Download - Bioinformatique et Biologie Structurale I/ – Principes et techniques

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

C/ Relations structure-fonction d’hélicases impliquées dans les cancers

X-PDAP activity

Selectivity : Pro ; ALA 10% AS ; GLY 1 % AS

Two families : S9B and S15 for the same enzyme activity

S9B : DPP-IV [eukaryotic and prokaryotic]

membrane-bound and soluble forms

S15 : PepX [prokaryotic]

cytoplasmic

X-Pro –AA3-AA4-AA5... AAn X-Pro + AA3-AA4-AA5... AAn -Ala- -Gly-

PepX

exopeptidase

Proline specific proteases : a rare group

- Many biologically active peptides contain an evolutionary conserved

proline residue as a proteolytic processing regulatory element

- Proline-specific proteases : important 'check-points' control

- Importance in some disease to inhibit such proteases

X-PDAP : S9b and S15 family evolutionarily distant enzymes DPP-IV and PepXImportance of the enzyme activity in prokaryotics

Proteases 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,…)

X-PDAP activity

The signature of the X-PDAP specificity in SC Clan enzymes

Structure / function relationships in X-PDAP enzymes

What makes the enzymes so specific?

- Clans, families of proteases and X-PDAP activity

- Structure of PepX, X-PDAP from Lactococcus lactis

- Comparison of bacterial and human X-PDAP structures

- Insights for drug design

- Conclusion

SC Clan

Almost 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 lactisDiffraction data and MIR analysis

Native HgNO3 Thiomersal

HgCl2-1 HgCl2-2

Space group P21212 P21212 P21212 P21212 P21212

Soaking concentration (mM) 1 23 1 2.5

Soaking time (hours) 22 192 24 46

Resolution (Å) 25 - 2.2 20 - 2.7 23 - 2.6 20 - 2.4 21 - 2.4

Number of observations 153738 62438 113924 62480 81848

Unique Reflections 46073 20661 28691 27354 32819

Multiplicity 3.3 3.0 4.0 2.3 2.5

Completeness (%) (last shell) 95.8 (95.3)

80.0 (80.9)

97.1 (98.7 )

74.3 (78.3)

87.0 (63.1)

Rsym (%) (last shell) 5.7 (17.0 ) 6.9 (16.6) 7.5 (15.3 ) 5.0 (12.0) 8.5 (22.7)

<I/I> 11.8 9.7 8.4 11.7 7.6

0.60 0.72 0.59 0.65 RCullis Centric Acentric 0.58 0.75 0.56 0.60

1.49 1.02 1.55 1.33 Phasing Power Centric Acentric 2.25 1.39 2.31 2.12 Mean figure of merit 0.64

Refinement data (20.0-2.2Å) and analysis of molecular model

Number of reflections 45043 rms deviation bonds (Å)/angles

(deg ) 0.006 / 1.2

Atoms protein/water molecules

6200 / 305 rms deviation dihedral angles (deg )

24.4

Rworking /Rfree (%) 18.4 / 22.3 rms deviation improper angles (deg )

0.74

Mean B factors (Å2) 12.4

Table I : Crystallographic data and refinement statistics.

10 % PEG 4000, 150mM NaCl

Ph 5.2 MES NaOH, 18°C

PepX prototype de la famille S15

- 4 domaines- hydrolase fold- éléments remarquables (peptide lasso, Boucle C-ter, …)

helical

N-terminal

catalytic

C-terminal

lasso

85 Å 25 Å

Rigolet, P. et al. (2002). Structure 10, 1383-1394

Enzymes families of SC Clan and related structures

Catalytic domain ( 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

SPAP[317

residues]

S33 family

CBPY[416

residues]

S10 family

POP[710

residues]

S9A family

DPP-IV[726

residues]

S9B family

CBPY[416

residues]

S10 family

228 CA(3.03 Å)17.5 %

POP[710

residues]

S9A family

180 CA(3.17 Å)15.3 %

154 CA(3.47 Å)17.6 %

DPP-IV[726

residues]

S9B family

207 CA(3.11 Å)10.8 %

189 CA(2.95 Å)11.9 %

451 CA(3.50 Å)19.8 %

PepX[763

residues]

S15 family

175 CA(3.02 Å)16.4 %

184 CA(3.30 Å)12.3 %

207 CA(2.63 Å)16.8 %

201 CA(3.15 Å)17.8 %

Cocaine Esterase

(COCE,)

1JU3 , 565 residues

40% Specific to PepX

between PepX and :

Prolyl Iminopeptidase

(XCPIP, Xant. campestris)

