Supporting Methods
Input structure energy minimization
Many protein X-ray crystal structures have minor clashes of atomic radii, improper
orientation of asparagine, glutamine, or histidine side-chains, or backbone dihedral
angles outside of acceptable ranges.1 All of these problems can adversely affect the
Rosetta score function. Thus every input X-ray crystal structure was repacked and
minimized with Rosetta. This process has three steps. First, the side-chain χ angles are
minimized to optimize local contacts, next a full rotamer packing step is done to relieve
any clashes that minimization could not solve. Finally, the side-chains, backbone and
rigid body orientation is minimized to obtain the structure used for further analysis.
Most minimized output structures had an RMSD of less than 1.5 Å to the native crystal
structure. The designed models were also run through this protocol to allow comparison
between the designs and native structures. An example command line for this protocol
is given below:
./min_pack_min.<exe>
-database <rosetta_database>
-l start_structs.list
-pack_first false
-no_rbmin false
-min_all_jumps true
-nstruct 50
-score12prime true
-out::pdb_gz true
-ndruns 5
-run::min_type dfpmin_armijo_nonmonotone
-use_input_sc true -ex1 -ex2 -extrachi_cutoff 1
-no_his_his_pairE true
-ignore_unrecognized_res
-no_optH false
Interface analysis protocol
The protein-protein interfaces were analyzed using the InterfaceAnalyzerMover 2. This
protocol takes a protein-protein complex as an input structure and then creates an
unbound structure. The interface energy, SASA, and other metrics are calculated based
on the differences between the bound and unbound structures.
./rosetta_scripts.<exe>
-l minimized_structs.list
-parser:protocol interface_analysis.xml
-score12prime true
-ignore_unrecognized_res
-no_his_his_pairE
-out:file:score_only IA.score.sc
-no_optH false -ex1 -ex2 -use_input_sc
-run::min_type dfpmin_armijo_nonmonotone
-extrachi_cutoff 1
-linmem_ig 10
-ignore_unrecognized_res
-atomic_burial_cutoff 0.01
-sasa_calculator_probe_radius 1.2
Where interface_analysis.xml is the following script:
<ROSETTASCRIPTS>
<SCOREFXNS>
<s12_prime weights="score12prime"/>
</SCOREFXNS>
<TASKOPERATIONS>
</TASKOPERATIONS>
<MOVERS>
<InterfaceAnalyzerMover name=fullanalyze scorefxn=s12_prime
packstat=1 pack_input=0 pack_separated=1 jump=1 tracer=0
use_jobname=1 resfile=0/>
</MOVERS>
<PROTOCOLS>
<Add mover_name=fullanalyze/>
</PROTOCOLS>
</ROSETTASCRIPTS>
Interface hot-spot calculation
Hotspot residues at designed and natural interfaces were found by mutating all
interface residues, except for glycine, to alanine and calculating Gbind with Rosetta. A
residue was judged to be a hotspot if the calculated was greater than +2.0 REU. Only
natural and designed heterodimers were used. The following command line was used to
run the protocol:
rosetta_scripts.default.macosclangrelease -l
heterodimer_pdbs.list -parser:protocol interface_hotspots.xml
The script interface_hotspots.xml is adapted from3, it also makes use of a score function
trained to recapitulate experimental Gbind4.
<ROSETTASCRIPTS>
<SCOREFXNS>
<interface_score weights=interface/>
</SCOREFXNS>
<TASKOPERATIONS>
<RestrictToInterfaceVector name=interface_task
jump=1/>
</TASKOPERATIONS>
<FILTERS>
<TaskAwareAlaScan name=ala_scan
task_operations=interface_task jump=1 repeats=3
scorefxn=interface_score repack=1 report_diffs=1
exempt_identities=GLY write2pdb=1 />
</FILTERS>
<MOVERS>
</MOVERS>
<PROTOCOLS>
<Add filter_name=ala_scan/>
</PROTOCOLS>
</ROSETTASCRIPTS>
Polar burial definition
Rosetta calculates solvent accessible surface area (SASA) using the Le Grand and Merz
method5 and keeps the SASA up to date as described by Leaver-Fay et al.6 The SASA for
a polar atom is sum of the SASA for that atom, plus the SASA for any bound hydrogens.
