Bioinformatique et Biologie Structurale I/ – Principes et techniques

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Bioinformatique et Biologie Structurale I/ – Principes et techniques A/ L’information structurale B/ Les différentes techniques de détermination de structure C/ Les nouveaux challenges de la biologie structurale II/ – Application à l’étude d’enzymes d’intérêt médical A/ Un bref aperçu de ce que l’on appelle « Drug design » B/ Recherche d’inhibiteurs d’aminopeptidases de Streptocoques
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Bioinformatique et Biologie Structurale I/ – Principes et techniques A/ L’information structurale B/ Les différentes techniques de détermination de structure C/ Les nouveaux challenges de la biologie structurale II/ – Application à l’étude d’enzymes d’intérêt médical - PowerPoint PPT Presentation

Transcript of Bioinformatique et Biologie Structurale I/ – Principes et techniques

  • Bioinformatique et Biologie Structurale

    I/ Principes et techniques

    A/ Linformation structurale B/ Les diffrentes techniques de dtermination de structureC/ Les nouveaux challenges de la biologie structurale

    II/ Application ltude denzymes dintrt mdical

    A/ Un bref aperu de ce que lon appelle Drug designB/ Recherche dinhibiteurs daminopeptidases de StreptocoquesC/ Relations structure-fonction dhlicases impliques dans les cancers

  • X-PDAP activitySelectivity :Pro ; ALA 10% AS ; GLY 1 % ASTwo families : S9B and S15 for the same enzyme activity

    S9B : DPP-IV [eukaryotic and prokaryotic] membrane-bound and soluble forms

    S15 : PepX [prokaryotic] cytoplasmic exopeptidase

    PepX

    X-Pro AA3-AA4-AA5... AAnX-Pro + AA3-AA4-AA5... AAn

    -Ala-

    -Gly-

  • X-PDAP activityProline specific proteases : a rare groupMany biologically active peptides contain an evolutionary conserved proline residue as a proteolytic processing regulatory elementProline-specific proteases : important 'check-points' controlImportance in some disease to inhibit such proteasesX-PDAP : S9b and S15 family evolutionarily distant enzymes DPP-IV and PepXImportance of the enzyme activity in prokaryoticsProteases and peptidases have been identified as critical virulence factors in numerous microbial pathogens ; may act on a variety of host proteins including serum and tissue components thus contributing to neutralization of the immune defense system and tissue invasion and destruction.

    DPP-IV is involved in various mammalian regulation processes and in serious human diseases (Diabetes type II,)

  • The signature of the X-PDAP specificity in SC Clan enzymesStructure / function relationships in X-PDAP enzymes

    What makes the enzymes so specific?

    - Clans, families of proteases and X-PDAP activityStructure of PepX, X-PDAP from Lactococcus lactisComparison of bacterial and human X-PDAP structuresInsights for drug designConclusion

  • SC ClanAlmost all enzymes are specialized in cleavages involving a proline residue

    Oligopeptidases [endo, release peptides]Iminopeptidases[exo, release PRO]Carboxypeptidases[release peptides]X-prolyl dipeptidyl aminopeptidases[exo, releases X-PRO] endo detected in the case of DDP-IV

  • http://merops.sanger.ac.uk/

  • S15 family - Alignments -+ legend figure 1 HERE!

    10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

    ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

    P22346 1 MRFNHFSIVDKNFDEQLAELDQLG-----FRWSVFWDEKKILKDFLIQ-----SPSD--MTALQATAELDVIEFLKSS-IELDWEIFWNIALQLLDFVPNFDFEIGKAFEYAKNSNLPQIEAEMT-TENIISAFYYLLCTRRKTGMILVE 136

    Q9CE1 1 MRFNHFSIVDKNFDEQLAELDQLG-----FRWSVFWDEKKILKDFLIQ-----SPTD--MTVLQANTELDVIEFLKSS-IELDWEIFWNITLQLLDFVPNFDFEIGKATEFAKKLNLPQRDVEMT-TETIISAFYYLLCSRRKSGMILVE 136

