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Making Directed Libraries using Rosetta

Solve E6AP-Ubc12 complex binding issue by mutating key phenylalanine residues and screening a mutation library for optimal pairs. Implement Protein Complementation Assay and analyze crystal structures.

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Making Directed Libraries using Rosetta

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  1. Making Directed Libraries using Rosetta Gurkan Guntas (Kuhlman Lab)

  2. PROBLEM • E6AP has several natural binding partners including UbcH7. • Most partners have a crucial phenylalanine at the interface. • The crystal structure of E6AP-UbcH7 complex has been solved. • Ubc12 does not normally bind E6AP, but recently it has been • engineered to bind E6AP. However, it binds to other proteins • in E6AP’s family. • GOAL • Design an orthogonal E6AP – Ubc12 pair

  3. STRATEGY • Mutating out the crucial phenylalanine in the engineered Ubc12 • (Ubc12*) that binds E6AP. • Compensating the loss of binding by introducing a library of • mutations into E6AP. • Screening the library to identify binding pairs.

  4. E6AP Ubc12* Protein Complementation Assay DHFR[1] DHFR[2] DHFR [1] and DHFR [2] fragments are the two halves of the monomeric enzyme Dihydrofolate Reductase. Its activity is required for (thymine synthesis) cell growth in the absence of any exogenous nucleotides. Michnick, SW Proc Natl Acad Sci U S A. 1998; 95(21):12141-6

  5. PCA control experiments

  6. Crystal structure of E6AP – UbcH7 complex Pavletich, NP Science. 1999; 286(5443):1321-6

  7. Targeting Ubc12 (F63R) • Protocol • Ubc12* sequence is threaded onto UbcH7 backbone. • All E6AP side chains proximal to the target arginine are removed. • (25 E6AP residues are set to alanine and the structure is repacked) • www.rosettadesign.med.unc.edu • 3. First round of analyze_interface identifies residues that form • hydrogen bonds with the arginine.

  8. A mutlist that introduces 99 double mutations (9 HECT residues * 11 amino acids that can form hydrogen bonds) was used to determine the best hydrogen bonding pairs. e.g. 99 2 694 A A S 63 D F R 2 694 A A E 63 D F R residue 63 of chain D is mutated from alanine to arginine

  9. -s X.pdb -Wpack_only -interface -linmem_ig -try_both_his_tautomers -soft_rep_design -mutlist mutlist1 -intout results -ex1 6 -ex2 6 -ex3 -ex4 -extrachi_cutoff 1 -fa_output -output_structure

  10. 23 residues form hydrogen bonds with the target arginine

  11. Using the set of hydrogen bonding pairs obtained from the first round, a second run of analyze interface was carried out to determine the triplets (2 E6AP side chains & target Arg) that form hydrogen bonds. The command line was the same as the first round. • Mutlist: • START • 224 • 3 • 694 A A S • 642 A A E • 63 D A R

  12. 642 Glu 694 Ser

  13. 642 Ser 694 Glu

  14. 659 Thr 698 Tyr

  15. Using each of the 46 models, fixed backbone designs were done • to pack around the hydrogen bonding residues. • Note: Nonpolar amino acids were preferred to design the • remaining alanine residues. • -design -fixbb -resfile X -s X.pdb -soft_rep_design • -multi_chain -try_both_his_tautomers -fa_ouput • -ex1 4 -ex2 4 -ex3 -ex4 -extrachi_cutoff 1 -profile • -ndruns 100

  16. Library size: 5.42 x 106

  17. Theoretical complexity: 5.54 x 108 Random library size: 1.55 x 1017

  18. Theoretical complexity: 5.54 x 108 Actual library size: 0.4 x 108 Probability of sampling a particular clone: 7 % Selection BL21 cells expressing Ubc12* F63R-DHFR[3] were transformed with the plasmid library and incubated on selective solid media at room temperature.

  19. Selection statistics Four colonies from Rd4 library were individually tested. All of them tested positive.

  20. Positive clones

  21. 659 Gln 694 Glu

  22. 659 Gln 698 Tyr

  23. Designing E6AP to bind Ubc12*-F63Q • DDMI protocol was used first to dock E6AP against Ubc12* F63Q • and then design the interface. • -design -dock_des_min_inter -dock_pert 1 1 5 -resfile Y -s X.pdb • -read_all_chains -series qq -protein X -chain _ -multi_chain • -try_both_his_tautomers -linmem_ig -ex1 -ex2 -exOH -extrachi_cutoff 1 • -tight_hb -set_interface_cutoff 7.0 -nstruct 1000 • ddg_bind_only was used to compute binding energies • -interface -Wpack_only -ddg_bind_only -soft_rep_design -l pdblist

  24. Library Size : 8.14 x 107 Actual library size: 5.4 x 107

  25. Selection results Rd2 surviving population sequenced

  26. Library Size : 8.14 x 107 Actual library size: 5.4 x 107

  27. Future work • Determining the affinity of E6AP mutants for Ubc12* F63R • More stringent Round 3 selection of the clones that seem to bind • Ubc12* F63Q • Using –grid_dock as an alternative to –dock_pert

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