A polar atom is defined as buried if the total SASA for that atom is less than 0.1 Å 2. If a
buried polar atom does not have a hydrogen-bonding partner, as defined as having a
hydrogen-bond energy of less than 0.0 REUs, then that atom is considered buried and
unsatisfied. A hydrogen bond is defined as buried if the SASA for the two involved polar
atoms is less than 3.0 Å2. Based on distances observed from low B-factor waters to
protein atoms7 we chose to use atomic radii from Reduce8 and a water probe radius of
1.2 Å to find buried polar atoms and hydrogen bonds.
References
1. Davis IW, Leaver-Fay A, Chen VB, Block JN, Kapral GJ, Wang X, Murray LW,
Arendall WB, Snoeyink J, Richardson JS, et al. (2007) MolProbity: all-atom contacts
and structure validation for proteins and nucleic acids. Nucleic acids research 35:W375–
83.
2. Lewis SM, Kuhlman BA (2011) Anchored design of protein-protein interfaces. PloS
one 6:e20872.
3. Meenan NAG, Sharma A, Fleishman SJ, Macdonald CJ, Morel B, Boetzel R, Moore
GR, Baker D, Kleanthous C (2010) The structural and energetic basis for high selectivity
in a high-affinity protein-protein interaction. Proceedings of the National Academy of
Sciences of the United States of America 107:10080–5.
4. Kortemme T, Baker D (2002) A simple physical model for binding energy hot spots in
protein-protein complexes. Proceedings of the National Academy of Sciences of the
United States of America 99:14116–21.
5. Le Grand SM, Merz KM (1993) Rapid Approximation to Molecular Surface Area via
the Use of Boolean Logic and Look-Up Tables. Journal Of Computational Chemistry
14:349–352.
6. Leaver-Fay A, Butterfoss G, Snoeyink J, Kuhlman B (2007) Maintaining solvent
accessible surface area under rotamer substitution for protein design. Journal Of
Computational Chemistry 28:1336–1341.
7. Matsuoka D, Nakasako M (2009) Probability distributions of hydration water
molecules around polar protein atoms obtained by a database analysis. The journal of
physical chemistry. B 113:11274–92.
8. Word JM, Lovell SC, Richardson JS, Richardson DC (1999) Asparagine and
Glutamine: Using Hydrogen Atom Contacts in the Choice of Side-chain Amide
Orientation. Journal Of Molecular Biology 285:1735–1747.
9. Lawrence MC, Colman PM (1993) Shape complementarity at protein/protein
interfaces. Journal of molecular biology 234:946–50.
10. Fleishman SJ, Whitehead TA, Ekiert DC, Dreyfus C, Corn JE, Strauch E-MM, Wilson
IA, Baker D (2011) Computational design of proteins targeting the conserved stem
region of influenza hemagglutinin. Science 332:816–821.
11. Karanicolas J, Corn JE, Chen I, Joachimiak LA, Dym O, Peck SH, Albeck S, Unger T,
Hu W, Liu G, et al. (2011) A de novo protein binding pair by computational design and
directed evolution. Molecular cell 42:250–60.
Supporting Figures
0.4
0.8
Frequency
0.3
designed
heterodimers
homodimers
0.2
0.1
0.4
24
00
22
00
20
00
18
00
16
00
14
00
12
00
0.2
10
00
80
0
0.0
60
0
Frequency
0.6
DSASA (Å2)
DSASA (Å2)
50
00
45
00
40
00
35
00
30
00
25
00
20
00
15
00
10
00
50
0
0.0
>
Figure S1: Size of designed and natural interfaces. The change in SASA upon binding
for natives and designs is shown. Inset: Designs only on a smaller scale. Successful
designs are highlighted by colored lines (red = GLhelix-4, blue = HB36, purple = HB80,
green = MID1, orange = βdimer1). Solid lines represent computational designs, dashed
lines represent the crystal structure of the design.
DGbind / DSASA
0.00
-0.01
-0.02
-0.03
-0.04
0
1000
2000
3000
DSASA (Å2)
Figure S2: Comparison of interface energy density (∆Gbind/∆SASA) designed interfaces
and crystal structures of successful designs. Successful designs are highlighted by
colored points (red = GLhelix-4, blue = HB36, purple = HB80, green = MID1, orange =
βdimer1). Solid squares represent the computational model, ×’s represent the crystal
structure of the design. Least squares fit is described in main text Figure 2.
A
0.4
Frequency
0.3
designed
native
native X-tal
0.2
0.1
0.
90
0.
85
0.
80
0.