    AAK75021 1 MRFNQYSYINFPKENVLSELKKCG-----FDLQNTANHKDSLETFLRRFFFTYQDTNYPLSILAADKKTDLLTFFQSE-DELTADIFYTVAFQLLGFSYLVDFED--SDVFRKETGFPIIYGD------LIENLYQLLNTRTKKGNTLID 136

    Q99Y58 1 MRYNQFSYIPTSLERAAEELKELG-----FDLDLQKTAKASLESFLRKLFFHYPDSDYPLSHLIAKNDMDALSFFQSE-QELSKEVFDLLALQVLGFIPGVDFTE--ADAFLDKLAFPIHFDET----EIIKHIHHLLATRCKSGMTLID 138

    AAK39633 1 MRYNQYSYTKASEEVMLDELARLG-----FTIQTTNSPKENLHHFLQKILFRYQDVNYVLSSWVADQKTDLLTFFQSD-KQLTEEVFYTVALQVLGFAPFVDFDD--VTAFCKEIHFPITYGN------ILENLYQLLNTRTKLGNTLID 136

    Q9RDW6 1 MKLNQFARLTPDIDQQLKELARIG-LPADPQAP-FADTAAAMYAAFFPEAYQPAAQQDKFAQVAVNSHQNLAEWLATKPTHMKRADFYNVALQLLGFEAFSDFDLSDPISFMTVTKLPSVAHDLLHTADLLQASYLLLTTRTKHLVSFLD 148

    P40334 1 MKYNQYAYVETSPEKATEELLAINFLPENYSSLSFSELLAVLTGNVLAEATTRQAKDAKLAEFAVDDQTDLAAFLLDTPTAITASQFANVALQLLGYHPNYDYSLTDPLTCGKKHALP-AFKDLTSKEELIFTFYRLLNTRSKNGQILLD 149

    Q59485 1 MKYNQYAYVETDFQQQVKELIDINFLPKNYQVWDFSSLLAKLVKNAIAEAKTDAAKNAKLAEFAVSDHQTLADFLKEKPTEIGTKQFYNVALQLLGYHVHYDYDFADPTGFMQRNALP-FLQDISDNQKLISAFYRLLNTRAKNGQILLD 149

    160 170 180 190 200 210 220 230 240 250 260 270 280 290 300

    ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

    P22346 137 HWVSEGLLPLDNH------YHFFNDKSLATFDSSLLEREVLWVESPVDSEQRGENDLIKIQIIRPKSTE---KLPVVMTASPYHLGINDKANDLALHDMNVELEEKTS-HEIHVEQKLPQKLSAKAKELPIVDKAPYRFTHGWTYSLNDY 276

    Q9CE1 137 HWVSEGLLPLDNH------YHFFNDKSLATFDSSLLEREVVWVESPVDTEQKGKNDLIKIQIIRPKSTE---KLPVVITASPYHLGINEKANDLALHEMNVDLEKKDS-HKIHVQGKLPQKRPSETKELPIVDKAPYRFTHGWTYSLNDY 276

    AAK75021 137 QLVSDGLIPEDND------YHYFNGKSLATFSNQDVIREVVYVESRVDTDQKGLSDLVKVSIIRPRFDG---KIPAIMTASPYHQGTNDKASDKALYKMEGELEVKLP-HKIELEKPQLNLVQPQGKAELIAEAEEKLTHINSSYTLNDY 276

    Q99Y58 139 DLVSQGMLTMDND------YHFFNGKSLATFDTSQLIREVVYVEAPLDTDQDGQLDLIKVNIIRPQSQK---PLPTLMTPSPYHQGINEVANDKKLYRMEKELVVKKR-RQITVEDRDFIPLETQPCKLPIGQNLESFSYIN-SYSLNDY 277

    AAK39633 137 QLVSEGFIPESND------YHFFNGKSLATFSSHEAIREVVYVESRVDTDGDGKPDLVKVSIIRPSYEG---QVPAVMTASPYHQGTNDKASDKALHNMNVDLSCKNP-RTITVQESSIQTIEPQGQASLVEKAEEKLGHIG-SYTLNDY 275

    Q9RDW6 149 DLANRGFFKDFQAQSSQPAHLLFNGKVQQVFDARQAVREVVWIESDVDSDHDGQRDLLEATIYRPKATDRGLKVPVLFTANPYFHGTNDVTAATHVPETVLAVKPHGASKAEVTAAPASKPKLPARPVTGETKQAAAYAEENSPYAFNDY 298

