Letter

https://doi.org/10.1038/s41586-018-0802-y

Programmable design of orthogonal protein
heterodimers
Zibo Chen1,2,3, Scott E. Boyken1,2, Mengxuan Jia4, Florian Busch4, David Flores-Solis5, Matthew J. Bick1,2, Peilong Lu1,2,
Zachary L. VanAernum4, Aniruddha Sahasrabuddhe4, Robert A. Langan1,2,3, Sherry Bermeo1,2,3, T. J. Brunette1,2,
Vikram Khipple Mulligan1,2, Lauren P. Carter1,2, Frank DiMaio1,2, Nikolaos G. Sgourakis5, Vicki H. Wysocki4 & David Baker1,2,6*

Specificity of interactions between two DNA strands, or between
protein and DNA, is often achieved by varying bases or side chainscoming off the DNA or protein backbone—for example, thebases participating in Watson–Crick pairing in the double helix,
or the side chains contacting DNA in TALEN–DNA complexes.
By contrast, specificity of protein–protein interactions usually
involves backbone shape complementarity1, which is less modular
and hence harder to generalize. Coiled-coil heterodimers are an
exception, but the restricted geometry of interactions across theheterodimer interface (primarily at the heptad a and d positions2)
limits the number of orthogonal pairs that can be created simply
by varying side-chain interactions3,4. Here we show that protein–protein interaction specificity can be achieved using extensive andmodular side-chain hydrogen-bond networks. We used the Crickgenerating equations5 to produce millions of four-helix backbones
with varying degrees of supercoiling around a central axis, identified
those accommodating extensive hydrogen-bond networks, and used
Rosetta to connect pairs of helices with short loops and to optimize

HBNet

samplingΔΦ1searchCN60,000,000 backbones

90º

e

d

Connect

f

0.5

0.0

chains

h

g

1.0

5

10

AB
8+

Relative intensity (%)

100

2,700,000 combinations

20

15

Elution volume (ml)

50

0
1,000

AB
9+

2,000

m/z

3,000

25 °C
75 °C
95 °C
25 °C end

–5

40

MRE

11 parameters

MRE (103 deg cm2 dmol–1)

R

...

N

–20
–35
20

60

Temperature (°C)

100

0

–40

200

i

2

log(I) (AU)

ZoffsetC

Systematicc2,251 unique networksb

Z

Normalized A280 (mAU)athe remainder of the sequence. Of 97 such designs expressed in
Escherichia coli, 65 formed constitutive heterodimers, and the
crystal structures of four designs were in close agreement withthe computational models and confirmed the designed hydrogenbond networks. In cells, six heterodimers were fully orthogonal, andin vitro—following mixing of 32 chains from 16 heterodimer
designs, denaturation in 5 M guanidine hydrochloride andreannealing—almost all of the interactions observed by native
mass spectrometry were between the designed cognate pairs. The
ability to design orthogonal protein heterodimers should enablesophisticated protein-based control logic for synthetic biology,
and illustrates that nature has not fully explored the possibilities
for programmable biomolecular interaction modalities.
Orthogonal sets of protein–protein and protein–peptide interactions
have important roles in biological systems6. It has proven difficult touse sequence redesign to create new specificities starting from naturally
occurring interacting proteins, such as toxin–antidote pairs7 (promiscuous binding has usually resulted8); the natural specificity results at

–1

–4

230

Wavelength (nm)260

F = 2.317
00.1

q (1/nm)0.2

Fig. 1 | Modular heterodimer
design. a, Individual helix
generation: the helical phase(Δɸ1), supercoil radius (R) and
offset along the Z-axis (Zoffset) were
exhaustively sampled; a total of 11
free parameters, because there is
no Zoffset for the first helix. b, Topdown view of a representative fourhelix backbone. c, Representativehydrogen-bond networks
identified using HBNet. d, Matchesof multiple HBNet-containing
heptads to a single full-length
backbone. e, Addition of loops toconnect the four helices into two
helix hairpins. f–i, SEC trace (f),circular dichroism spectra and(inset) temperature melt (g), native
MS spectrum (h) and SAXS
(i; black, experimental SAXS data;
red, spectra computed from thedesigned backbones; q, scatteringvector) profiles of the design
DHD37_ABXB. Experiments were
performed once. A280, absorbanceat 280 nm; mAU, milli-absorbanceunits; MRE, molar residue
ellipticity.

0.3

1

Department of Biochemistry, University of Washington, Seattle, WA, USA. 2Institute for Protein Design, University of Washington, Seattle, WA, USA. 3Graduate Program in Biological Physics,
Structure, and Design, University of Washington, Seattle, WA, USA. 4Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, USA. 5Department of Chemistry andBiochemistry, University of California Santa Cruz, Santa Cruz, CA, USA. 6Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA. *e-mail: dabaker@uw.edu
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RESEARCH Letter
a

b

c

e

f

2

log(I) (AU)

d

–1

–4

g

F = 3.020

0.1

log(I) (AU)0.2

q (1/nm)

6

0.3

3

0
0

F = 2.24
0.1

0.2

q (1/nm)0.3

Fig. 2 | Structural characterization of designed heterodimers.
a–e, Crystal and NMR structures (white) superimposed on design models
with monomers coloured green and purple; coloured cross-sections ofbackbones (left) indicate locations of designed hydrogen-bond networks(middle panels). Solid and dashed red boxes compare networks in designmodel and crystal structure, respectively. Green and black boxes denoteadditional hydrogen-bond network and hydrophobic packing layers,respectively. a, DHD_131, 2.4 Å resolution with 1.0 Å Cα r.m.s.d.
b, DHD37_1:234, 3.3 Å resolution with 1.4 Å r.m.s.d. c, DHD_127,

1.8 Å resolution with 1.7 Å r.m.s.d. d, DHD_15, 3.4 Å resolution with
0.9 Å r.m.s.d.; hydrogen-bond networks were not well-resolved. e, NMR
ensemble (white) of DHD13_XAAA superimposed onto the design model;
the assigned side-chain–side-chain NMR distance constraints were notsufficient to define hydrogen-bond networks. f, g, Backbones and designedhydrogen-bond networks of DHD_39 and DHD_120. Experimental SAXS
data (black) are similar to spectra computed from the designed backbones(red).

least in part from complementary variation in backbone conformation9. Orthogonal sets of 2–4 interacting coiled-coil pairs have beencreated and experimentally validated10,11, including the widely usedSYNZIPs12–18, but interaction promiscuity has again hampered thedesign of larger orthogonal sets.
Guided by the example of the DNA double helix, we hypothesizedthat large sets of designed heterodimers could be generated by incorporating asymmetric buried hydrogen-bond networks into regularly
repeating backbone structures. We generated helical bundle heterodimers in which each monomer is a helix–turn–helix starting fromfour-helix backbones produced using a generalization of the Crickcoiled-coil parameterization5,19. For each of the four helices, we exhaustively sampled the helical phase (Δɸ1), supercoil radius (R) and offset

along the Z-axis (Zoffset) (Fig. 1a), restricting the supercoil phases of thehelices to 0, 90, 180 and 270°, and the supercoil twist (ω0) and helicaltwist (ω1) to the ideal values for either a two-layer left-handed supercoil(ω0 = −2.85 and ω1 = 102.85), or a five-layer untwisted bundle (ω0 = 0
and ω1 = 100)20 (Extended Data Fig. 1a–d). This yielded 27 million
untwisted and 60 million left-handed supercoiled backbones for bothparallel and antiparallel orientations of opposing helices (Fig. 1b,
Extended Data Fig. 1g).
To identify the modular hydrogen-bond network equivalents to DNA
base pairs, we used Rosetta HBNet21 to design buried hydrogen-bondnetworks in the central repeat units of each backbone, and obtained
2,251 hydrogen-bond networks involving at least four side-chain
residues with all heavy-atom donors and acceptors participating in