75
0.
70
0.
65
0.
60
0.
55
0.
50
0.0
Shape complementarity
B
0.4
Frequency
0.3
designed
native
native X-tal
0.2
0.1
0.
85
0.
80
0.
75
0.
70
0.
65
0.
60
0.
55
0.
50
0.
45
0.0
RosettaHoles score
Figure S3: Packing quality measure of the design models, Rosetta minimized natural
interfaces and crystal structures of native proteins. Two independent measures of
packing quality are shown; (A) the shape complementarity score9 for interface the
interface and (B) the RosettaHoles score for residues at the interface. For each metric a
value of 1.0 represents perfect packing, while lower values represent packing defects.
Dashed lines represent the crystal structures of the design models (red = GLhelix-4, blue
= HB36, HB80 = purple, green = MID1, orange = βdimer1).
0.4
Frequency
0.3
natural heterodimers
designed heterodimers
0.2
0.1
0.0
0
1
2
3
4
5
6
Hotspots / 1000 Å2
Figure S4: Number of hotspot residues at natural and designed interfaces per 1,000 Å2
(SASA) of interface area. A hotspot residue is defined as one such that mutation of that
residue to alanine gives a Gbind > +2.0 REU.
0.25
0.20
Frequencey
Fleishman set
Our lab set
0.15
0.10
0.05
0.
58
0.
55
0.
52
0.
49
0.
46
0.
43
0.
40
0.
37
0.
34
0.
31
0.
28
0.
25
0.00
DSASA(Polar)/DSASA
Figure S5: Fraction of polar interface area of two sets of designed protein interfaces.
The Fleishman et al. (Nature 2011) set represents proteins designed to bind to influenza
HA. Our lab set represents the designs included in Supporting Information Table II and.
A
0.4
Frequency
0.3
designed
native
0.2
0.1
0.0
0
1
2
3
4
5
6
2
No. Unsatisfied / 1000 Å
B
0.5
Frequency
0.4
designed
native
0.3
0.2
0.1
0.
28
0.
24
0.
20
0.
16
0.
12
0.
08
0.
04
0.
00
0.0
Buried side-chain EH-bond / DGbind
Figure S6: Buried polar atoms and buried hydrogen bonds at interfaces of design
models. Values for successful design models are shown in solid lines (red = GLhelix-4,
blue = HB36, HB80 = purple, green = MID1, orange = βdimer1). A) The number of
buried polar atoms without a hydrogen-bonding partner per 1,000 Å2 of interface area.
B) The total energy of a buried, side-chain involved hydrogen bond at the interface as a
fraction of total binding energy (∆Gbind). HB36 and HB80 acquired an additional buried
hydrogen bond due to mutations introduced by affinity maturation.
Supporting Information Table I
PROJECT
-strand
homodimer
experimental
result
success
definition
failure
failure
failure
failure
failure
failure
betadimer2
betadimer3
betadimer4
oligomer
oligomer
oligomer
oligomer
monomer
monomer
dimer KD = 1
M
dimer low
expression
monomer
monomer
1JL1_anti_des1
1JL1_parl_des1
1sc0_des1
no binding
no binding
dimer
failure
failure
failure