    P40334 150 VMAGKGYFTQFWGEGK---FMFFNGKSLPVFDTSQVIREVVYVQSDLDTDGDGKGDLLPVTVFRPVESQDQLKVPALYTASPYFGGIIDNVKTNHNVDENLTDATTWT-NPKYVAKPLVKSPAPS----DQDVPATELATGQSSYGLNEY 291

    Q59485 150 VMAGKGYFTQFWGQNK---FKFFNGKSIPVFDTNKVIREVVYVETDLDTDHDGKSDLIQVTVFRPEETNKGLKVPALYTASPYFGGIIANEKRNHNVDENLSDSTEWN-DPQYVHSPIVKAEKPD----GSSRPATEEAVHKSSYPLNEY 291

    310 320 330 340 350 360 370 380 390 400 410 420 430 440 450

    ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

    P22346 277 FLTRGFASIYVAGVGTRSSDGFQTSGDYQQIYSMTAVIDWLNGRARAYTSRKKTHEIKASWANGKVAMTGKSYLGTMAYGAATTGVEGLELILAEAGISSWYNYYRENGLVRSPGGFPGEDLDVLAALTYSRNLDGADFLKGNAEYEKRL 426

    Q9CE1 277 FLTRGFASIYVAGVGTRGSNGFQTSGDYQQIYSMTAVIDWLNGRTRAYTSRKKTHEIKATWANGKVAMTGKSYLGTMAYGAATTGVDGLEVILAEAGISSWYNYYRENGLVRSPGGFPGEDLDVLAALTYSRNLDGADYLKGNDEYEKRL 426

    AAK75021 277 FLPRGFANLYVSGVGTKDSTGFMTNGDYQQIEAYKNVIDWLNGRCRAFTDHTRQRQVKADWSNGKVATTGLSYLGTMSNGLATTGVDGLEVIIAEAGISSWYNYYRENGLVTSPGGYPGEDFDSLAELTYSRNLLAGDYIRGNEAHQADL 426

    Q99Y58 278 FLARGFANIYVSGVGTAGSTGFMTSGNYAQIESFKAVIDWLNGRATAYTSHSKTHQVRADWANGLVCTTGKSYLGTMSTGLATTGVDGLAMIIAESAISSWYNYYRENGLVCSPGGYPGEDLDVLTELTYSRNLLAGDYLRHNDRYQELL 427

    AAK39633 276 LLPRGFANLYVSGVGTKDSEGMMTSGDYQQIEAYKNVIDWLNGRCRAFTDHTRQREIKATWSNGKVATTGISYLGTMSNGLATTGVDGLEVIIAEAGISSWYNYYRENGLVTSPGGYPGEDFESLTELTYSRNLLAGEYLRHNQAYQAYL 425

    Q9RDW6 299 FLARGFAVVYSAGVGTRYSDGFRTIGGPEETDGAVAVIEWLTGKRRAFTNRTDGVAIKAWWSSGKVAMTGKSYLATLAIAGATTGVEGLKTIVADAGISSWYDYYRENGLVVAPGGYQGEDADVLAVDTFSRQKSGGDMIRLKKAWEQHL 448

    P40334 292 LLARGFASVFSGAIGNRHGDGIRITGSPEETISQKEVIEWLTGDRVAYTDRTRRFETKASWCSGNVGMTGRSYLGTLQIAIATTGVKGLKTVVSEAAISSWYDYYREHGLVVAPSECQGEDMDKLAEVCQSNLWDGG-NFTAKKAYEAEQ 440

    Q59485 292 MLARGFASVFAGAIGTRGSDGVRITGAPEETESAAAVIEWLHGDRVAYTDRTRTVQTTADWCNGNIGMTGRSYLGTLQIAIATTGVKGLKTVVSEAAISSWYDYYREHGLVIAPEACQGEDLDLLAETCQSNLWDAGSYLKIKPEYDKMQ 441

