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CARBOXYPEPTIDASE - Metalloprotease with Zn - Released as pro-protease
- a/b topology
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+ PRO-CARBOXYPEPTIDASE Activation Segment N C Activation by
cleavage + C N Activation Segment Activation by Trypsin limited proteolysis. Active enzyme: carboxypeptidase
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PRO-CARBOXYPEPTIDASES
Porcine Pro Carboxypeptidase B PCPBp Pro Carboxypeptidase A1 PCPA1p Bovine PCPA1b
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Human Pro Carboxypeptidase A2
Model of Human Pro-Carboxypeptidase A2 ( PCPA2h ) Human Pro Carboxypeptidase A2 Model of PCPA2h obtained by Distance Restraints from CPA and the Activation Domain. Catasús et al. (1995) J.Biol.Chem. 270, RMSD = Backbone: 2.25 Å All: 3.06 Å Experimental PCPA2h structure is in red
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SEQUENCE ALIGNMENT OF PRO-CARBOXYPEPTIDASES
-ETFVGDQVLEIVPSNEEQIKNLLQLEAQEHLQLDFWKSPTTPGETAHVRVPFVNVQAVKVFLESQGIAYSIMIEDVQVL PCPA2h KEDFVGHQVLRITAADEAEVQTVKELEDLEHLQLDFWRGPGQPGSPIDVRVPFPSLQAVKVFLEAHGIRYRIMIEDVQSL PCPA1b KEDFVGHQVLRISVDDEAQVQKVKELEDLEHLQLDFWRGPARPGFPIDVRVPFPSIQAVKVFLEAHGIRYTIMIEDVQLL PCPA1p LDKENEEMLFNRRRERSGN-FNFGAYHTLEEISQEMDNLVAEHPGLVSKVNIGSSFENRPMNVLKFSTGG-DKPAIWLDA PCPA2h LDEEQEQMFASQSRARSTNTFNYATYHTLDEIYDFMDLLVAEHPQLVSKLQIGRSYEGRPIYVLKFSTGGSNRPAIWIDL PCPA1b LDEEQEQMFASQGRARTTSTFNYATYHTLEEIYDFMDILVAEHPALVSKLQIGRSYEGRPIYVLKFSTGGSNRPAIWIDS PCPA1p GIHAREWVTQATALWTANKIVSDYGKDPSITSILDALDIFLLPVTNPDGYVFSQTKNRMWRKTRSKVSGSLCVGVDPNRN PCPA2h GIHSREWITQATGVWFAKKFTEDYGQDPSFTAILDSMDIFLEIVTNPDGFAFTHSQNRLWRKTRSVTSSSLCVGVDANRN PCPA1b GIXSRXWITQASGVWFAKKITENYGQNSSFTAILDSMDIFLEIVTNPNGFAFTHSDNRLWRKTRSKASGSLCVGSDSNRN PCPA1p WDAGFGGPGASSNPCSDSYHGPSANSEVEVKSIVDFIKSHGKVKAFIILHSYSQLLMFPYGYKCTKLDDFDELSEVAQKA PCPA2h WDAGFGKAGASSSPCSETYHGKYANSEVEVKSIVDFVKDHGNFKAFLSIHSYSQLLLYPYGYTTQSIPDKTELNQVAKSA PCPA1b WDAGFGGAGASSSPCAETYHGKYPNSEVEVKSITDFVKNNGNIKAFISIXSYSQLLLYPYGYKTQSPADKSELNQIAKSA PCPA1p AQSLRSLHGTKYKVGPICSVIYQASGGSIDWSYDYGIKYSFAFELRDTGRYGFLLPARQILPTAEETWLGLKAIMEHVRD PCPA2h VEALKSLYGTSYKYGSIITTIYQASGGSIDWSYNQGIKYSFTFELRDTGRYGFLLPASQIIPTAQETWLGVLTIMEHTLN PCPA1b VAALKSLYGTSYKYGSIITVIYQASGGVIDWTYNQGIKYSFSFELRDTGRRGFLLPASQIIPTAQETWLALLTIMEHTLN PCPA1p
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Automated Models of PCPA2h
MODELLER RMSD = Backbone: 1.20 Å All: 1.89 Å COMPOSER Backbone: 1.31 Å All: 2.03 Å
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Detection of mismodelled regions
Largest deviations found in the Distance Map Plot between the model obtained by Distance Restraints # and the experimental structure # Catasús et al.,1995) J.Biol.Chem. 270, Difference < 1 Å Difference < 2 Å Difference < 3 Å Difference > 4 Å residue number
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C-cap of the a-helix connecting segment