1c1y_tf
1c1y_v
1c1y_wy
no binding
no binding
no binding
failure
failure
failure
30 M
nonspecific
failure
>50μM
>50μM
failure
failure
2d4x_0028
2d4x_0320
2fz4_design
2onu_design
20 μM
40 μM
> 100 μM
aggregates
weak
failure
failure
failure
1g2r_334
oligomer
no
expression
dimer
failure
Design model
1cc8_3742
1cc8_0966
1cc8_1894
1cc8_4097
1cc8_LeuZipper
1cc8_4579
betadimer1
RalA
binder
design
Rac1
binder
design
Ubiquitin
binder
design
Znmediated
heterodime
r
Znmediated
homodimer
1JJV.ubq.11_0005
1Y8C.ubq.123_000
2
2ODV.ubq.9_0003
1rz4_436
1yzm_329
strong
PDB ID
3zy7
weak
failure
failure
failure
strong
3v1c
2qov_414
KD=30 nM
oligomer
oligomer
monomer/dime
r - poor
solubility
always
monomer
monomer w/o
zinc, tet
with zinc
ubch7_10266
helical_peptide
no binding
no binding
2il5_335
1he9_180
2a90_308
2d4x_557
UbcH7 and
Ankyrin
designs
Ubc12 Zn
Ankyrin
designs
PAK1
binder
design
-pix
anchor
design
G-tail Cterm helix
0097_D108A_D99G
aggregates
with zinc
aggregates
with zinc
1i2t_233
spider_roll
1i2t_1212
1i2t_3533
s032
s037
mbp_17
mbp_42
1mn8_17567
1mn8_4957
no binding
KD = 100 μM
330 μM
not soluble
not soluble
160 μM
no binding
no binding
not soluble
not soluble
bpix1
bpix2
bpix3
100 μM
155 μM
148 μM
non-specific
binding
non-specific
binding
non-specific
binding
non-specific
binding
0032_I141A
gtail1
gtail2
gtail3
gtail4
3v1e
failure
failure
failure
failure
failure
failure
failure
failure
failure
no
weak
failure
failure
failure
failure
failure
failure
failure
failure
failure weakened
failure
failure
failure
failure
failure
failure
GoLoco
extension
1255
1680
4091
aa_0273
aa_0951
aa_0971
aa_0976
Successful designs from other
Pdar/Prb
Design_11
HA binder
HB36
HB80
no binding
no binding
no binding
no binding
KD = 800 nM
no binding
no binding
labs:
KD < 10 nM
wrong
orientation
Evolved to
KD = 4 nM
Evolved to
KD = 1 nM
failure
failure
failure
failure
strong
failure
failure
2xns
weak
3q9n
strong
3r2x
strong
4eef
The design models for the designed binders to HA can be found in the supplemental
information in Fleishman et al.10 Models for Pdar/Prb from Karanicolas et al.11 are
available on Proteipedia:
http://www.proteopedia.org/wiki/index.php/User:John_Karanicolas/de_novo_protein_binding_pairs_
by_computational_design
Supporting Information Table II
Heterodimer (Chains)
1ACB (E,I)
1AVA (A,C)
1AVW (A,B)
1AXI (B,A)
1AY7 (A,B)
1B27 (A,D)
1BLX (A,B)
1BND (A,B)
1BPL (B,A)
1BRB (E,I)
1BRL (A,B)
1BVN (P,T)
1C1Y (A,B)
1CGI (E,I)
1CSE (E,I)
1CT4 (E,I)
1CXZ (A,B)
1D4V (B,A)
1D4X (A,G)
1D6R (A,I)
1DFJ (E,I)
1DHK (A,B)
1DS6 (A,B)
1DTD (A,B)
1DZB (A,X)
1E44 (B,A)
1E96 (B,A)
1EAI (A,C)
1EAY (A,C)
1EFV (A,B)
1EM8 (A,B)
1EUC (B,A)
1EUV (A,B)
1F2T (A,B)
1F34 (A,B)