    460 470 480 490 500 510 520 530 540 550 560 570 580 590 600

    ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

    P22346 427 AEMTAALDRKSGDYNQFWHDRNYLINTDKVKADVLIVHGLQDWNVTPEQAYNFWKALPEG-HAKHAFLHRGAHIYMNSWQSIDFSETINAYFVAKLLDRDLNLN--LPPVILQENSKDQVWTMMNDFG-ANTQIKLPLGKTAVS------ 566

    Q9CE1 427 AEMTTALDRKSGDYNQFWHDRNYLINSDQVRADVLIVHGLQDWNVTPEQAYNFWQALPEG-HAKHAFLHRGAHIYMNSWQSIDFSETINAYFSAKLLDRDLNLN--LPPVILQENSKEQVWSAVSKFG-GDDQLKLPLGKTAVS------ 566

    AAK75021 427 EKVKAQLDRKTGDYNQFWHDRNYLLNAHKVKAEVVFTHGSQDWNVKPLHVYQMFHALPTH-IHKHLFFHNGAHVYMNNWQSIDFRESINALLTKKLLGQETDFQ--LPTVIWQDNTAPQTWLSLDNFGGQENCETFSLGQEEQ------- 566

    Q99Y58 428 NQQSQALDRQSGDYNQFWHDRNYLKNAHQIKCDVVYTHGLQDWNVKPRQVYEIFNALPST-INKHLFLHQGEHVYMHNWQSIDFRESMNALLCQKLLGLANDFS--LPEMIWQDNTCPQNWQERKVFG-TSTIKELDLGQELL------- 566

    AAK39633 426 DQQRKDLERETGDYNQFWHDRNYLIHADKVKAEVVFTHGSQDWNVKPLHVYNMFHALPAH-IKKHLFFHNGAHVYINNWQSIDFRESMNALLSKKLLGHSSDFD--LPPVIWQDNSQAQNWMSLDDFGNQEDYSHFHLGKGSQ------- 565

    Q9RDW6 449 AQMTRDQDRTTGAYTAWWDARNYRKNAANVKADVVLIHGLNDWNVKPKNAIRFWQAIADLPIQKKLILHQGQHVYVHNVRSLDFLDMMNLWLTHELLGVANQAEQVLPNVLVQDNVTPQTWSAYSDFAN---PAAEHVTTTAN--LKTDF 593

    P40334 441 AELLAAQDRATGQYSDFWESRNYRHHTDGIKCSWISVHGLNDWNVKPKNVYKIWQKVKQLPVKSHLFLHQGPHYNMNNLVSIDFTDLMNLWFVHELLEVENGAYEQWPKVMIQDNLEADEWHAESDWASDLGQASLYLPTADGDLSTVEN 590

    Q59485 442 KQLREKEDRNTGQYSDFWEARNYRHHADGIKCSWISVHGLNDWNVKPKNVYKIWQLVKKMPMKHHLFLHQGPHYNMNNLVSIDFTDLMNLWFVHELLGIENNAYNQWPTVMIQDNLQADKWHEEPDWSNDLGQEKIYYPTDEGELFQDGN 591

    610 620 630 640 650 660 670 680 690 700 710 720 730 740 750

    ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

    P22346 566 --FAQFDNNYDDETFKKYSKDFNVFKKDLFENKANEAVIDLELPSMLTIN-----GPVELELRLKLNDTKGFLSAQILDFGQKKRLEDKVRVKDFKVLDRGRNFMLDDLVELPLV--ESPYQLVTKGFTNLQNQ-SLLTVSDLKADEWFT 706

    Q9CE1 566 --FAQFDNHYDDESFKKYSKDFNVFKKDLFENKANEAVIDLELPSELTIN-----GPIELEIRLKLNDSKGLLSAQILDFGPKKRLEDKARVKDFKVLDRGRNFMLDDLVELPLV--ESPYQLVTKGFTNLQNK-DLLTVSDLKADEWFT 706

    AAK75021 566 ----AIQNQYPDKDFERYGKTYQTFNTELYQGKANQITINLPVTKDLHLN-----GRAQLNLRIKSSTNKGLLSAQLLEFGQKKYLQPYPAILSARTIDNGRYHMLENLCELPFR--PEAQRVVTKGYLNLQNRNDLLLVEDITADEWMD 705

