Transcript Making Directed Libraries Using Rosetta

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
E6AP
DHFR[1]
Ubc12*
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
PCA control experiments
Partner 1
E6AP -DHFR [1]
None
Partner 2
Ubc12* -DHFR [2]
Ubc12* -DHFR [2]
Cell growth
w.t.
+++++
F63Y
++++
F63W
++++
F63L
+++++
F63D
-
F63E
-
F63Q
-
F63N
-
F63H
+++
F63K
-
F63R
-
F63L
-
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 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
-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
23 residues form hydrogen bonds with the target arginine
Ehbond
635
639
642
Asn
-0.31
His
Ehbond
655
His
-0.26
-0.42
Asp
-0.77
Asp
-0.80
Glu
-1.18
Glu
-0.06
Gln
-1.15
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
Gln
-1.21
Tyr
-0.70
Glu
-2.11
Ser
-0.90
Thr
-0.94
659
698
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 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
628
635
638
639
642
653
A N
M H
L D
L
A
F
Y
S
A
S
T
H
D
E
L
A
M
Q
E
S
T
L
V
I
L
S
L
L
L
Library size: 5.42 x 106
655
659
682
690
694
695
698
M H
V D
E
V
Q
T
E
V
I
E
A
Y
E
S
A
Y
S
A
I
Y
M I
I
I
F
Y
S
I
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
Theoretical complexity: 5.54 x 108
Random library size: 1.55 x 1017
Q
F
Y
Stop
3
13
2
17
11
2
6
6
2
6
6
2
4
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.
Selection statistics
Library
size
µg DNA
# of
# of
transformed transformants 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
Four colonies from Rd4 library were individually tested.
All of them tested positive.
Positive clones
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
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
I
W
T
M
D
L
H
A
K
E
M
Q
Library Size : 8.14 x 107
S
G
Actual library size: 5.4 x 107
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
Selection results
Library
size
µg DNA
# of
# of
transformed transformants survivors
%
survival
Round 1:
5.4 x 107
2.6
?
1000
?
Round 2:
2.1 x 105
0.5
?
>106
?
Rd2 surviving
population 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
S
S
S
I
A
V
F
S
A
V
V
V
I
T
L
G
F
R
E
G
M
A
M
T
A
L
L
L
V
V
I
I
Q
W
T
M
D
L
H
A
K
E
M
Q
Library Size : 8.14 x 107
S
G
Actual library size: 5.4 x 107
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
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