Mm Of Endoglucanases

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  • Mm Of Endoglucanases

    1. 1. Molecular Modeling Of Endoglucanases August, 2007
    2. 2. INTRODUCTION
    3. 3. Cellulase <ul><li>Enzyme catalyzes the beta-(1,4)-glycosidic bonds between glucose units </li></ul>
    4. 4. Cellulases: Types of Action <ul><li>In nature, organisms produce battery of cellulases : </li></ul><ul><li>Exoglucanase </li></ul><ul><li>Endoglucanase </li></ul><ul><li>Cellobiohydrolase </li></ul><ul><li>Glucosidase </li></ul>CBM Cellulosome Scheme Cohensins Dockerins Catalytic domains
    5. 5. Application of Cellulases <ul><li>Pulping in Paper industry </li></ul><ul><li>Biopolishing of cotton in the Textile industry </li></ul><ul><li>Cellulose conversion for Ethanol-production industry </li></ul>
    6. 6. GH Classification <ul><li>Glycoside hydrolase (GH) refers to enzymes that catalyse the hydrolysis of carbohydrates, e.g. </li></ul><ul><ul><li>cellulase </li></ul></ul><ul><ul><li>mannanase </li></ul></ul><ul><ul><li>amylase </li></ul></ul><ul><ul><li>xylanase </li></ul></ul><ul><li>Classify into over 100 families by Henrissat et al. (cazy database) </li></ul>
    7. 7. GH families <ul><li>Each family (e.g. family 5) </li></ul><ul><li>has the same overall structure (TIM barrel) </li></ul><ul><li>result with inversion/retention of anomeric carbon configuration (retention) </li></ul><ul><li>catalytic residues (GLU/ASP) </li></ul>
    8. 8. Pyrococcus horikoshii <ul><li>Thermophilic archaeon living in the range of 88-104°C </li></ul><ul><li>isolated in hyperthermal vent in Japan </li></ul><ul><li>Encodes hyperthermostable enzymes (e.g. beta-glucosidase) </li></ul><ul><li>Gene homologous to GH5 endoglucanase </li></ul>
    9. 9. EGPh <ul><li>Endoglucanse from P. horikoshii (EGPh) </li></ul><ul><li>Hyperthermostable (Ando et al) </li></ul><ul><ul><li>T m 95.5 ° C </li></ul></ul><ul><li>Shows activity towards crystalline cellulose </li></ul>
    10. 10. Protein Engineering <ul><li>Computational Methods </li></ul><ul><ul><li>Homology Modeling </li></ul></ul><ul><ul><li>Molecular Dynamics Simulation </li></ul></ul><ul><li>Lab project: protein expression and assay by Jorge Del-Aguila </li></ul>
    11. 11. Homology Modeling <ul><li>Target: the sequence to be modeled </li></ul><ul><li>Template: the sequence with a structure </li></ul><ul><li>Requires template structure with at least 30% sequence identity with the target sequence. </li></ul>Amino acid sequence Protein 3D structure
    12. 12. Molecular Dynamics (MD) Simulation <ul><li>Solves Newton’s equation of motions </li></ul><ul><li>Predict atomic velocities and locations </li></ul>Protein Structure Protein Dynamics
    13. 13. METHODS
    14. 14. Overview <ul><li>Modeling of EGPh model </li></ul><ul><li>Model Validation </li></ul><ul><li>Molecular Dynamics Simulations of EGPh </li></ul><ul><li>Analysis of MD </li></ul>
    15. 15. Programs and Tools <ul><li>MODELLER: homology modeling </li></ul><ul><li>VMD: visualization and tools </li></ul><ul><li>NAMD: running parallel MD simulation </li></ul><ul><li>GROMOS: analysis </li></ul><ul><li>PROPKA: pKa prediction </li></ul><ul><li>PROCHECK: protein stereochemistry </li></ul><ul><li>PROSA: protein fold </li></ul>