    Q99Y58 566 ----LIDNHYGEDEFKAYGKDFRAFKAALFKGKANQALIDILLEEDLPIN-----GEIVLQLKVKSSENKGLLSAQILDYGKKKRLGDLPIALTQSSIDNGQNFSREPLKELPFR--EDSYRVISKGFMNLQNRNNLSSIETIPNNKWMT 705

    AAK39633 565 ----EIRNRYSDEDYNRFAKSYQVFKNELFEGKTQQITLDWTLEQDLFIN-----GPAKLKLRLKSSTNKGLISAQLLDYGPAKRLTPIPSLLEPRVMDNGRYYMLDNLMELPFA--DTPHRVITKGFLNLQNRTDLLTVEEVVPNQWME 704

    Q9RDW6 594 ESATDQFSDHATATFTAQHDTSASFEKAIITPNSAYVNSRLWLTQPVLEHDRVLEGIPHLELTLAVDAPTGILSVRLVDLGKAKRFEENAATVGASGLQLGFDFKTTDIVEFKPANKETPSKLITLGHINLQNPKNAYEVQTITPGQFFH 743

    P40334 591 GTGQLTFTDLGGTEFKKAGISETDWEYQFISGEEKWAKASLRFESEEFLHPTTLVGRPKVRVRVAANKTVGQLSVALVDLGTRQRLTATPKIFARGNQPFGYRFGADSLQEFVPD-KATKAKLITKAHMNLQNYQDMKQPSKLEAGQFVD 739

    Q59485 592 GKAQKSFTDVGGIEFKKAGISESDWQYKFICGDEKWAKPSLRFETDEFTHPTTIVGRPEVKVRVSASLPKGEISVALVELGERQRLTATPKFLMHGGQELGYRFGTDTLQEFVPD-KKTKAKLITKAHMNLQNFKDMKKPEAIDADKFYD 740

    760 770 780 790 800

    ....|....|....|....|....|....|....|....|....|....|....|..

    P22346 707 IKFELQPTIYHLEKADKLRVILYSTDFEHTVRDNRKVTYEIDLSQSKLIIPIESVKN 763

    Q9CE1 707 LKFELQPTIYHLEKADKLRVILYSTDFEHTVRDNRKVTYEIDLSQSKLIIPIESVKK 763

    AAK75021 706 VQFELQPTIYKLKEGDTLRLVLYTTDFEITIRDNTDYHLTVDLAQSMLTLPC----- 757

    Q99Y58 706 VRLPLQPTIYHLEKGDTLRVILYTTDFEHTVRDNSNYALTIDLSQSQLIVPIASN-- 760

    AAK39633 705 LSFELQPTIYKLKKGDQLRLVLYTTDFEHTVRDKTDYHLSVDMEHSSLSLPHKKS-- 759

    Q9RDW6 744 VSLDLQPTHYHLPAGRQLALIIHGADMAQTIRPTKVTHYQLDLAKSTLTLPFRI--- 797

    P40334 740 LEFELQPTYYTLPAGAKLGLIIYSTDQGMTKRPLETEDYTVDLAGTALLLYRK---- 792

    Q59485 741 LDFLLQPTYYTIPSGSKLALIIYSTDQGMTKRPLEDETYTIDLANTEIKFYEK---- 793

  • Structure Resolution of PepX from Lactoccocus lactis10 % PEG 4000, 150mM NaClPh 5.2 MES NaOH, 18C

  • PepX prototype de la famille S15- 4 domaines- a/b hydrolase fold- lments remarquables (peptide lasso, Boucle C-ter, )85 25 Rigolet, P. et al. (2002). Structure 10, 1383-1394

  • Enzymes families of SC Clan and related structuresCatalytic domain (a/b hydrolase fold) in green, N-ter domain in red, C-ter domain in blue and helical domain in orange

  • Comparison of sequence and structures of SC Clan enzymes

  • Cocaine Esterase(COCE,)1JU3 , 565 residues40% Specific to PepXbetween PepX and :Prolyl Iminopeptidase(XCPIP, Xant. campestris) 1JU3 , 313 residues66% Specific to PepX

    Prolyl Oligopeptidase(POP, porcine muscle)1JU3 , 710 residues64% Specific to PepX

    Structure comparisons

  • Structure-based sequence aligmentOnly 4 conserved sequences can notably be distinguished between the two sequences of PepX and DPP-IV: - sequence NxxxAxxGxSYxG around the active serine ;- sequence LxxHGxxDxNVxxxxQxxxxxKAL around the active aspartic acid ;- short sequence HxxxxxS after the active histidine ;- sequence AxAxxSxWxxY before the Pos1 subsite of the X-PDAP signature.

