 30 minutes under identical conditions as described in figure 3D and 3E. ?? ?? 90 o MPP-MDH IDE-N IDE-C IDE-E111Q/Insulin Pitrilysin Supplemental figure 10 Structural comparison of insulin B chain-bound IDE with substrate-free E. coli pitrilysin (pdb code: 1Q2L) and Yeast mitochondria processing peptidase (MPP, pdb code:1HR9). Four structurally homologous domains are colored green, blue, yellow, and red, respectively. Insulin and Zn ion are colored orange and magenta. Domains of MPP ? and ? subunit are colored based on the corresponding domains of IDE. Peptide substrate from the N-terminal signal peptide of Malate dehydrogenase (MDH) and zinc ion are colored orange and magenta, respectively. It is worth noting that the opening of MPP is completely different from pitrilysin. Pitri-N Pitri-C MALDI-ToF mass spectrometry analysis of the degradation of insulin and A? (1-42) by IDE mutants. To perform the reactions, 10 µl buffer (20 mM HEPES, pH7.2, 1mM TCEP) was incubated with 5 µg IDE protein (5µl of 1 mg/ml protein solution) at room temperature for 5 minutes. The reaction was started by adding 5 µg insulin or 15 µg A? (1-42) into the above mixture and then incubated for one hour at 37 ºC. The reaction was stopped by the addition of 5 µl TFA(10%) . The 5 µg bacitracin was then added to serve as a recovery standard of mass spectrometry. The reaction solution (0.5 µl ) was mixed with 0.5 µl matrix (?-cyano-4-hydroxycinn) and directly spotted on the metal plate as described in supplemental figure 7. The estimated molecular weight of bacitracin, insulin B chain and A? (1-42) are 1,423 daltons, 3,431 daltons and 4,514 daltons, respectively. Date presented are representative of two independent experiments. This experiment demonstrates that, under identical reaction condition all three double cysteine IDE mutants could degrade both insulin and A?(1-42) more effectively than wild type IDE. This suggests that all three IDE mutants under reduced condition (+ TCEP) are substantially more active than wild type IDE. This result is consistent with our finding using fluorogenic substrate V. Supplemental figure 11 Rela tiv e In tensit y (%) Mass (m/z) 3429 1423 1423 3429 1423 1423 1423 3429 Substrate + WT (5 µg) Substrate + D426C/K899C (5 µg) Substrate + N184C/Q828C (5 µg) Substrate + S132C/E817C (5 µg) Insulin Substrate alone 1423 4515 1422 4515 1423 1423 1422 A? (1-42) Mass (m/z) Rela tiv e In tensit y (%) Bacitracin Insulin B chain Bacitracin A? (1-42) +TCEP -TCEP WT D426C/K899C N184C/Q828C S132C/E817C WT D426C/K899C N184C/Q828C S132C/E817C 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Void 670kDa 158kDa 44kDa 17kDa Supplemental figure 12 Oligomeric state of wild type IDE and its mutants. Each protein (5 mg) was mixed with 0.6 ml buffer (20 mM Tris pH 8.0, 100 mM NaCl with/without 1 mM TCEP) on ice for 30 minutes and then loaded onto the superose 6 size-exclusion column (GE healthcare). The elusion was run by AKTA FPLC (GE healthcare) with a flow rate of 0.3 ml/min and fractions were collected every two minutes. The peak fractions are then run on 13% SDS-PAGE gel and the gel was stained by Coomassie staining. The gel-filtration standard (Biorad) was run under identical conditions. The molecular weight standard and fraction number are indicated above. Compared with wild type IDE, we did not observe an obvious change in the elusion profile of all three double cysteine IDE mutants. This indicates that no clear change occurs upon oligomerization of these three IDE mutants. Thus, the change of oligomerization state is unlikely to be the cause of the elevated proteolytic activity of these three IDE mutants. Supplemental figure 13 Identification