    16. 16. Part 1 Homology Modeling
    17. 17. Homology Modeling <ul><li>Steps: </li></ul><ul><li>Template Selection </li></ul><ul><li>Sequence Alignment </li></ul><ul><li>Model Building </li></ul><ul><li>Model Refinement and Evaluation </li></ul>
    18. 18. 1. Template Selection <ul><li>Blast Search </li></ul><ul><li>Against pdb database </li></ul><ul><li>Target sequence (gi: 14590997) </li></ul>
    19. 19. Template Selected <ul><li>Crystal Structure of the endoglucanase from Acidothermus cellulolyticus </li></ul><ul><li>(EGAc) </li></ul><ul><ul><li>Wild type (pdb code: 1ece) </li></ul></ul><ul><ul><li>Y245G mutant (pdb code: 1vrx) </li></ul></ul><ul><ul><li>2.4A resolution </li></ul></ul><ul><ul><li>358 amino acids </li></ul></ul>
    20. 20. Homology Modeling <ul><li>Steps: </li></ul><ul><li>Template Selection </li></ul><ul><li>Sequence Alignment </li></ul><ul><li>Model Building </li></ul><ul><li>Model Refinement and Evaluation </li></ul>
    21. 21. Structural-based Multiple Sequence Alignment
    22. 22. Locations of Conserved Residues <ul><li>Only 8 residues are conserved among all GH5 sequences: </li></ul><ul><li>Arg62, His116, Asn161, Glu162 (proton donor), His238, Tyr 240, Glu282 (nucleophile), also W319 </li></ul>cellotetraose E282 R62 N161 H238 Y240 H116 E162
    23. 23. Alignment of the endoglucanases sequences from Acidothermus cellulolyticus (1ece) [358 residues] and Pyrococcus horikoshii (EGPh) [458 residues] with ClustalW. Extra loop?
    24. 24. <ul><li>Template sequence: 458aa </li></ul><ul><li>Target sequence: 358aa </li></ul><ul><li>N-terminal and C-terminal of EGPh (gi:14590997) amino acids removed </li></ul><ul><li>Coz </li></ul><ul><ul><li>No homology regions found in 1ece </li></ul></ul><ul><ul><li>Not close to active site </li></ul></ul>
    25. 25. EGPh Model loop loop
    26. 26. Alignment from MODELLER (Align2d method)
    27. 27. Modeling for insertions in the loops <ul><li>Any secondary structure/motif? </li></ul><ul><li>Secondary Structure Prediction performed by PSIPRED </li></ul>
    28. 28. Secondary Structure Prediction by PSIDPRED
    29. 29. Homology Modeling <ul><li>Steps: </li></ul><ul><li>Template Selection </li></ul><ul><li>Sequence Alignment </li></ul><ul><li>Model Building </li></ul><ul><li>Model Refinement and Evaluation </li></ul>
    30. 30. Model Building <ul><li>Program: MODELLER </li></ul><ul><li>Target Sequence: EGPh (gi:14590997) </li></ul><ul><li>Template Structure: A. cellulolyticus (pdb: 1ece) </li></ul><ul><li>Generation of 100 models </li></ul><ul><li>Selection using the lowest DOPE score </li></ul><ul><li>Dope scores </li></ul><ul><ul><li>Compares models created by MODELLER </li></ul></ul><ul><li>Range -46514 to -45629 </li></ul>
    31. 31. Homology Modeling <ul><li>Steps: </li></ul><ul><li>Template Selection </li></ul><ul><li>Sequence Alignment </li></ul><ul><li>Model Building </li></ul><ul><li>Model Refinement and Evaluation </li></ul>