  • PepX Dimeric structureImportant for activityGlobular shapeInvolve principally N-ter and helical domainsCanal acces to catalytic residuesThe two active sites are far away from each other and independently accessible to the substrateHydrophylic interface; labile contacts

  • Engel, M., Hoffmann, T., Wagner, L., Wermann, M., Heiser, U., Kiefersauer, R., Huber, R., Bode, W., Demuth, H.U. and Brandstetter H. (2003). Proc. Natl. Acad. Sci. USA 100, 5063-5068.DPP-IV Dimeric structureDPP-IV

  • PepX Electrostatic propertiesAcidic surface (also seen for other proteases of Lact. lactis ; adaptation to a particular cellular environment)

  • Rasmussen HB, Branner S, Wiberg FC, Wagtmann N. (2003). Nature Struct. Biol. 10, 19-25. DPP-IV Electrostatic propertiesDPP-IV

  • Differences between the two enzymesNearly the same lenght (PepX 763 and DPP-IV 766) Different folds (moreover 4 versus 3 domains)Both dimer but of different quaternary organizationDPP-IV is integrated in the plasmatic membrane whereas PepX is an cytoplasmic enzymeSubstrate selection processes via an N-ter propeller domain whereas via dimeric domain in PepX.

  • Comparison of the specificity sites of PepX and DPP IVEvolution conserved thus a particular arrangement of residues, perhaps the most efficient, ensuring XPDAP activity with a high specificity.

  • Comparison of the specificity sites of PepX and DPP IVEvolution conserved thus a particular arrangement of residues, perhaps the most efficient, ensuring XPDAP activity with a high specificity. Oxyanion hole

  • Comparison of the specificity sites of PepX and DPP IVEvolution conserved thus a particular arrangement of residues, perhaps the most efficient, ensuring XPDAP activity with a high specificity. Signature X-PDAP activity

  • Comparison of the specificity sites of PepX and DPP IVEvolution conserved thus a particular arrangement of residues, perhaps the most efficient, ensuring XPDAP activity with a high specificity. ?

  • Equivalent residues in compared enzymes of the clan SC.Structural signature of the XPDAP activityRigolet P. et al. . (2005). FEBS J. 272; 2050-2059.

    LabelPepX[family S15]DPP-IV[ family S9B]POP[family S9A]SPAP[family S33]CBPY[family S10]Catalytic triadSerS348S630S554S113S146AspD468D708D641D268D338HisH498H740H680H296H397Residues implicated in positioning of the substrate proline in the active sitePos1Y380Y662W595--Pos2L401Y666F476E232W312Pos3W377W659-L141-Pos4I374V656V580F139L178Residues stabilizing the binding of the substrate in the specificity pocketStb1-R125R643-Y256Stb2N470N710R643A270I340Oxyanion holeOxa1Y349Y631N555W114Y147Oxa2Y210Y547Y473--Residues responsible for the exopeptidase activityExo1F393E205---Exo2E396E206---Other residues postulated to play a role in enzyme specificityOtherV471V711V644C271C341

  • Exploiter diffrences :

    R125 DPP-IV L401 PepX / Y666 DPPIVRecherche dinhibiteurs spcifiquesTester inhibiteurs de DPPIV (ceux de petite taille) Structure de PepX avec un inhibiteur de DPP-IV

  • Table 2: Inhibition experiments realized with PepX from Lactococcus lactis. Recherche dinhibiteurs spcifiques

    EnzymePepXDPP-IVCompoundIC50KIIC50KISI avaline-pyrrolidide30 mM9.3 mM4 mM b2 mM c7.5diprotin A260 mM71.5 mM8 mM d4.6 mM e32.5diprotin B600 mM118.45 mM

  • Valine-PyrrolidideInhibition comptitiveIC50 = 30 mMKI = 9 mMRigolet P., Xi, X.G., Rety, S. and Chich, J.F. (2005). FEBS J. 272; 2050-2059. KI = 9 mMRecherche dinhibiteurs spcifiques

  • Table 3: Docking simulations of valine-pyrrolidide in the X-PDAP enzymes.Recherche dinhibiteurs spcifiquesTable 2: Inhibition experiments realized with PepX from Lactococcus lactis.