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Letter RESEARCH

Relative intensity (%)

50

11+

e
100

Relative intensity (%)

6xHis

6xHis

Network A

3,000

4,500

m/z

14+

13+

m/z
X (Hydrophobic)

15A 37A 13B 37B
vs. 9B vs. 9A vs. 9B vs. 9A
Intact complex
Scaffold
Scaffold + 9b + 13b
Scaffold + 9b + 37b
Scaffold + 13b + 37b
Scaffold + 9b
9b
Scaffold + 13b
13b
Scaffold + 37b
37b

100

50

3,000

0

f

15+

0
1,500

g

10+

14+
0
1,500

d

0.5

4,500

h

50

0
0

1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000

m/z

1.2

i

1.2

0.6

0.6

0

OD600

6xHis

13+
OD600

6xHis

– Heterodimerizer+ Heterodimerizer

c 1.0

12+

100

Relative intensityb

Relative intensity (%)

a0Fig. 3 | New functionality from DHD combinations. a, Induced
dimerizer formed from b component of DHD13_XAAA (dark blue)
fused to b component of DHD37_ABXB (dark green) with an intervening
flexible linker. The a components of the two heterodimers (light blue
and light green) are brought into close proximity by the heterodimerizer.
b, Native MS of purified DHD13_XAAA:DHD37_ABXB heterotrimer
complex; no heterodimers or monomers were observed. Molecular mass:
designed 37,133 Da; observed 37,132 Da. c, Y2H data on four induced
dimerization systems. Yellow, without heterodimerizer fusion; green, withheterodimerizer fusion. Red dashed line indicates background growthwith unfused activation domain and DBD. Data are mean ± s.d. fromthree biological repeats. d, 9_a (pink), 13_XAAA_a (light blue) and

37_ABXB_a (light green) were covalently linked to form a scaffold,recruiting 9_b (red, hexahistidine tagged), 13_XAAA_b (dark blue) and
37_ABXB_b (dark green). e, Native MS of purified scaffold complex; noheterotrimers, heterodimers or monomers were observed. Molecularmass: designed 52,979 Da; observed 52,979 Da. f, SID of the 11+ peakin e; no cross binding between b monomers is detected. g, The backboneof 2L4HC2_23 can accommodate hydrogen-bond networks at four
heptad positions. h, Native MS mixing data of four variants generated
by hydrogen-bond-network shuffling; the interactions are orthogonal.
i, Y2H data of four hydrogen-bond shuffling variants. Two biologicallyindependent experiments were performed for b, e, f, h, i.

hydrogen bonds, and connecting all four helices (Fig. 1c, Extended Data
Fig. 2, Supplementary Table 1). We then identified all of the geometrically compatible placements of these hydrogen-bond networks in eachbackbone (Fig. 1d), selected backbones that accommodated at least two
networks, and connected pairs of helices with short loops (Fig. 1e).
Low-energy sequences were identified using RosettaDesign22 calculations in which the hydrogen-bond networks were held fixed. Designs
with fully satisfied hydrogen-bond networks and tight hydrophobic
packing were selected for experimental characterization, excluding
those with networks with C2 symmetry to disfavour homodimerization of monomers (Extended Data Fig. 1e, f). Designed heterodimers(DHDs) are referred to by numbers with monomers labelled a or b; forexample, DHD15_a refers to monomer a of design DHD15.
Of the 97 selected designs (Supplementary Table 2), 94 were wellexpressed in E. coli with both monomers co-purifying by Ni-affinity
chromatography (only one monomer contains a hexahistidine tag;
Supplementary Table 3). For 85 of these 94, the dominant species
observed in size-exclusion chromatography (SEC) had the expected size
(Fig. 1f). Thirty-nine of these 85 were exclusive heterodimers at 15 μM
by native mass spectrometry (MS)23,24 (Fig. 1h), 13 were heterodimers
with a minor population of heterotetramers, and 13 formed heterodimers with one monomer (but never both) also present as a homodimer arising from unbalanced expression in E. coli (Supplementary
Tables 4–6). Native MS experiments with serially diluted samples
suggested that the DHDs have affinities in the nanomolar range(Supplementary Table 7). Three designs characterized by circulardichroism spectroscopy were found to be all α-helical and stable at
95 °C (Fig. 1g, Extended Data Fig. 3).
We investigated the extent to which the heterodimer set could be
expanded by permuting the hydrogen-bond networks in the different helical repeat units, and by permuting the backbone connectivity.
Assigning each unique network a letter, DHD37_XBBA indicates avariant in which the second, third and fourth repeat units have hydrogenbond networks B, B and A, and the first heptad has exclusively hydrophobic residues in the core, whereas DHD103_1:423 indicates a heterodimer in which one monomer consists of the first helix of DHD103
and the other monomer consists of helices 2–4 (Extended Data Fig. 4).
Thirteen of fourteen hydrogen-bond-network-permuted variants andnine of ten ‘3 + 1’ backbone-permuted heterodimers (generated fromfive starting ‘2 + 2’ heterodimers) ran as single peaks on SEC andwere constitutive heterodimers by native MS (Fig. 2b, Supplementary
Tables 8, 9). Using the hydrogen-bond network permutation approach,we were also able to generate a set of four orthogonal homodimers(Fig. 3g–i, Extended Data Fig. 5, Supplementary Table 10).
Small-angle X-ray scattering (SAXS) spectra collected for 44
designs that were constitutive heterodimers by native MS are consistent with the design models (Figs. 1i, 2f, g, Extended Data Fig. 6,
Supplementary Table 11). The nuclear magnetic resonance (NMR)
structure of DHD13_XAAA closely matched the design model: theroot mean square deviation (r.m.s.d.) over all main-chain α-carbon(Cα) atoms was 2 Å between the designed structure and thelowest-energy NMR model (Fig. 2e). The X-ray crystal structures of
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RESEARCH Letter
b

ab
ba
aa
bb

0.3

e

DNA binding domain

0.6

OD600

1.2

150_a
150_b
154_a
154_b
155_a
155_b
37_BBBB_a
37_BBBB_b
37_ABXB_a
37_ABXB_b
162_a
162_b
6_a
6_b
13_XAAA_a
13_XAAA_b

d

0.8
6.0

4.5

3.0

OD600

1.5

0.9

c

Activation domain

Colony intensity

DHD_6
DHD_6_XA
DHD_6_AX
DHD_13_XAAA
DHD_15
DHD_20
DHD_27
DHD_37_ABXB
DHD_37_BBBB
DHD_39
DHD_65
DHD_70
DHD_103
DHD_112
DHD_148
DHD_149
DHD_150
DHD_153
DHD_154
DHD_155
DHD_157
DHD_161
DHD_162
DHD_165

150_a
150_b
154_a
154_b
155_a
155_b
37_BBBB_a
37_BBBB_b
37_ABXB_a
37_ABXB_b
162_a
162_b
6_a
6_b
13_XAAA_a
13_XAAA_b

a

0.4

0

1.5

–

+

0

TBS

5 M GdnHCl, 75 ºC

37_ABXB_a
37_ABXB_b
37_ABXB_15N_a

AmAc

1.0

0.5

Relative intensity

37_ABXB_15N_b
37_BBBB_a
37_BBBB_b
15_a
15_b
37_XAAXA_a
37_XAAXA_b
101_a
101_b
126_a
126_b
147_a
147_b
116_a
116_b
131_a
131_b
130_a
130_b
13_2:341_a
13_2:341_b
13_1:234_a
13_1:234_b
37_3:124_a
37_3:124_b
94_3:143_a
94_3:143_b
13_XAAA_a
13_XAAA_b
37_ABXB_15N_a

37_ABXB_15N_b
37_BBBB_a
37_BBBB_b
15_a
15_b
37_XAAXA_a
37_XAAXA_b
101_a
101_b
126_a
126_b
147_a
147_b
116_a
116_b
131_a
131_b
130_a
130_b
13_2:341_a
13_2:341_b
13_1:234_a
13_1:234_b
37_3:124_a
37_3:124_b
94_3:143_a
94_3:143_b
13_XAAA_a
13_XAAA_b