1JU3 , 313 residues


Prolyl Oligopeptidase

(POP, porcine muscle)

1JU3 , 710 residues


Structure comparisons

Structure-based sequence aligment

Only 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 structure

- Important for activity- Globular shape

- Involve principally N-ter and helical domains

- Canal acces to catalytic residues

- The two active sites are far away from each other and independently accessible to the substrate

- Hydrophylic 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 structure

DPP-IV

PepX Electrostatic properties

Acidic 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 properties

DPP-IV

Differences between the two enzymes

- Nearly the same lenght (PepX 763 and DPP-IV 766)

- Different folds (moreover 4 versus 3 domains)

- Both dimer but of different quaternary organization

- DPP-IV is integrated in the plasmatic membrane whereas PepX is an

cytoplasmic enzyme

- Substrate selection processes via an N-ter propeller domain whereas via

dimeric domain in PepX.

Comparison of the specificity sites of PepX and DPP IV

Evolution conserved thus a particular arrangement of residues,

perhaps the most efficient, ensuring XPDAP activity with a high specificity.




Oxyanion hole




Signature

X-PDAP activity




?

LabelPepX

[family S15]DPP-IV

[ family S9B]POP

[family S9A]SPAP

[family S33]CBPY

[family S10]

Catalytic triad

Ser S348 S630 S554 S113 S146

Asp D468 D708 D641 D268 D338

His H498 H740 H680 H296 H397

Residues implicated in positioning of the substrate proline in the active site

Pos1 Y380 Y662 W595 - -

Pos2 L401 Y666 F476 E232 W312

Pos3 W377 W659 - L141 -

Pos4 I374 V656 V580 F139 L178

Residues stabilizing the binding of the substrate in the specificity pocket

Stb1 - R125 R643 - Y256

Stb2 N470 N710 R643 A270 I340

Oxyanion hole

Oxa1 Y349 Y631 N555 W114 Y147

Oxa2 Y210 Y547 Y473 - -

Residues responsible for the exopeptidase activity

Exo1 F393 E205 - - -

Exo2 E396 E206 - - -

Other residues postulated to play a role in enzyme specificity

Other V471 V711 V644 C271 C341

Equivalent residues in compared enzymes of the clan SC.

Structural signature of the XPDAP activity

Rigolet P. et al. . (2005). FEBS J. 272; 2050-2059.

- Exploiter différences :

R125 DPP-IV

L401 PepX / Y666 DPPIV

Recherche d’inhibiteurs spécifiques

-Tester inhibiteurs de DPPIV

(ceux de petite taille)

- Structure de PepX avec un inhibiteur de DPP-IV

Enzyme PepX DPP-IV

Compound IC50 KI IC50 KI SI a

valine-pyrrolidide 30 M 9.3 M 4 M b 2 M c 7.5

diprotin A 260 M 71.5 M 8 M d 4.6 M e 32.5

diprotin B 600 M118.45 M

Table 2: Inhibition experiments realized with PepX from Lactococcus lactis.


Valine-Pyrrolidide

Inhibition compétitive

IC50 = 30 M

KI = 9 M

Rigolet P., Xi, X.G., Rety, S. and Chich, J.F. (2005). FEBS J. 272; 2050-2059.

KI = 9 M


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

(Å)

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

Table 3: Docking simulations of valine-pyrrolidide in the X-PDAP enzymes.


Enzyme PepX DPP-IV

Compound IC50 KI IC50 KI SI a

valine-pyrrolidide 30 M 9.3 M 4 M b 2 M c 7.5

diprotin A 260 M 71.5 M 8 M d 4.6 M e 32.5

diprotin B 600 M118.45 M

Table 2: Inhibition experiments realized with PepX from Lactococcus lactis.


Nouveaux dérivés partant de la valine-pyrrolidide …

… Nous les avons synthétisés ….

… Nous les testons actuellement ….

Drug design: autres stratégies

-boucles C-TER

- interface de dimérisation

- Mutagenèse dirigée : PHE 80

Quelle est la fonction des domaines Nter et Cter?

- Qu’apportent-ils vraiment à la catalyse ?

- Rôle présenté chez DPPIV

- Pas de réelle interprétation ni chez aAeH ni chez Coce

- Rôle supposé chez PepX, qui possède à la fois un Nter et un Cter

- Siège d'une deuxième fonction ?

• P. Rigolet INRA – LURE – ENS Cachan• J. F. Chich INRA• M. M. Delage INRA• I. Mechin EMBL

Contributions

Collaboration avec CHIMISTES de l’ENS-Cachan

Equipe de Joane XIE

Synthèse organique d'inhibiteurs potentiels