    EnzymePepXDPP-IVCompoundIC50KIIC50KISI avaline-pyrrolidide30 mM9.3 mM4 mM b2 mM c7.5diprotin A260 mM71.5 mM8 mM d4.6 mM e32.5diprotin B600 mM118.45 mM

    Docking simulations of valine-pyrrolidide in X-PDAP enzymes

    Enzyme

    Source

    Cluster rank

    Robustness

    Mean Docked Energy

    (Kcal/mol)

    KI b

    (M)

    Reference RMSD c ()

    DPP-IV a

    human

    1

    91 %

    -8.4

    1.50

    1.36

    PepX

    Lactococcus lactis

    1

    83 %

    -7.2

    9.60

    1.50

    PepX

    Streptococcus gordonii

    2

    80 %

    -7.5

    7.10

    1.70

  • Recherche dinhibiteurs spcifiquesNouveaux drivs partant de la valine-pyrrolidide Nous les avons synthtiss . Nous les testons actuellement .

  • Drug design: autres stratgies- Mutagense dirige : PHE 80

  • Quelle est la fonction des domaines Nter et Cter?Quapportent-ils vraiment la catalyse ?Rle prsent chez DPPIVPas de relle interprtation ni chez aAeH ni chez CoceRle suppos chez PepX, qui possde la fois un Nter et un CterSige d'une deuxime fonction ?

  • ContributionsP. RigoletINRA LURE ENS CachanJ. F. ChichINRAM. M. DelageINRAI. MechinEMBL

    Le fichier a t copier depuis word select all est important) et collage special en tant quimage sous Powerpoint!Comme dhabitude cette table a t copier puis coller (collage spcial) sous forme dimage! On peut ensuite la redimensionner DANS TOUS LES SENS souhait!!!!Un de nos objectifs, comme il a t rappel est d'intgrer l'information structurale aux projets du laboratoire.Notre dmarche s'appuiera sur notre exprience en ce domaine et intgrera tant que faire se peut les nouvelles techniques utilises en cristallographie des protines. Comme ce fut le cas pour l'tude structurale de cette aminopeptidase bactrienne, rpondant au nom de PepX, que j'ai rsolu une rsolution de 2.2 A, compose de quatre domaines distincts. Un domaine catalytique, en vert sur la figure, un domaine impliqu dans la liaison au substrat en orange sur la figure, un domaine N terminal impliqu dans l'interface du dimre et enfin un domaine C-tal dont la fonction reste prciser.Nous verrons dans quelques instants que les hlicases de la famille RecQ, s'organisent elles aussi en plusieurs domaines impliqus chacun dans une des fonctions de l'enzyme. Animation : 60 images moyenne rsolution (cartoonD) 180 ms entre chaque => 5.5 images par seconde. Fichier moovix13.gif, taille 1.3 Mo.

    Malgr ces diffrences, l'volution a conserv pratiquement le mme site catalytique chez les deux enzymes. Peut-tre le plus performant pour catalyser la libration d'un dipeptide contenant une proline, acide amin bien particulier, en 2me position de la squence. Cette surprenante conservation de l'arrangement spatial des rsidus les plus impliqus dans la catalyse (pour seulement 13% d'identit de squence) autorise l'essai des inhibiteurs de l'enzyme humaine, bien tudie sur l'enzyme bactrienne o aucun inhibiteur spcifique n'est connu ce jour.Le dveloppement d'inhibiteurs spcifiques de PepX pourra ensuite tre envisag sur la base des diffrences structurales localises entre les deux sites actifs de l'enzyme humaine et de l'enzyme bactrienne. C'est ainsi que l'Arginine prsente ici seulement chez l'enzyme humaine devrait empcher un inhibiteur s'insrant dans PepX d'entrer dans le site actif de DPP-IV. D'autres cibles peuvent aussi tre exploites.