37_ABXB_a
37_ABXB_b

0

Fig. 4 | All-against-all orthogonality assessment. a, Y2H for 21
heterodimers shows heterodimer formation with little homodimer
formation. First letter at bottom indicates monomer fused to activation
domain; second letter indicates monomer fused to DBD. b, Y2H all-byall testing of eight pairs of heterodimers; colours indicate growth. Red
boxes indicate designed cognate heterodimer pairs, dashed black box
indicates a set of six orthogonal heterodimers. c, Off-target binding of
DHD15_a and DHD13_XAAA_b, in the absence (yellow) or presence(green) of DHD15_b and DHD13_XAAA_a. Data are mean ± s.d. Red

dashed line indicates background growth with unfused activation domain
and DBD. d, e, All-against-all orthogonality of 16 pairs of heterodimers
assessed by native MS mixing assay. Red boxes indicate designed cognate
pairs. Exchange of unlabelled and partially 15N-labelled DHD37_ABXB
results in a distribution of overlapping species with low individual signal
intensities. Two (a, b) or three (c) biologically independent or three (e)technically independent experiments were performed. AmAc, ammonium
acetate.

DHD131, DHD37_1:234, DHD127 and DHD15 have backbone Cα
atom r.m.s.d. values to the design models ranging from 0.95 to 1.7 Å.
The extensive five-residue buried hydrogen-bond network of DHD131(involving two serines, an asparagine, a tyrosine and a tryptophan) is
nearly identical in the crystal structure, with an additional bridging
water molecule (Fig. 2a). The two designed hydrogen-bond networks
in DHD37_1:234, which contain buried histidine and tyrosine aromaticside chains that sterically disfavour homodimers, are in close agreementwith the crystal structure (Fig. 2b). In DHD127, the histidines in thetwo hydrogen-bond networks adopt a rotamer that differs from thedesign model (Fig. 2c), making a hydrogen bond with a water molecule. A crystal structure of DHD15 at pH 7.0 is similar to the designmodel (Fig. 2d), whereas a structure at pH 6.5 is of a domain-swapped,

heterotetramer conformation; native MS at pH 6.5 suggests that thedesigned heterodimer—rather than the heterotetramer—is dominant
in solution (Extended Data Fig. 7a–c).
We built three induced dimerization systems by fusing one monomer from each of two different heterodimers via a flexible linker, andtesting whether the remaining two monomers from each pair could be
brought together by the fusion (Fig. 3a). In each case, the three components copurified by Ni-nitrilotriacetic acid (Ni-NTA) chromatography (one monomer has a hexahistidine tag), and native MS showed
they formed constitutive heterotrimers (Fig. 3b); no partial complexes(heterodimers) were observed. Surface-induced dissociation (SID)
MS25,26 resulted in binary complexes consisting of the heterodimerizer
bound to either one of the monomers (Supplementary Tables 12, 13),

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Letter RESEARCH
indicating that the interaction between monomers is mediated by thedimerizer fusion. In yeast two-hybrid (Y2H) assays with monomers
from two different heterodimers fused to the DNA binding domain(DBD) and transcriptional activation domain, expression of the heterodimerizer fusion as a separate polypeptide chain increased signal
substantially over background (Fig. 3c).
We constructed synthetic scaffolding systems27 by covalently linking
the a subunits of three DHDs via flexible linkers (Fig. 3d), and coexpressing this scaffold and the three separate b subunits, one with ahexahistidine tag, in E. coli. Native MS of the purified sample revealed
a heterotetramer of all four proteins (Fig. 3e), with SID producing subcomplexes consisting of the scaffold with one or two of the three b
subunits bound (Fig. 3f); no association between b subunits without
the scaffold was detected. The scaffold plus monomer assembly is stable
at 95 °C and has a guanidine denaturation midpoint of 4 M (Extended
Data Fig. 7h, i).
By generating interfaces with many polar groups that are energetically costly to bury without geometrically matched hydrogen-bonding
interactions, our design protocol implicitly disfavours non-cognate
interactions (explicit negative design to disfavour non-cognate interactions is computationally intractable, given the very large numberof possible off-target binding modes). We investigated the interaction specificity of the DHDs in cells using Y2H experiments. For 24
designs, strong interactions were observed by Y2H with the two partners fused to the DBD and activation domain, but not when either
partner was fused to both domains; the designed heterodimers, but
not the homodimers, form in cells (Fig. 4a). The 24 monomers in 12of these designs were crossed in an all-by-all Y2H experiment; interactions were observed for all cognate pairs, and 27 of the 552 possible
non-cognate interactions (Extended Data Fig. 8). Orthogonality was
higher for an eight-DHD subset: of 240 possible non-cognate interactions, only four were observed (Fig. 4b; the interacting polar residues
are depicted schematically in Extended Data Fig. 9). Coexpressionof unfused monomers eliminated off-target interactions (Fig. 4c);
the cognate interactions are evidently stronger than the non-cognate
interactions.
To probe all-by-all interactions specificity of the designed monomers when allowed to associate freely in a single pot, purified DHDs
were mixed, denatured in 5 M guanidine hydrochloride (GdnHCl) at
75 °C (Fig. 4d), and allowed to reanneal by dialysis. An 15N-labelledvariant was added as a control for subunit exchange under denaturingconditions; the hybrid labelled–unlabelled complexes expected if fullexchange was taking place were observed in all cases (Extended Data
Fig. 10). The resulting mixture was analysed by online ion exchange
chromatography coupled to high-resolution native MS (Supplementary
Table 14). Sixteen designs (15 unique pairs and the 15N-labelled control) with the highest cognate specificity were pooled together. The
native MS results on the 32-chain mixture are notable (Fig. 4e). All
16 designed pairs were recovered, and of the 512 non-cognate binary
complexes possible, only 6 were observed. No non-cognate trimers or
higher-order oligomers were observed. Several of the orthogonal pairs
were generated using hydrogen-bond network shuffling and backbone
permutation: the orthogonality of the former is due to positioning of
the hydrogen-bond networks as the backbone is fixed, and the orthogonality of the latter, to the connectivity of the chain as the sequence is
identical. The differences in orthogonality observed in the native MS
mixing and Y2H experiments are likely to stem from the dependenceof the former on relative affinity (all monomers are present, and only
the lowest-energy complexes form), and the latter, on absolute affinity(only two monomers are present at a time).
Our results demonstrate that the domain of unbounded sets oforthogonal heterodimeric biomolecules constructed from a single
repeating backbone is not limited to nucleic acids. Interaction specificity arises from extensive buried hydrogen-bond networks such as the
fully connected Tyr-Ser-Trp-Asn-Ser crystallographically confirmed
network in Fig. 2a, and heterogeneity in the size of the residues atthe designed interface (Extended Data Fig. 8d–i), analogous to thecontribution of steric effects to the specificity of Watson–Crick basepairing. The power of native MS to determine interaction specificity
in complex mixtures is highlighted by the 32-chain mixing experimentin Fig. 4e; of the large number of possible oligomeric complexes thatcan be formed from these chains (528 two-chain species, 5,984 threechain species, and so on), only the 15 designed heterodimers and 6
off-target interactions were observed. The relatively simple encodingof specificity in DNA gave birth to a broad spectrum of new technology, from DNA origami28 to artificial circuits29. Our large set oforthogonal interactions—together with the retention of specificity
in the fused monomer systems (the induced dimerizer and scaffoldof Fig. 3), and the interaction strength hierarchy illustrated by the
cognate interaction competition experiment (Fig. 4c)—open the door
to protein-based cellular control circuits with faster response times
and better integration with signalling inputs and outputs than current
nucleic-acid-based circuitry.