Distance Map Plot for Automated Models COMPOSER MODELLER Mismodelled region : C-cap of the a-helix connecting segment residue number
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How to Test a Model ? pseudo-energy residue number Mismodelled
connecting segment ! Difference between the experimental structure and the model of PCPA2h pseudo-energy residue number
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Secondary Structure Prediction and Multiple
Alignment of Pro-Carboxypeptidases EEEEE HHHHHHHHHHHHHHH EEEE EEEEE HHHHHHHHHH EEEEHHHHHHHH PCPA2h PHD -ETFVGDQVLEIVPSNEEQIKNLLQLEAQEHLQLDFWKSPTTPGETAHVRVPFVNVQAVKVFLESQGIAYSIMIEDVQVL PCPA2h KEDFVGHQVLRITAADEAEVQTVKELEDLEHLQLDFWRGPGQPGSPIDVRVPFPSLQAVKVFLEAHGIRYRIMIEDVQSL PCPA1b KEDFVGHQVLRISVDDEAQVQKVKELEDLEHLQLDFWRGPARPGFPIDVRVPFPSIQAVKVFLEAHGIRYTIMIEDVQLL PCPA1p HHHHHHHHHHHHHH PCPA2h PHD LDKENEEMLFNRRRERSGN-FNFGAYHTLEEISQEMDNLVAEHPGLVSKVNIGSSFENRPMNVLKFSTGG-DKPAIWLDA PCPA2h LDEEQEQMFASQSRARSTNTFNYATYHTLDEIYDFMDLLVAEHPQLVSKLQIGRSYEGRPIYVLKFSTGGSNRPAIWIDL PCPA1b LDEEQEQMFASQGRARTTSTFNYATYHTLEEIYDFMDILVAEHPALVSKLQIGRSYEGRPIYVLKFSTGGSNRPAIWIDS PCPA1p GIHAREWVTQATALWTANKIVSDYGKDPSITSILDALDIFLLPVTNPDGYVFSQTKNRMWRKTRSKVSGSLCVGVDPNRN PCPA2h GIHSREWITQATGVWFAKKFTEDYGQDPSFTAILDSMDIFLEIVTNPDGFAFTHSQNRLWRKTRSVTSSSLCVGVDANRN PCPA1b GIXSRXWITQASGVWFAKKITENYGQNSSFTAILDSMDIFLEIVTNPNGFAFTHSDNRLWRKTRSKASGSLCVGSDSNRN PCPA1p WDAGFGGPGASSNPCSDSYHGPSANSEVEVKSIVDFIKSHGKVKAFIILHSYSQLLMFPYGYKCTKLDDFDELSEVAQKA PCPA2h WDAGFGKAGASSSPCSETYHGKYANSEVEVKSIVDFVKDHGNFKAFLSIHSYSQLLLYPYGYTTQSIPDKTELNQVAKSA PCPA1b WDAGFGGAGASSSPCAETYHGKYPNSEVEVKSITDFVKNNGNIKAFISIXSYSQLLLYPYGYKTQSPADKSELNQIAKSA PCPA1p AQSLRSLHGTKYKVGPICSVIYQASGGSIDWSYDYGIKYSFAFELRDTGRYGFLLPARQILPTAEETWLGLKAIMEHVRD PCPA2h VEALKSLYGTSYKYGSIITTIYQASGGSIDWSYNQGIKYSFTFELRDTGRYGFLLPASQIIPTAQETWLGVLTIMEHTLN PCPA1b VAALKSLYGTSYKYGSIITVIYQASGGVIDWTYNQGIKYSFSFELRDTGRRGFLLPASQIIPTAQETWLALLTIMEHTLN PCPA1p
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a-Helix C-cap of the Connecting Segment
a Schellman Motif G (C’) N (C’’) S (Ccap ) R (C1) E (C2) R (C3) R (C5) R (C4) Schellman Motif Profile C´´ » C3 / C´ Gly C3 C2 C1 Ccap C’ C’’ L Q E H G I A R A K G V M K K L G K hydrogen bond hydrophobic interaction N R R R E R S G N F N hydrogen bonds C3 Ccap C’’ ^ ^ ^ Schellman Motif found in PCPA2H
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Refinement of the Model
Extension of the a-Helix C-cap of the Connecting Segment RMSD (backbone) DR = 2.25 Å Composer = 1.09 Å Modeller = 0.96 Å Composer = 1.31 Å Modeller = 1.20 Å
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Checking the Refinement of the Model
ab initio TEST by Knowledge-Based Energy pseudo-energy residue number Difference between the modified model and the original. IMPROVEMENT
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Checking the Refinement of the Model
Distance Map Plot residue number COMPOSER MODELLER
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