of major cleavage sites of Amylin (A) and Glucagon (B). The degradation experiment was performed in the presence of 100 mM HEPES (pH 7.2), 2 mM MnCl 2 , 2.5 µM IDE and 82 µM amylin or 92 µM glucagon at 37ºC. The reaction was stopped by mixing 10 µl reaction mixture with 10 µl 500 mM EDTA and 20 µl 0.1% TFA. The solution was then subjected to MALDI-TOF analysis as described in supplemental figure 6. The peaks indicated by red arrows were analyzed. The cleavage sites of glucagon by IDE is consistent with partially mapped sites (Baskin, F. K., et al., Sites of cleavage of glucagon by insulin-glucagon protease, Biochem. Biophys. Res. Commun., 67:163-169, 1975; Rose, K., et al., Insulin proteinase liberates from glucagon a fragment known to have enhanced activity against Ca 2+ + Mg 2+ -dependent ATPase, Biochem. J., 256:847-851, 1988.) User mass DB mass mass difference peptide position 1049.600 1049.516 -0.084 (R)RAQDFVQW(L) 18-25 1293.700 1293.641 -0.059 (R)RAQDFVQWLM(N) 18-27 1449.700 1449.742 0.042 (S)RRAQDFVQWLM(N) 17-27 1508.900 1508.731 -0.169 (R)RAQDFVQWLMNT 18-29 1664.700 1664.833 0.133 (S)RRAQDFVQWLMNT 17-29 1992.700 1991.909 -0.791 HSQGTFTSDYSKYLDSR(R) 1-17 2125 3088 4051 5014199 1162 3905.3 1954.8 100 50 0 50 999 1602 2205 2808 3411 4014 1642.2 1992.8 100 1641.7 100 3905.2 50 1000 1603 2206 2809 3412 4015 3904.7 50 1067.6 1168.6 1508.7 1742.5 2523.2 2144.41867.9 1752.8 799 1241 1684 2126 2569 3011 100 1508.9 799 1241 1684 2126 2569 3011 1449.7 1293.7 1049.6 100 1508.5 1664.7 1992.7 50 100 1449.5 1241 1684 2126 2569 3011799 Mass (m/z) Relativ e int ensit y (%) A B Mass (m/z) User mass DB mass mass difference peptide position 1641.700 1641.784 0.084 KCNTATCATQRLANF(L) 1-15 1642.200 1641.784 -0.416 KCNTATCATQRLANF(L) 1-15 1992.800 1992.977 0.177 (L)ANFLVHSSNNFGAILSSTN(V) 13-31 Relativ e int ensit y (%) T=0 hour T=1 hour T=19 hours T=0 min T=30 mins T=60 mins 50 Supplemental figure 14 Oligomerization of IDE molecules. (A) Stereo view of the IDE dimer and the crystal packing of IDE dimers. IDE-N and IDE-C are colored green and blue, respectively. Two IDE-E111Q molecules form a dimer through the interaction of IDE-C (dashed red box) in one asymmetric unit. (B) List of close contacts between two IDE molecules in an IDE dimer, which are largely mediated through domain 3 and the C-terminus of IDE. (C) List of close contacts between two IDE dimers depicted by crystal packing of an IDE dimer. This interaction could lock IDE in the closed conformation, which would prevent substrate entry and product release. This could explain why an IDE tetramer is less active than an IDE monomer or dimer (Song et al J. Biol. Chem 278:49789, 2003). IDE-N IDE-N IDE-C IDE-C A B C Atoms from 1st IDE molecule Atoms from 2nd IDE molecule Distance (Angstrom) ---------------------------------------------------------- Glu 692A OE2 ... Glu 692B OE2 ... 2.90 Asp 706A OD2 ... Arg 722B NH1 ... 2.51 Asp 706A OD2 ... Arg 722B NH2 ... 2.63 Arg 711A NE ... Gln 718B OE1 ... 3.14 Arg 722A NH1 ... Asp 706B OD2 ... 3.00 Lys 756A NZ ... Asp 706B OD2 ... 2.86 Arg 767A NH1 ... Leu 1002B O ... 2.79 Arg 767A NH2 ... Lys 999B O ... 2.83 Lys 999A O ... Arg 767B NH2 ... 3.02 Arg 1000A NH2 ... Val 1008B O ... 3.10 Arg 1000A O ... Leu 1007B N ... 2.83 Leu 1002A O ... Arg 767B NH1 ... 2.84 Leu 1004A N ... Leu 1004B O ... 2.94 Leu 1004A O ... Leu 1004B N ... 2.92 Leu 1007A N ... Arg 1000B O ... 2.82 Atoms from 1st IDE dimer Atoms from 2nd IDE dimer Distance (Angstrom) ---------------------------------------------------------- Tyr 121B OH ... Trp 409C N ... 3.29 Lys 123B NZ ... Asp 416C OD1 ... 3.07 Arg 164B NH1 ... Glu 408C OE1 ... 3.03 Arg 164B NH2 ... Gln 412C NE2 ... 