Making Directed Libraries using Rosetta

Gurkan Guntas

(Kuhlman Lab)

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

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.

Protein Complementation Assay

DHFR[1]

DHFR[2]

E6AP

Ubc12*

DHFR [1] and DHFR [2] fragments are the two halves of themonomeric enzyme Dihydrofolate Reductase.

Its activity is required for (thymine synthesis) cell growthin the absence of any exogenous nucleotides.

Michnick, SW Proc Natl Acad Sci U S A. 1998; 95(21):12141-6

Partner 1 Partner 2 Cell growth

E6AP -DHFR [1]

Ubc12* -DHFR [2] w.t. +++++

F63Y ++++

F63W ++++

F63L +++++

F63D -

F63E -

F63Q -

F63N -

F63H +++

F63K -

F63R -

None Ubc12* -DHFR [2] F63L -

PCA control experiments

Crystal structure of E6AP – UbcH7 complex

Pavletich, NP Science. 1999; 286(5443):1321-6

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.

A mutlist that introduces 99 double mutations (9 HECT residues *11 amino acids that can form hydrogen bonds) was used to determinethe best hydrogen bonding pairs.

e.g.992

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

-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

Ehbond Ehbond

635 Asn -0.31 655 His -0.26

His -0.42 Asp -0.77

Asp -0.80 Glu -1.18

Glu -0.06 659 Gln -1.15

639 Ser -0.80 Thr -1.02

Thr -1.04 Glu -1.04

His -0.89 690 Glu -1.18

Asp -0.74 694 Glu -0.93

Glu -0.26 Ser -0.99

642 Gln -1.21 698 Tyr -0.70

Glu -2.11

Ser -0.90

Thr -0.94

23 residues form hydrogen bonds with the target arginine

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

E6AP residues hbonding with Arg Total delta(Ehbond)

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

642 Glu 694 Ser

642 Ser 694 Glu

659 Thr 698 Tyr

5. Using each of the 46 models, fixed backbone designs were doneto 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

628 635 638 639 642 653 655 659 682 690 694 695 698

A N S S Q M H Q V E E S I

M H A T E V D T I A S A Y

L D H S E E Y A

L D T V Y

A E L

F L V

Y A I

M

L L S L L M I I I F Y S I

Library size: 5.42 x 106

628 635 638 639 642 653 655 659 682 690 694 695 698

A N S S Q M H Q V E E S I

M H A T E V D T I A S A Y

L D H S E E Y A F

E D T V A D Y N

L E L Q K S D

A L V L P Stop Stop

F A I

Y M A

S I K

T N P

I K Stop

V V

P P

Q

F

Y

Stop

3 13 2 17 11 2 6 6 2 6 6 2 4

Theoretical complexity: 5.54 x 108

Random library size: 1.55 x 1017

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 transformedwith the plasmid library and incubated on selective solid media atroom temperature.

Library size

µg DNA transformed

# of transformants

# of survivors

% survival

Round 1: 4 x 107

2 4 x 108 1000 -2000 0.0005

Round 2: 2 x 106

0.3 0.6 x 108 600 0.001

Round 3: 1 x 106

0.3 0.6 x 108 105 - 106 1.7

Selection statistics

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

628 635 638 639 642 653 655 659 682 690 694 695 698

M F A I I M Q Q I S E A N

M F A I I M Q Q I S E A Y

L F S L L M H Q I A D A Y

L F S L I M Q Q I S E A N

L L S L L M I I I F Y S I

Positive clones

deltaEtotal delta(Ehbond)

655 His 694 Glu -1.01 -0.34

659 Gln 694 Glu -1.16 -1.30

659 Gln 698 Tyr -1.64 -1.92

659 Gln 694 Glu

659 Gln 698 Tyr

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

634 635 638 639 642 653 655 659 660 661 682 690 694

T Allaa S L L L T T S T I Y Y

A A S S S I A V F S

V V I T L G F

R G M A M T A

L L V V I L

I W T M H

D A

K

E

M

Q

S

G

P

N

H

16 21 6 7 1 6 5 2 4 1 2 2 6

V L S L L M I I S Q I F Y

Library Size : 8.14 x 107

Actual library size: 5.4 x 107

Library size

µg DNA transformed

# of transformants

# of survivors

% survival

Round 1: 5.4 x 107

2.6 ? 1000 ?

Round 2: 2.1 x 105

0.5 ? >106 ?

Selection results

Rd2 survivingpopulation sequenced

634 635 638 639 642 653 655 659 660 661 682 690 694

T Allaa S L L L T T S T I Y Y

A A S S S I A V F S

V V V I T L G F

R E G M A M T A

L L L V V I L

I Q W T M H

D A

K

E

M

Q

S

G

P

N

H

16 21 6 7 1 6 5 2 4 1 2 2 6

V L S L L M I I S Q I F Y

Library Size : 8.14 x 107

Actual library size: 5.4 x 107

Future work

• Determining the affinity of E6AP mutants for Ubc12* F63R

• More stringent Round 3 selection of the clones that seem to bindUbc12* F63Q

• Using –grid_dock as an alternative to –dock_pert