    32. 32. MODEL REFINEMENT <ul><li>Energy minimization and MD were carried out to refine the selected model </li></ul><ul><li>2ns MD </li></ul>
    33. 33. Evaluation of Models <ul><li>PROSA </li></ul><ul><ul><li>Z-score (using knowledge-based potentials) to assess the quality of protein structure </li></ul></ul><ul><ul><li>Sippl, M. J. Proteins., 17, pp. 355-362,(1993) </li></ul></ul><ul><li>PROCHECK </li></ul><ul><ul><li>Stereochemistry </li></ul></ul>
    34. 34. Z-score: -8.48 EGAc crystal structure (pdb code:1ece) EGPh model Z-score: -8.17
    35. 35. https://prosa.services.came.sbg.ac.at/prosa.php EGAc crystal structure (pdb code:1ece) EGPh model
    36. 36. Z-score (68.pdb) -8.17
    37. 37. Z-score (after 2ns MD) -9.1
    38. 38. Z-score(after 2ns&Bmin) -9.32
    39. 39. DOPE profile before and after MD
    40. 40. Evaluation of Models <ul><li>Dope scores </li></ul><ul><ul><li>Compares models created by MODELLER </li></ul></ul><ul><li>PROCHECK </li></ul><ul><ul><li>Stereochemistry </li></ul></ul><ul><li>PROSA </li></ul><ul><ul><li>Z-score (using knowledge-based potentials) to assess the quality of protein structure </li></ul></ul><ul><ul><li>Energy profile for each position in the model </li></ul></ul>
    41. 41. 100% 314 Total 37+23+2 Gly+Pro+end residues 85.4% 2 68 Most favored regions 12.1% 35 Additional allowed regions 1.6% 5 Generously allowed regions 1.0% 3 Disallowed regions % # Residues in
    42. 43. <ul><li>Talk about 328 to 330 movement </li></ul>
    43. 44. Y 264 W 233 W 337 F 29 EGPh Y 245 W 213 W 319 F 29 EGAc
    44. 45. -2 -1 +1 +2
    45. 46. Nucleophile -E 282 E 162 – proton donor
    46. 47. Accepted Model <ul><li>Insert a model with substrate, label cysteine locations, binding site, catalytic residues, binding platform </li></ul>
    47. 49. Part 2 MD simulations of EGPh & EGAc
    48. 50. Molecular Dynamics Protocol <ul><li>1. Preparation </li></ul><ul><ul><li>Obtain pdb file </li></ul></ul><ul><ul><li>Add hydrogen (check pKa) </li></ul></ul><ul><ul><li>Solvate the molecule in a box </li></ul></ul><ul><ul><li>Add ions </li></ul></ul><ul><li>2. Energy Minimization </li></ul><ul><li> </li></ul><ul><li>3. Heating and Equilibrium </li></ul><ul><li>4. Production Run(s) </li></ul>
    49. 51. Preparation Obtain pdb file Step 1 Check pKa & add H atoms accordingly Step 2 Solvate the protein in a water box Step 3 Add ions (Na + or Cl - )
    50. 52. Molecular Dynamics Protocol <ul><li>1. Preparation </li></ul><ul><ul><li>Obtain pdb file </li></ul></ul><ul><ul><li>Adding hydrogen (check pKa) </li></ul></ul><ul><ul><li>Solvate the molecule in a box </li></ul></ul><ul><ul><li>Add ions </li></ul></ul><ul><li>2. Energy Minimization </li></ul><ul><li> </li></ul><ul><li>3. Heating and Equilibrium </li></ul><ul><li>4. Production Run(s) </li></ul>
    51. 54. EGPh EGAc
    52. 59. Thermostability <ul><li>Increase by </li></ul><ul><li>Higher number and special locations of Pro/Arg </li></ul><ul><li>Lower # of Gly </li></ul><ul><li>More aromatic, hydrophobic, charge-mediated interactions (salt bridges); more hydrogen bonds </li></ul><ul><li>Smaller accessible surface area </li></ul>