Online content

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data, statements of data availability and associated accession codes are available athttps://doi.org/10.1038/s41586-018-0802-y.

Received: 6 June 2018; Accepted: 8 November 2018;
Published online xx xx xxxx.
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oligomeric assembly in membranes. J. Am. Chem. Soc. 128, 4170–4171(2006).
19. Grigoryan, G. & Degrado, W. F. Probing designability via a generalized model ofhelical bundle geometry. J. Mol. Biol. 405, 1079–1100 (2011).
20. Huang, P.-S. et al. High thermodynamic stability of parametrically designed
helical bundles. Science 346, 481–485 (2014).
21. Boyken, S. E. et al. De novo design of protein homo-oligomers with modularhydrogen-bond network-mediated specificity. Science 352, 680–687 (2016).
22. Leaver-Fay, A. et al. ROSETTA3: an object-oriented software suite for thesimulation and design of macromolecules. Methods Enzymol. 487, 545–574(2011).
23. Ruotolo, B. T. & Robinson, C. V. Aspects of native proteins are retained in
vacuum. Curr. Opin. Chem. Biol. 10, 402–408 (2006).
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24. Sahasrabuddhe, A. et al. Confirmation of intersubunit connectivity and topology
of designed protein complexes by native MS. Proc. Natl Acad. Sci. USA 115,
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Reviewer information Nature thanks G. Grigoryan, C. Robinson and the otheranonymous reviewer(s) for their contribution to the peer review of this work.

Acknowledgements We thank Rosetta@Home volunteers for contributing
computing resources; A. Kang for protein crystallization support; B. Sankaran
for assistance with diffraction data collection; K. Lau and B. Groves forassistance with Y2H assays; S. Rettie for MS support; S. Ovchinnikov for help
with TMalign; M. Marty, M. Bern and A. Norris for assistance with native MS;
S. Pennington for making media for Y2H assays; the SIBYLS mail-in SAXS
program, supported by the DOE BER IDAT grant (DE-AC02-05CH11231) and
ALS-ENABLE (P30 GM124169) for SAXS; and A. Keating, G. Rocklin and
N. Woodall for feedback on the manuscript. Additional funding and computing
resources are listed in the Supplementary Information.

Additional information
Extended data is available for this paper at https://doi.org/10.1038/s41586018-0802-y.
Supplementary information is available for this paper at https://doi.org/
10.1038/s41586-018-0802-y.
Reprints and permissions information is available at http://www.nature.com/
reprints.
Correspondence and requests for materials should be addressed to D.B.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.

Author contributions Z.C., S.E.B. and D.B. designed the research. Z.C. and D.B.
wrote the manuscript. M.J., F.B., Z.L.V., A.S. and V.H.W. performed native MS
experiments and analysed data. L.P.C. prepared proteins for NMR experiments.
D.F.-S. and N.G.S. performed NMR experiments. Z.C. wrote the heptad stacking
code. S.E.B. improved the HBNet method. V.K.M. wrote the parametric backbone
generation code. T.J.B. wrote the loop closure code. Z.C. and S.E.B. carriedout design calculations, and R.A.L. and S.B. helped. Z.C., M.J.B., P.L. and F.D.
solved crystal structures. All authors discussed results and commented on the
manuscript.
Competing interests Z.C., S.E.B., R.A.L., S.B. and D.B. are inventors on
US provisional patent application no. 62755264 and patent application
WO2017173356A1. D.B. and S.E.B. hold equity in Lyell Immunopharma.

N A t U r e | www.nature.com/nature© 2018 Springer Nature Limited. All rights reserved.

Letter RESEARCH
Methods

No statistical methods were used to predetermine sample size. The experiments
were not randomized and the investigators were not blinded to allocation duringexperiments and outcome assessment.
Computational design. Systematic sampling of parametric helical backbones. We
used a generalization of the Crick coiled-coil parameters5 to independently sample
all four helices of the heterodimers supercoiled around the same axis, as previously
described19–21. The supercoil twist (ω0) and helical twist (ω1) were coupled andideal values were used20 with ω0 and ω1 held constant among the helices. A lefthanded supercoil results from ω0 = −2.85 and ω1 = 102.85, and a straight bundle
with no supercoiling from ω0 = 0 and ω1 = 100. The supercoil phases (Δɸ0) forthe helices were fixed at 0°, 90°, 180° and 270°, respectively. The offset along the
Z-axis (Zoffset) for the first helix was fixed to 0 as a reference point, with the restof the helices independently sampling from −1.51 Å to 1.51 Å, with a step size of
1.51 Å. All helices sampled helical phases (Δɸ1) independently, from 0° to 90°, witha step size of 10°. Two of the helices with a Δɸ0 separation of 180° sampled the radiusfrom Z-axis (R) from 5 Å to 8 Å, while the other two sampled from 7 Å to 10 Å,
all with a step size of 1 Å. Each helix is set to have 35 residues to accommodate
5 heptad repeats. After removing redundant sample points from the overlapping
regions of radius sampling, the supercoiled helical bundles contained more than
60 million unique backbones, and the straight helical bundles contained more than
27 million unique backbones.
HBNet search. For each parametrically generated backbone, HBNet21 was used
to search the middle heptad for hydrogen-bond networks that connect all four
helices, contain at least four side chains contributing hydrogen bonds, have all
heavy atom donors and acceptors satisfied, and span the intermolecular interface.
Symmetry was not enforced during the HBNet search. For buried interface positions, only non-charged polar amino acids were considered; for residues that wereat the boundary between the protein core and surface, all polar amino acids wereconsidered. A subsequent Rosetta design calculation was performed to optimizehydrophobic packing, with atom pair restraints from HBNet being put on the newly
identified hydrogen-bond networks. Finally, a minimization step and side-chain
repacking step were performed without atom pair restraints on hydrogen-bonding
residues to evaluate how well the networks remained intact in the absence of theconstraints. Designs with at most five alanines in the middle heptad and no buried
unsatisfied polar heavy atoms were selected for downstream design.
Generating combinations of HBNets with heptad stacking. The purpose of this stepis to identify five-heptad backbones (full backbones) that can accommodate atleast two HBNets. Instead of generating one-heptad backbones and full backbones
separately, searching for HBNets in the one-heptad backbones and aligning themto all full backbones, we reasoned that the heptad stacking method would remain
the same if we simply searched for HBNets in the middle heptad on all full backbones, extracted the middle heptads, and aligned them to all full backbones. We
therefore extracted the middle heptads containing HBNets, generated all variantsof chain ordering, and did pairwise alignment of middle heptads to full backbones
using TMalign30. All alignments with r.m.s.d. less than 0.3 were identified and fullbackbones that could accommodate at least two middle heptads were selected forfinal design.
Connecting parametric helical backbones. Helical backbones are connected with short
2–5-residue loops such that the r.m.s.d. of each loop is less than 0.4 to a 9-residue
stretch in a native protein. The distance and directionality between helices limit
what loops can connect, so our closure extends and shrinks helices by up to threeresidues. We then superimpose all short loops from the PDB onto the first andlast two helical residues. The loops with the lowest stub-r.m.s.d. are minimized
using the Rosetta score function onto the helical endpoints to ensure near-perfectclosure. Loop quality is assessed by measuring the distance in r.m.s.d. to the closest
nine stretch in the PDB. The loop with the lowest r.m.s.d. is returned as the solution. We repeat this procedure to connect all helices and report the solution withthe lowest r.m.s.d.
Design calculations. Backbones were regularized using Cartesian space minimization in Rosetta to alleviate any torsional strain introduced by heptad stacking.
Two consecutive Rosetta packing rounds were performed with increasing weighton the repulsive energy to optimize hydrophobic packing, while constraining thehydrogen-bond network residues. A FastDesign step was subsequently used withina generic Monte Carlo mover to optimize secondary structure shape complementarity, while allowing at most 8% alanine, three methionines and three phenylalanines in the protein core. The last step of minimization and side-chain repacking
to identify the movement of HBNets without atom pair constraints is the same as
described in ‘HBNet search’ above.
Selection criteria and metrics used to evaluate designs. Designs were selected using
the following criteria: change in polar surface area upon binding (dSASA_polar)
greater than 800 Å; secondary structure shape complementarity (ss_sc)score greater than 0.65; holes score around HBNets less than −1.4; no buried
unsatisfied heavy atoms; at least one buried bulky polar side chain per monomer.

