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
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
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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
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
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
66% Specific to PepX
Prolyl Oligopeptidase
(POP, porcine muscle)
1JU3 , 710 residues
64% Specific to PepX
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 ;