1F5Q (A,B)
1F5R (A,I)
1F60 (A,B)
1FCD (A,C)
1FFG (B,A)
1FIN (A,B)
Homodimer (Chains)
1A4I (A,B)
1A4U (A,B)
1AA7 (A,B)
1AD1 (A,B)
1ADE (A,B)
1AFW (A,B)
1ALK (A,B)
1AOR (A,B)
1AQ6 (A,B)
1AUO (A,B)
1BBH (A,B)
1BD0 (A,B)
1BH5 (A,B)
1BJW (A,B)
1BMD (A,B)
1BXG (A,B)
1C6X (A,B)
1CBK (A,B)
1CDC (A,B)
1CHM (A,B)
1CNZ (A,B)
1COZ (A,B)
1CQS (A,B)
1D1G (A,B)
1DOR (A,B)
1DPG (A,B)
1DQP (A,B)
1DQT (A,B)
1DVJ (A,B)
1EAJ (A,B)
1EBL (A,B)
1EHI (A,B)
1EKP (A,B)
1EN5 (A,B)
1EN7 (A,B)
1EOG (A,B)
1EV7 (A,B)
1EWZ (A,C)
1EXQ (A,B)
1EYV (A,B)
1EZ2 (A,B)
1FR2
1FT1
1FYH
1G4U
1G4Y
1GL4
1GPW
1GUA
1H1S
1H2A
1H2S
1H31
1HE1
1HL6
1HX1
1I1R
1IAR
1IBR
1ITB
1J7D
1JIW
1JKG
1JLT
1JQL
1JTD
1JTP
1JTT
1JW9
1K9O
1KA9
1KI1
1KSH
1KTZ
1KU6
1KXP
1KXV
1L4Z
1LFD
1LUJ
1LW6
1M4U
1M9E
1MA9
1MEE
1MG9
(B,A)
(B,A)
(A,B)
(S,R)
(R,B)
(A,B)
(A,B)
(A,B)
(A,B)
(L,S)
(A,B)
(A,B)
(C,A)
(A,B)
(A,B)
(A,B)
(B,A)
(B,A)
(B,A)
(B,A)
(P,I)
(B,A)
(B,A)
(A,B)
(A,B)
(A,L)
(A,L)
(B,D)
(E,I)
(F,H)
(B,A)
(A,B)
(B,A)
(A,B)
(D,A)
(A,C)
(A,B)
(B,A)
(A,B)
(E,I)
(A,L)
(A,D)
(A,B)
(A,I)
(B,A)
1F13
1F17
1F4Q
1F6D
1F89
1FC5
1FJH
1FL1
1FP3
1FUX
1FWL
1FYD
1G0S
1G1A
1G1M
1G64
1G8T
1GD7
1GGQ
1H8X
1HDY
1HJ3
1HJR
1HQO
1HSJ
1HSS
1I0R
1I2W
1I4S
1I8T
1IPI
1IRI
1J30
1JD0
1JMV
1JOG
1JP3
1JR8
1JV3
1JYS
1K3S
1K6Z
1K75
1KGN
1KIY
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,C)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
1N0L
1NF3
1NLV
1NPE
1NQI
1NRJ
1NW9
1O6S
1OHZ
1OKK
1ONQ
1OO0
1OP9
1OPH
1OR0
1OXB
1P2J
1P2M
1P5V
1PDK
1PPF
1PQZ
1PVH
1Q40
1QAV
1R0R
1R8S
1RE0
1RJ9
1RKE
1S0W
1S1Q
1SCJ
1SGD
1SHW
1SLU
1SMP
1SPB
1STF
1SVX
1TA3
1TE1
1TMQ
1TX4
1UBK
(A,B)
(A,C)
(A,G)
(A,B)
(B,A)
(B,A)
(B,A)
(A,B)
(A,B)
(D,A)
(A,B)
(A,B)
(B,A)
(B,A)
(B,A)
(A,B)
(A,I)
(A,B)
(A,B)
(A,B)
(E,I)
(A,B)
(A,B)
(B,A)
(B,A)
(E,I)
(E,A)
(B,A)
(A,B)
(A,B)
(A,C)
(A,B)
(A,B)
(E,I)
(B,A)
(B,A)
(A,I)
(S,P)
(E,I)
(B,A)
(B,A)
(B,A)
(A,B)
(A,B)
(L,S)
1KSO
1L5X
1LBQ
1LHP
1LHZ
1LNW
1LQ9
1M0W
1M3E
1M4I
1M6P
1M7H
1M98
1M9K
1MI3
1MJH
1MKB
1MNA
1N1B
1N2A
1N2O
1N80
1NA8
1NFZ
1NU6
1NW1
1NWW
1NY5
1O4U
1OAC
1ON2
1OR4
1ORO
1OTV
1OX8
1P3W
1P43
1P6O
1PE0
1PJQ
1PN0
1PN2
1PP2
1PT5
1Q8R
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,C)
(A,B)
(R,L)
(A,B)
(A,B)
1UGH
1UJZ
1US7
1USU
1UUZ
1UZX
1V74
1VG0
1WQ1
1YCS
1YVN
2HBE
2KIN
2NGR
2SIC
2SNI
2TEC
3FAP
3YGS
4SGB
(E,I)
(B,A)
(B,A)
(A,B)
(D,A)
(A,B)
(A,B)
(A,B)
(G,R)
(B,A)
(A,G)
(B,A)
(A,B)
(B,A)
(E,I)
(E,I)
(E,I)
(A,B)
(P,C)
(E,I)
1QFH
1QHI
1QMJ
1QR2
1QXR
1QYA
1R5P
1R7A
1R8J
1R9C
1REG
1RN5
1RQL
1RVE
1RYA
1S2Q
1S44
1SCF
1SMT
1SO2
1SOX
1TLU
1TRK
1UC8
1V26
2DAB
2GSA
2HHM
2NAC
2SQC
3LYN
3SDH
7AAT
8PRK
9WGA
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(X,Y)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)
(A,B)