Selected designs were then visually inspected for good packing of hydrophobicside chains, especially the interdigitation of isoleucine, leucine and valine. Surface
tyrosines were added at non-interfering positions to aid protein concentration
measurement by recording optical density at 280 nm (OD280). Surface charge
residues for a few of the designs were redesigned to shift the theoretical isoelectric
point away from buffer pH.
Calculations of r.m.s.d. Crystal structures and the corresponding design models
were superimposed with TMalign using all heavy atoms. From this alignment,
r.m.s.d. was calculated across all α-carbon atoms, and also across heavy atoms of
the hydrogen-bond network residues.
Logistic regression. Designs were first scored with various filters in Rosetta withthe filter values reported. Experimental results and Rosetta filter values were used
as inputs to a logistic regression method31 to find correlations between computational metrics and experimental observations.
Visualization and Figures. All structural images for figures were generated using
PyMOL32.
Buffer and medium recipes. TBM-5052: 1.2% (wt/vol) tryptone, 2.4% (wt/vol)yeast extract, 0.5% (wt/vol) glycerol, 0.05% (wt/vol) d-glucose, 0.2% (wt/vol)d-lactose, 25 mM Na2HPO4, 25 mM KH2PO4, 50 mM NH4Cl, 5 mM Na2SO4,
2 mM MgSO4, 10 μM FeCl3, 4 μM CaCl2, 2 μM MnCl2, 2 μM ZnSO4, 400 nM
CoCl2, 400 nM NiCl2, 400 nM CuCl2, 400 nM Na2MoO4, 400 nM Na2SeO3, 400 nM
H3BO3. Lysis buffer: 20 mM Tris, 300 mM NaCl, 20 mM imidazole, pH 8.0 at room
temperature. Wash buffer: 20 mM Tris, 300mM NaCl, 30 mM imidazole, pH 8.0 atroom temperature. Elution buffer: 20 mM Tris, 300 mM NaCl, 250 mM imidazole,
pH 8.0 at room temperature. Buffer W: 100 mM Tris-HCl pH 8.0, 150 mM NaCland 1 mM EDTA. Buffer E: buffer W containing 2.5 mM d-desthiobiotin. TBS
buffer: 20 mM Tris pH 8.0, 100 mM NaCl.
Construction of synthetic genes. For the expression of heterodimers, both monomers were encoded in the same plasmid, separated by a ribosome binding sequence(GAAGGAGATATCATC). Synthetic genes were ordered from Genscript anddelivered in pET21-NESG E. coli expression vector, inserted between the NdeI and
XhoI sites. For the pET21-NESG constructs, a hexahistidine tag and a tobacco etchvirus (TEV) protease cleavage site (GSSHHHHHHSSGENLYFQGS) were addedin frame at the N terminus of the second monomer. A stop codon was introduced
at the 3′ end of the second monomer to stop expression of the C-terminal hexahistidine tag in the vector. For purification with Strep-tactin resin, a streptavidin tag(SAWSHPQFEKGGGSGGGSGGSAWSHPQFEKSGENLYFQGS) coding sequence
was cloned in-frame 5′ of the first monomer sequence.
For the co-expression of three and four proteins from the same plasmid(induced dimerization and synthetic scaffold designs), synthetic genes were cloned
in the pRSFDuet-1 expression vector. The first (in the case of three proteins) or
first two (in the case of four proteins) genes were cloned between NcoI and HindIIIsites, with a ribosome binding site separating the two genes in the latter case. The
last two genes were cloned between NdeI and XhoI sites, separated by a ribosome
binding site. A hexahistidine tag and a TEV protease cleavage site coding sequence
were cloned in-frame 5′ of the last gene.
Genes for Y2H studies were cloned into plasmids bearing the GAL4 transcription activation domain (poAD) and the GAL4 DNA-binding domain (poDBD).
Protein expression. Plasmids were transformed into chemically competent E. coli
expression strains BL21(DE3)Star (Invitrogen) or Lemo21(DE3) (New England
Biolabs) for protein expression. Single colonies were picked from agar plates following transformation and growth overnight, and 5-ml starter cultures were grown
at 37 °C in Luria-Bertani (LB) medium containing 100 μg/ml carbenicillin (forpET21-NESG vectors) or kanamycin (for pRSFDuet-1 vectors) with shaking at
225 r.p.m. for 18 h at 37 °C. Starter cultures were diluted into 500 ml TBM-5052
containing 100 μg/ml carbenicillin or kanamycin, and incubated with shaking at
225 r.p.m. for 24 h at 37 °C.
For expression of 13C15N- or 15N-labelled proteins, the plasmids were transformed into the Lemo21(DE3) E. coli expression strain and plated on M9/glucose
plates containing 50 μg/ml carbenicillin. For the starter culture, a single colony wasused for inoculation of 50 ml LB medium with 50 μg/ml carbenicillin in a 250-ml
baffled flask, and incubated with shaking at 225 r.p.m. for 18 h at 37 °C. Starter
culture (10 ml) was then transferred to a 2-l baffled flask containing 500 ml Terrific
Broth (Difco), with 25 mM Na2HPO4, 25 mM KH2PO4, 50 mM NH4Cl, 5 mM
Na2SO4, and 100 μg/ml carbenicillin. The culture was grown at 37 °C to an OD600
of approximately 1.0, then centrifuged at 5,000 relative centrifugal force (r.c.f.) for
15 min to pellet the cells. The Terrific Broth medium was removed, and the cells
were washed briefly with 30 ml of phosphate buffered saline (PBS). The cells werethen transferred to a fresh 2-l baffled flask containing 500 ml labelled medium(25 mM Na2HPO4, 25 mM KH2PO4, 50 mM 15NH4Cl, 5 mM Na2SO4, 0.2% (w/v)
13
C glucose), and 100 μg/ml carbenicillin. The cells were allowed to grow at 37 °C
for 2 h, before isopropyl β-d-1-thiogalactopyranoside (IPTG; Carbosynth) was
added to 1 mM and the temperature was reduced to 18 °C. The labelled glucose
and NH4Cl were obtained from Cambridge Isotopes.

© 2018 Springer Nature Limited. All rights reserved.

