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. Protein Complementation Assay DHFR[1] DHFR[2] E6AP Ubc12* 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. Partner 1 Partner 2Cell growth E6AP -DHFR [1]Ubc12* -DHFR [2] w.t.+++++ F63Y++++ F63W++++ F63L+++++ F63D- F63E- F63Q- F63N- F63H+++ F63K- F63R- NoneUbc12* -DHFR [2] F63L- PCA control experiments

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

7. Targeting Ubc12 (F63R) Protocol 1.Ubc12* sequence is threaded onto UbcH7 backbone. 2.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. E hbond 635Asn-0.31655His-0.26 His-0.42Asp-0.77 Asp-0.80Glu-1.18 Glu-0.06659Gln-1.15 639Ser-0.80Thr-1.02 Thr-1.04Glu-1.04 His-0.89690Glu-1.18 Asp-0.74694Glu-0.93 Glu-0.26Ser-0.99 642Gln-1.21698Tyr-0.70 Glu-2.11 Ser-0.90 Thr-0.94 23 residues form hydrogen bonds with the target arginine

11. 4.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. E6AP residues hbonding with ArgTotal delta(E hbond ) 642 Glu 694 Ser-3.08 642 Gln 694 Ser-2.38 642 Thr 694 Glu-2.53 642 Ser 694 Glu-2.49 635 His 642 Glu-2.40 639 His 659 Thr-2.25 639 Asp 690 Glu-1.83 655 Glu 694 Glu-1.79 659 Glu 698 Tyr-1.69 659 Gln 698 Tyr-1.92 659 Thr 698 Tyr-1.63

13. 642 Glu 694 Ser

14. 642 Ser 694 Glu

15. 659 Thr 698 Tyr

16. 5.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

17. 628635638639642653655659682690694695698 ANSSQMHQVEESI MHATEVDTIASAY LDHSEEYA LDTVY AEL FLV YAI M LLSLLMIIIFYSI Library size: 5.42 x 10 6

18. 628635638639642653655659682690694695698 ANSSQMHQVEESI MHATEVDTIASAY LDHSEEYAF EDTVADYN LELQKSD ALVLPStop FAI YMA SIK TNP IK VV PP Q F Y 3132171126626624 Theoretical complexity: 5.54 x 10 8 Random library size: 1.55 x 10 17

19. Theoretical complexity: 5.54 x 10 8 Actual library size: 0.4 x 10 8 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.

20. Library size µg DNA transformed # of transformants # of survivors % survival Round 1: 4 x 10 7 24 x 10 8 1000 -20000.0005 Round 2: 2 x 10 6 0.30.6 x 10 8 6000.001 Round 3: 1 x 10 6 0.30.6 x 10 8 10 5 - 10 6 1.7 Selection statistics Four colonies from Rd4 library were individually tested. All of them tested positive.

21. 628635638639642653655659682690694695698 MFAIIMQQISEAN MFAIIMQQISEAY LFSLLMHQIADAY LFSLIMQQISEAN LLSLLMIIIFYSI Positive clones deltaE total delta(E hbond ) 655 His 694 Glu-1.01-0.34 659 Gln 694 Glu-1.16-1.30 659 Gln 698 Tyr-1.64-1.92

22. 659 Gln 694 Glu

23. 659 Gln 698 Tyr

25. 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

26. 634635638639642653655659660661682690694 TAllaaSLLLTTSTIYY AASSSIAVFS VVITLGF RGMAMTA LLVVIL IWTMH DA K E M Q S G P N H 162167165241226 VLSLLMIISQIFY Library Size : 8.14 x 10 7 Actual library size: 5.4 x 10 7

27. Library size µg DNA transformed # of transformants # of survivors % survival Round 1: 5.4 x 10 7 2.6?1000? Round 2: 2.1 x 10 5 0.5?>10 6 ? Selection results Rd2 surviving population sequenced

28. 634635638639642653655659660661682690694 TAllaaSLLLTTSTIYY AASSSIAVFS VVVITLGF REGMAMTA LLLVVIL IQWTMH DA K E M Q S G P N H 162167165241226 VLSLLMIISQIFY Library Size : 8.14 x 10 7 Actual library size: 5.4 x 10 7

29. 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