    53. 60. Comparison of Amino Acids Composition in EGAc and EGPh Charged residues: 55 vs. 80 3.99 15 5.87 21 Ala (A) 5.85 22 6.42 23 Val (V) 6.65 25 5.31 19 Ile (I) 7.18 27 8.38 30 Leu (L) 1.86 7 1.40 5 Met (M) 6.12 23 5.87 21 Pro (P) Hydrophobic 4.79 18 3.35 12 Phe (F) 9.84 37 9.22 33 Gly (G) Residues Mol% # Mol% # EGPh (total 376aa) EGAc (total 358aa)
    54. 61. # 80 7.18 27 7.26 26 Asp (D) Charged 5.05 19 1.96 7 Glu (E) 2.66 10 3.63 13 Arg (R) 6.38 24 2.51 9 Lys (K) 6.12 23 6.70 24 Asn (N) 1.86 7 5.59 20 Gln (Q) 1.06 4 1.12 4 Cys (C) 4.79 18 7.26 26 Ser (S) 5.32 20 5.31 19 Tyr (Y) Polar 5.05 19 5.03 18 Trp (W) 5.32 20 6.15 22 Thr (T) 2.93 11 1.68 6 His (H) Residues Mol% # Mol% # EGPh (total 376aa) EGAc (total 358aa) # 55
    55. 63. Salt Bridges
    56. 64. CONCLUSION <ul><li>EGPh sequence belong to subfamily 1 </li></ul><ul><li>Model reveals the catalytic center of EGPh is structurally conserved barrel </li></ul><ul><li>refined model has a (β/ α ) 8 barrel structure </li></ul><ul><li>Uncertainty in modeling the loops that follows β­4 and β­5 </li></ul><ul><li>Substrate subsites at least from -2 to +2 </li></ul><ul><li>Higher number of charged residues in EGPh; extensive ionic network </li></ul>
    57. 65. References
    58. 66. Extras
    59. 67. RMSD (Root mean square deviation) <ul><li>Numerical measure of the difference between two structures </li></ul>
    60. 68. Root Mean Square Deviation <ul><li>RMSD (structure vs. time) </li></ul><ul><li>RMSF (C-alpha averages) </li></ul>
    61. 69. 12 Families with endoglucanse activity Asp (experimental)  Not known  Glu  Asp (experimental)  Not known Glu (experimental)  Glu (experimental)  Glu (experimental)  Glu (experimental)  Glu (experimental)  Asp (experimental)  Glu (experimental)  Catalytic Proton Donor Fold 7-fold β -propeller --- Fold ( α / α ) 6 --- Not available Fold ( β / α ) 8 Fold -jelly roll Fold ( α / α ) 6 Fold ( α / α ) 6 Fold -jelly roll --- Fold ( β / α ) 8 Fold Asp (experimental)  I endoglucanase; etc 74 Not known  -- endoglucanase 61 Not known  I endoglucanase; cellobiohydrolase. 48 Clan M Asp (experimental)  I endoglucanase.  45 Not known I endoglucanase; xyloglucanase 44 Glu (experimental)  R mannanase; -1,3-xylanase 26 Clan A Glu (experimental)  R endoglucanase; xyloglucan hydrolase ; -1,3-1,4-glucanase 12 Clan C Asp (experimental)  I endoglucanase; cellobiohydrolase; -glucosidase 9 Asp (inferred)  I Chitosanase; Cellulase; Licheninase; Endo-1,4--xylanase 8 Clan M Glu (experimental)  R endoglucanase; reducing end-acting cellobiohydrolase 7 Clan B Asp (experimental)  I Endoglucanase;cellobiohydrolase 6 Glu (experimental)  R Cellulase (EC 3.2.1.4); Glucan 1,3--glucosidase (EC 3.2.1.58); Endo-1,4--xylanase (EC 3.2.1.8); Cellulose 1,4--cellobiosidase (EC 3.2.1.91), etc 5 Clan A Catalytic Nucleophile/Base Mechanism Known Activity CAZY Family
    62. 70. Ligand: cellutetraose Catalytic Site
    63. 71. Classification <ul><li>Exoglucanase </li></ul><ul><li>Endoglucanase * </li></ul><ul><li>Glucosidase </li></ul><ul><li>Cellobiohydrolase </li></ul>

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