RESEARCH Letter
Affinity purification. Cells were collected by centrifugation for 15 min at 5,000
r.c.f. at 4 °C and resuspended in 20 ml lysis buffer. Lysozyme, DNase, and EDTAfree cocktail protease inhibitor (Roche) were added to the resuspended cell pellet
before sonication at 70% power for 5 min. For immobilized metal affinity chromatography, lysates were clarified by centrifugation at 4 °C and 18,000 r.p.m. forat least 30 min and applied to Ni-NTA (Qiagen) columns pre-equilibrated withlysis buffer. The column was washed twice with five column volumes (CV) of
wash buffer, followed by 5 CV of elution buffer. For Strep tag purification, elution fractions from immobilized metal affinity chromatography were applied toStrep-Tactin Superflow resin (IBA) pre-equilibrated in buffer W. The column waswashed with 5 CV Buffer W, before applying 3 CV buffer E to elute proteins off thecolumn. The mass and purity of eluted proteins were confirmed using electrosprayionization mass spectrometry on a Thermo Scientific TSQ Quantum Access mass
spectrometer.
Size-exclusion chromatography. N-terminal hexahistidine tags and streptavidin
tags were cleaved with TEV protease overnight at room temperature, at a ratio of
1 mg TEV to 100 mg protein. Prior to addition of TEV, buffer was exchanged intolysis buffer. After TEV cleavage, the sample was passed over an additional Ni-NTA
column and washed with 1.5 CV of lysis buffer, and flowthrough was collected
and further purified by SEC using a Superdex 75 10/300 increase column (GEHealthcare) in TBS buffer.
Circular dichroism measurements. Circular dichroism (CD) wavelength scans(260–195 nm) and temperature melts (25–95 °C) were performed using an AVIV
model 420 CD spectrometer. Temperature melts were carried out at a heating rateof 4 °C/min and monitored by the change in ellipticity at 222 nm; protein samples
were diluted to 0.25 mg/ml in PBS pH 7.4 in a 0.1-cm cuvette. GdmCl titrations
were performed on the same spectrometer with automated titration apparatus in
PBS pH 7.4 at 25 °C, with a protein concentration of 0.025 mg/ml in a 1-cm cuvettewith stir bar. Each titration consisted of at least 40 evenly distributed GdmCl concentration points with 1-min mixing time for each step. Titrant solution consistedof the same concentration of protein in PBS + GdmCl.
Nuclear magnetic resonance. SEC-purified 13C15N-labelled protein was concentrated to >1 mM and buffer exchanged into 50 mM NaCl, 20 mM sodium
phosphate, 10% D2O, 0.01% NaN3 at pH 6.3. Sample was loaded into a 5.0-mm
Shigemi tube and four NMR experiments were recorded and analysed: 2D transverse relaxation optimized spectroscopy-heteronuclear single quantum coherence,
4D HNCH nuclear Overhauser effect spectroscopy (NOESY), 4D HNCH total
correlated spectroscopy and 4D HCCH NOESY. The data were acquired with a
non-uniform sampling (NUS) scheme and subsequently reconstructed with the
SMILE program in nmrPipe. For the NOESY experiments, the mixing time was
120 ms and for the NUS protocol the data were recorded with 0.3% and 3.0% ofsparsity for the HNCH and HCCH experiments, respectively. The final spectra
were loaded and analysed on Sparky 3.115.
The spin systems were identified using supervised NMR data analysis and 148
residues were successfully assigned (93.67%). The completeness in terms of protons
assigned was 87.4% (Supplementary Table 4). For the structural determination,
3,423 peaks were extracted from the NOESY data. Twenty-four long-range contacts(|i − j | > 4) were manually assigned with the 4D HNCH NOESY experiment and
29 in 4D HCCH. Owing to the lack of stereospecific assignments (ambiguousdata), the NOE contacts were considered as non-stereospecific assignments forthe methyl groups of Leu and Val residues. Those contacts were principally locatedat the beginning, centre and end of both sequences. The assignments, chemical
shifts and proton–proton constraints were used for RASREC or AutoNoe structural
calculations in ROSETTA 3. Summaries of refinement statistics are provided in
Supplementary Table 16.
Crystallization of protein samples. Purified protein samples were concentrated
to approximately 20 mg/ml in 25 mM Tris pH 8.0 and 150 mM NaCl. Samples
were screened with a 5-position deck Mosquito crystal (ttplabtech) with an active
humidity chamber, using the following crystallization screens: JCSG+ (Qiagen),
Crystal Screen (Hampton Research), PEG/Ion (Hampton Research), PEGRx HT(Hampton Research), Index (Hampton Research) and Morpheus (MolecularDimensions). The optimal conditions for crystallization of the different designs
were found as follows: OPHD_37_N3C1, 0.15 M potassium bromide and 30% w/v
polyethylene glycol monomethyl ether 2000; OPHD_127, 0.12 M ethylene glycols,
0.1 M buffer system 3 pH 8,5, and 50% v/v precipitate mix 1 from the Morpheus
screen; OPHD_15, 0.2 M ammonium sulfate, 0.1 M BIS-TRIS pH 6.5, 18% v/v
polyethylene glycol 400; OPHD_15, 0.1 M imidazole pH 7.0, and 25% v/v polyethylene glycol monomethyl ether 550; OPHD_131, 0.2 M ammonium acetate, 0.1 M
HEPES pH 7.5, 25% w/v polyethylene glycol 3,350. Crystals were obtained after
1–14 days by the hanging drop vapour diffusion method with the drops consistingof a 1:1, 2:1 or 1:2 mixture of protein solution and reservoir solution.
X-ray data collection and structure determination. The crystals of the designed
proteins were looped and placed in the corresponding reservoir solution, containing 20% (v/v) glycerol if the reservoir solution did not contain cryoprotectant,and flash-frozen in liquid nitrogen. The X-ray datasets were collected at the Advanced
Light Source at Lawrence Berkeley National Laboratory with beamlines 8.2.1 and8.2.2. Datasets were indexed and scaled using either XDS33 or HKL200034. Initialmodels were generated by the molecular-replacement method with the program
PHASER35 within the Phenix software suite36, using the design models as the initial
search models. Efforts were made to reduce model bias through refinement withsimulated annealing using Phenix.refine37, or, if the resolution was sufficient, byusing Phenix.autobuild38 with rebuild-in-place set to false, simulated annealing
and prime-and-switch phasing. Iterative rounds of manual building in COOT39
and refinement in Phenix were used to produce the final models. Owing to thehigh degree of self-similarity inherit in coiled-coil-like proteins, datasets forthe reported structures suffered from a high degree of pseudo-translational
non-crystallographic symmetry, as reported by Phenix.Xtriage, which complicated structure refinement and may explain the higher-than-expected R values
reported. The r.m.s.d. values of bond lengths, angles and dihedrals from ideal
geometries were calculated with Phenix36. The overall quality of all final models
was assessed using the program MOLPROBITY40. Summaries of diffraction data
and refinement statistics are provided in Supplementary Table 17.
Small-angle X-ray scattering. Samples were purified by SEC in 25 mM Tris
pH 8.0, 150 mM NaCl and 2% glycerol; fractions preceding the void volume of
the column were used as blanks for buffer subtraction. Scattering measurements
were performed at the SIBYLS 12.3.1 beamline at the Advanced Light Source. The
X-ray wavelength (λ) was 1.27 Å, and the sample-to-detector distance was 1.5 m,
corresponding to a scattering vector q (q = 4π sin θ/λ, where 2θ is the scattering
angle) range of 0.01 to 0.3 Å−1. A series of exposures, in equal sub-second timeslices, were taken of each well: 0.3-s exposures for 10 s resulting in 32 frames per
sample. For each sample, data were collected for two different concentrations totest for concentration-dependent effects; ‘low’ concentration samples ranged from
2 to 3 mg/ml and ‘high’ concentration samples ranged from 5 to 7 mg/ml. Data
were processed using the SAXS FrameSlice online serve and analysed using theScÅtter software package41,42. FoXS43,44 was used to compare design models toexperimental scattering profiles and calculate quality of fit (χ) values.
Yeast two-hybrid assay. For each pair of binders tested, chemically competent cells of yeast strain PJ69-4a (MATa trp1-901 leu2-3,112 ura3-52 his3-200
gal4(deleted) gal80(deleted) LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ)
were transformed with the appropriate pair of plasmids containing DBDs or
activation domains, using the LiAc/SS carrier DNA/PEG method45. In the caseof induced dimerization, the heterodimerizer was cloned downstream of one ofthe ‘monomer proteins’, separated by a p2a and nuclear locolization sequence(GSGATNFSLLKQAGDVEENPGPGDKAELIPEPPKKKRKVELGTA). The p2a
sequence ensures translational cleavage to make the heterodimerizer a separate
protein from the monomer protein. The selection of transformed yeast cells was
performed in synthetic dropout (SDO) medium lacking tryptophan and leucine
for 48 h with shaking at 1,000 r.p.m. at 30 °C. The resulting culture was diluted
1:100 and grown for 16 h in fresh SDO medium lacking tryptophan and leucine,
before being transferred to a 96-well plate and diluted 1:100 into SDO medium
containing 100 mM 3-amino-1,2,4-triazole (3-AT), lacking tryptophan, leucine
and histidine (5 mM 3-AT in the case of induced dimerization). The culture wasincubated with shaking at 1,000 r.p.m. at 30 °C. As it is necessary to bring the
DBD and the transcription activation domain into proximity for the growth ofyeast cells in medium lacking histidine, binding of two proteins was indicatedby the growth of yeast cells46,47. The optical density of yeast cells was recorded
after 48 h. For Y2H assay on agar plates, the 1:100 diluted overnight culture wastransferred onto a Nunc OmniTray (Thermo Fisher) using a 96 Solid Pin MultiBlot Replicator (V&P Scientific), with the agar lacking tryptophan, leucine andhistidine, and containing 100 mM 3-AT. The plates were imaged daily until day
5 to monitor the sizes of colonies. Images were analysed by the ColonyArea48
package on ImageJ.
Native MS assessment of heterodimer affinity. Samples were buffer-exchanged
into 200 mM ammonium acetate using Micro Bio-Spin 6 columns (Bio-Rad).
Protein concentrations were determined spectroscopically by a NanoDrop 2000c(Thermo Fisher Scientific). After dilution with 200 mM ammonium acetate, proteins were allowed to equilibrate for 24 h at 4 °C. Mass spectra were subsequently
recorded by nanoESI-MS using an Exactive Plus EMR Orbitrap instrument(Thermo Fisher Scientific) modified to incorporate a quadrupole mass filter andallow surface-induced dissociation25,26,49.
Native MS of individual heterodimers. Sample purity and integrity were first
analysed using a self-packed buffer exchange column50 (P6 polyacrylamide gel,
BioRad), coupled online to an Exactive Plus EMR Orbitrap instrument (Thermo
Fisher Scientific) modified to incorporate a quadrupole mass filter and allow
surface-induced dissociation. For online buffer-exchange, 200 mM ammonium
acetate, pH 6.8 (AmAc) was used as a mobile phase. Samples that showed specificdimer formation and a good correlation with the theoretical monomer or dimer
masses were selected for mixing experiments.