3.18 Thr 878B OG1 ... Glu 457C OE1 ... 2.53 Glu 880B OE1 ... Glu 458C OE1 ... 2.79 Glu 880B OE2 ... Lys 327C NZ ... 2.67 Lys 933B NZ ... Thr 55C OG1 ... 3.20 Supplemental figure 15 The electron density map representing ?-sheet (top) and ?-helix (bottom) regions of IDE from SAD phases is contoured at 1.0 ? level. The backbone is colored cyan and atoms oxygen, nitrogen, carbon and sulfur are colored red, blue, cyan, and orange, respectively. Supplemental figure 16 A The comparison of MALDI-TOF mass spectrometry of insulin-bound IDE-E111Q complex in the absence (A) and presence (B) of TCEP. The expected molecular weight of intact insulin and insulin B chain is 5812 Da and 3431 Da, respectively. IDE-E111Q/insulin protein complex was isolated using S-200 gel-filtration chromatography (left). To know the state of insulin in the IDE-E111Q/insulin crystals, IDE-E111Q/insulin crystals were washed and dissolved by buffer (20 mM Tris, 50 mM NaCl). Crystals grown without TCEP diffract poorly. The mass spec experiment was done by the same procedure as described in supplemental figure 7. 5810 3431 999 2805 4611 6417 8223 10030 Mass (m/z) 0 50 100 5811 Relativ e int ensit y (%) 50 100 0 999 2805 4611 6417 8223 10030 3432 3432 999 2805 4611 6417 8223 10030 Relativ e int ensit y (%) 0 50 100 B - TCEP 3432 Mass (m/z) 999 2805 4611 6417 8223 10030 0 50 100 + TCEP IDE-E111Q/insulin protein IDE-E111Q/insulin crystals Supplemental Table 1 Data collection and phasing statistics Crystal name Se-IDE-Insulin B chain-Zn 2+ Beamline APS, 19-ID Space group P6 5 Wavelength ( Å) 0.9793445 Unit cell (Å) a c 263.37 90.53 Resolution (Å) 30-2.6 Unique reflections 110,675 (10,963) Completeness (%) 100(100) Redundancy 22.5(20.2) Rsym(%) 9.9(64.2) I/? 36.6(5.4) Se sites 34 out of 52 Figure of merit 0.506 Figure of merit after density modification 0.801 Supplemental Table 2 Data collection and refinement statistics Data collection Crystal name IDE-Insulin B-Zn 2+ IDE-Insulin B IDE-A? (1-40) IDE-Amylin IDE-Glucagon Beamline APS, 14-BM-C APS, 19-ID APS, 19-ID APS, 19-ID APS, 19-ID Space group P6 5 P6 5 P6 5 P6 5 P6 5 Unit cell (Å) a c 262.53 90.50 262.25 90.61 262.43 90.71 262.28 91.11 262.72 90.79 Resolution (Å) 50-2.25 30-2.2 30-2.1 30-2.6 30-2.5 Unique reflections 166,009 (13,956) c 175,528 (15,806) c 194,459 (15,723) c 108,791 (10,738) c 123,701 (11,802) c Completeness (%) 98.0 (82.9) c 97.2 (87.9) c 94.3 (76.9) c 99.2 (98.9) c 99.6 (95.6) c Redundancy a 10.5 (7.1) c 8.4 (5.7) c 7.6 (4.4) c 5.6 (5.2) c 10.6 (7.5) c R sym (%) b 8.6 (63.8) c 6. (34.7) c 7. (55.0) c 10.4 (36.6) c 10.1 (49.5) c I / ? 27.3 (2.6) c 26.5(4.6) c 24.2 (2.3) c 19.0 (5.2) c 23.6 (3.2) c Refinement R cryst (%) d 20.6 20.5 20.3 19.6 19.8 R free (%) e 23.3 22.5 22 22.5 22.5 Rmsd bond (Å) 0.007 0.009 0.008 0.007 0.007 Rmsd angle (º) 1.3 1.3 1.3 1.3 1.3 Number of protein atoms ligand atoms solvent molecules metal atoms 15,948 12 787 2 15,914 12 1044 0 15,884 12 1066 0 16,001 12 696 0 15,931 12 695 0 Ramachandran plot (%) Favorable region Allowed region Generously allowed region Disallowed region 90.3 9.7 0 0 90.7 9.3 0 0 91.3 8.6 0.1 0 90.1 9.8 0.1 0 89.7 10.2 0.1 0 B-factors (Å 2 ) IDE Substrate solvent 36.7 59.9 44.5 31.2 41.6 40.9 33.5 44.0 45.2 29.9 44.5 38.4 33.1 54.7 41.1 a N obs /N unique. b R sym = hkl i I i (h k l)- / hkl i I i (h k l) for n independent reflections and i observations of a given reflection; is the average intensity of the i observations. c The outer resolution shell. d R work = hkl F obs - F calc / hkl F obs . e R free , calculated the same as for R work but on the 10% data excluded from the refinement calculation. 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