© 2018 Springer Nature Limited. All rights reserved.

Letter RESEARCH
Native MS mixing assay and data analysis. In the mixing experiment, heterodimers were mixed in equimolar ratio of 10 μM. GdnHCl was added to a final
concentration of 5 M and the mixture was incubated at 75 °C for 30 min to ensurecomplete denaturation. To allow relative quantification of exchanged species, acontrol mixing experiment was performed in which the denaturation and refolding steps were omitted. The mixtures were then dialysed against 150 mM AmAc
solution for refolding and subsequent formation of protein–protein interactions.
Eight microlitres of sample was injected on a ProPac WCX-10 column and separately, a ProPac WAX-10 column (Thermo Scientific) and separated using a Dionex
UltiMate 3000 HPLC (Thermo Scientific) by a salt gradient elution from 20 mM
AmAc to 1,000 mM AmAc over a period of 55 min. The eluting proteins weredetected online using a modified Exactive Plus EMR Orbitrap mass spectrometer.
LC–MS analysis was performed for mixtures in full MS mode (no collision voltage
applied) and all-ion fragmentation (MSMS) mode with high energy collisioninduced dissociation (HCD) 100 V and surface-induced dissociation (SID) 85
V, respectively51. Details of instrument settings are in Supplementary Table 15.
Data were deconvoluted using Xcalibur (Thermo Scientific), UniDec52 and Intact
Mass (Protein Metrics53). The detailed deconvolution parameters are listed in
Supplementary Table 15. The deconvoluted mass lists from Intact Mass weresearched against a theoretical mass list of all possible monomers to tetramers
combinations. Only one trimeric species was found, which corresponds to the
cognate 13_XAAA_b + 13_2:341_a + 13_1:234_a heterotrimer formation, whichconstitutes the 13_XAAA design with the two helices of its a monomer comingfrom 13_2:341_a and 13_1:234_a. Dimers were identified using the full MS runs
and MSMS runs with both subunits being detected at the same retention time. The
mass tolerance was set to 2 Da and intensity tolerance was set to 1% of the highest
intensity. The relative intensity was calculated using the equation

I (N M )I (N M )Ir (NaMb) = DN a b + DN a b
2IN (NaNb)
2IN (MaMb)
in which Ir (NaNb) is the relative intensity of a dimer NaNb identified in the mixing
experiment. IDN (NaNb) is the intensity of the NaNb species in the run involving
denaturation and refolding. IN (NaNb) and IN (MaMb) are the intensities of the
cognate pairs NaNb and MaMb in the run that skipped denaturation and refolding.
The native MS mixing workflow is shown in Extended Data Fig. 10.
Native MS of higher-order hetero-oligomers. Samples were buffer exchanged into
200 mM ammonium acetate using Micro Bio-Spin 6 columns (Bio-Rad). Twenty
per cent (v/v) 200 mM triethylammonium acetate (Sigma) was added for charge
reduction. SID was performed on an in-house modified SYNAPT G2 HDMS
(Waters) with a SID device incorporated between a truncated trap travelling wave
ion guide and the ion mobility cell25. The following instrument parameters wereused: sampling cone, 20 V; extraction cone, 2 V; source temperature, 20 °C; trap
gas flow, 2 ml/min; trap bias, 45 V. The SID settings are listed in Supplementary
Table 15.
Reporting summary. Further information on research design is available in
the Nature Research Reporting Summary linked to this paper.
Code availability. All program code is in Rosetta or can be downloaded from theGithub repository at https://github.com/uagaug/DeNovoHeterodimers.

Data availability

Coordinates and structure files have been deposited in the Protein Data Bank
with accession codes: 6DMP (DHD13_XAAA), 6DKM (DHD131), 6DLC
(DHD37_1:234), 6DLM (DHD127), 6DMA (DHD15 heterodimer) and 6DM9
(DHD15 heterotetramer). The native MS spectra generated and analysed during

the current study are available at http://files.ipd.uw.edu/pub/de_novo_heterodimers_2018/180813_native_ms_raw.zip. Raw X-ray diffraction images have been
deposited at https://proteindiffraction.org/. All source data are available upon request.
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RESEARCH Letter

Extended Data Fig. 1 | Overview of different topologies designed.
a–d, Overall topologies on the left and example HBNets on the right.
a, A left-handed supercoiled backbone, with each monomer being helix
hairpins. b, A backbone-permuted ‘3 + 1’ design; one monomer is a single
helix and the other is a three-helix bundle. c, A left-handed supercoiled
backbone, with each monomer being a three-helix bundle. d, A straight,
untwisted backbone, with each monomer being a helix hairpin.

e, Hydrogen-bond pairing in DNA bases. Top, A–T base pairing. Bottom,
C–G base pairing. Green arrows point from hydrogen-bond donors toacceptors. f, Two examples of hydrogen-bond pairing in designed proteinhydrogen-bond networks. g, Top-down view of antiparallel twisted (top)and parallel untwisted (bottom) backbones sampled in this study.
h, Comparison of a designed protein heterodimer (right) with B-form
DNA (left) on the same scale.

© 2018 Springer Nature Limited. All rights reserved.

Letter RESEARCH

Extended Data Fig. 2 | Example HBNets resulting from the systematic search. a, Overlay of 50 backbones with different Crick parameters for eachhelix. b, Example hydrogen-bond networks from the systematic search, each involving at least four residues and contacting all four helices.

© 2018 Springer Nature Limited. All rights reserved.

RESEARCH Letter

Extended Data Fig. 3 | Thermal and chemical denaturation of DHDs.
a, b, CD spectra for thermal denaturation of DHD_15 and DHD_20,
respectively. Top, wavelength scan at 25 °C, 75 °C, 95 °C and final 25 °C.
Designs were α-helical and stable up to 95 °C. Bottom, CD temperaturemelts, monitoring absorption at 222 nm as temperature was increased
from 25 °C to 95 °C. c, GdnHCl denaturation of DHD_127 measured
by CD monitoring absorption at 222 nm. All CD experiments were
performed once.

© 2018 Springer Nature Limited. All rights reserved.

Letter RESEARCH

Extended Data Fig. 4 | Backbone and hydrogen-bond network
permutations. a, On a 2 + 2 backbone (left), two loops were designed
to connect the four helices into a single monomer in two different ways
(middle), after which four different cut points were introduced to generate
four possible backbone-permuted heterodimers of a single helix anda three helix bundle (3 + 1 heterodimers, right). For example, 2:134
refers to a heterodimer in which the original helix 2 is a single helix, andhelices 1, 3 and 4 were connected into a three-helix bundle. b, Hydrogenbond network permutation. Each unique network was assigned a letter(networks ‘A’ and ‘B’ in this case), with the hydrophobic packing assigned X.
The backbone on the left reads ‘ABXB’; its first heptad accommodates
network A, its second and fourth heptad accommodate network B, and its
third heptad accommodates hydrophobic packing only (X).

© 2018 Springer Nature Limited. All rights reserved.

RESEARCH Letter

Extended Data Fig. 5 | Biophysical characterization of hydrogen-bondnetwork-permuted homodimers. a, SEC traces of all six homodimer
designs. b–g, SAXS profiles of hydrogen-bond network-permuted

homodimer designs. Black, experimental SAXS data; red, spectra
computed from the designed backbones. Two (a) or one (b–g) biologicallyindependent repeats were performed.

© 2018 Springer Nature Limited. All rights reserved.

Letter RESEARCH

Extended Data Fig. 6 | SAXS profiles of all tested DHDs. Black,experimental SAXS data; red, spectra computed from the designed
backbones. a, SAXS profiles with χ values smaller than 6. b, SAXS profiles

with χ values greater than 6. All tested designs showed close agreement to
expected radius of gyration (Rg) and maximum distance (dmax).

© 2018 Springer Nature Limited. All rights reserved.

RESEARCH Letter

Extended Data Fig. 7 | Crystal structure of the domain-swapped
DHD_15 and biophysical characterization of higher-order oligomers.
a, Crystal structure of DHD_15 at pH 6.5, with 2.25 Å resolution.
b, Superposition of design models (in colour) onto both halves of thecrystal structure (in white), with backbone r.m.s.d. of 1.83 Å. c, Native
MS study of DHD_15 at different pH values indicates that heterodimers,
rather than heterotetramers, are dominant in solution. d–g, SEC traces of
the induced dimerization DHD_9-13 fusion (d), DHD_15-37 fusion (e),DHD_13-37 fusion (f), and the scaffolding complex in Fig. 3d (g; the peakat around 15 ml corresponds to the fully assembled complex, followed bya peak representing an excess of individual components). h, CD thermalmelt curves for the scaffolding complex in Fig. 3d. Wavelength scan was
performed at 25 °C, 75 °C, 95 °C and final 25 °C. Design was α-helical andstable up to 95 °C. i, CD chemical denaturation profile of the scaffolding
complex in Fig. 3d. Two (c–g) or one (h, i) biologically independent
repeats were performed.

© 2018 Springer Nature Limited. All rights reserved.

Letter RESEARCH

Extended Data Fig. 8 | See next page for caption.

© 2018 Springer Nature Limited. All rights reserved.

RESEARCH Letter
Extended Data Fig. 8 | Y2H all-against-all assay of 16 DHDs. a, Y2H
assay with cell growth on agar plates containing 100 mM 3-AT, lacking
tryptophan, leucine and histidine. Plates were imaged on day 5. Yellow,
no growth on agar plates; light blue, weak growth forming non-circularcolonies; dark blue, strong growth. b, Y2H result by growing yeast
culture in liquid medium containing 100 mM 3-AT, lacking tryptophan,leucine and histidine. OD600 values were measured on day 2 to evaluate
cell growth. c, An additional set of DHDs tested by Y2H showing
improved orthogonality. d, Distribution of OD600 values for non-cognate
interactions in b. The majority of cells grew to OD600 < 0.4, indicating
weak interactions for non-cognate binding. e–g, Box plots of various
properties for designs that assembled to off-target oligomeric states bynative MS (failure) and that assembled into constitutive heterodimers(success). n = 88; 25th, 50th and 75th percentiles are shown in the box
with the centre being median, extended to 1.5 × interquartile range(IQR) beyond the box. e, The number of buried bulky polar residues
correlates strongly with design success. f, Successful designs tend to havea bigger polar interface surface area. g, Designs with better hydrophobic
packing (as reported by the Rosetta filter value Average Degree on Ile,

Leu and Val residues) tend to have a higher chance of being constitutive
heterodimers as assessed by native MS. h, Contribution of bulky residues
and hydrogen-bond networks to specific dimer formation. dSASA_polarmeasures interface hydrophilicity and correlates positively with the surface
area of hydrogen-bond networks at the interface. Bulky polar residues
in core counts the total number of buried bulky residues that participate
in hydrogen-bond networks. Constitutive heterodimer formation (bluecircles) or off-target oligomer formation (red circles) were determinedwith native MS. Filter cutoff values of dSASA_polar > 970 Å2 and morethan one polar bulky residue buried in the core includes most of thesuccessful designs and excludes most of the design failures. i, On the basisof the Y2H data in b, all 32 monomers from the 16 pairs were categorized
as being specific (blue, has ≤1 non-cognate binding), or non-specific (red,has >1 non-cognate binding). With application of secondary structure
prediction scores (PsiPred54) and Rosetta centroid energy score perresidue as filters, designs with higher PsiPred values and lower Rosetta
centroid score per residue are more specific (green box). Two independent
experiments were performed (a–c).

© 2018 Springer Nature Limited. All rights reserved.

Letter RESEARCH

Extended Data Fig. 9 | Hydrogen-bond network sequence motifs of theset of six orthogonal pairs in Y2H experiments. Green patches mark thelocations of hydrogen-bond network-forming residues on the backbones.
Letters along the backbones indicate residue identities.

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RESEARCH Letter

Extended Data Fig. 10 | See next page for caption.

© 2018 Springer Nature Limited. All rights reserved.

Letter RESEARCH
Extended Data Fig. 10 | The workflow of native MS mixing
experiments. a, Protein samples were characterized using online desalting
coupled to native MS and deconvoluted using UniDec software. Proteinsshowing expected masses were mixed in equimolar ratio, and the final
mix was divided into two parts: in the experimental group (DN), proteins
were denatured by 5 M GdnHCl at 75 °C and refoled into 150 mM AmAc;
in the control mixing experiment (N), denaturation and refolding steps
were omitted. Sample mixtures in each group were further equally divided
into three parts that were individually injected on LC–MS with cationexchange and anion exchange, respectively, coupled with CID or SID.
LC–MS analysis was performed for mixtures in full MS mode and MSMS
mode with HCD and SID, respectively. Data were deconvoluted using
Intact Mass. The deconvoluted mass lists from Intact Mass were searchedagainst a theoretical mass list of all possible monomer, dimer, trimer andtetramer combinations. Dimers were identified using the full MS runs
and MSMS runs with both subunits being detected at the same retention
time. b, In the control mixing experiment (N), after mixing all 16 proteinsin solution without the denaturation and renaturation steps, no exchange
among proteins were observed. c, CD data for a mixture of purified
DHDs in PBS (red) or 5 M GdnHCl and 75 °C (blue). Protein mixture was
fully denatured under the latter conditions. d, A mixing experiment of
DHD_37_ABXB and 15N-labelled DHD_37_ABXB with (red) or without(black) the denaturation and refolding steps. MS peaks merged after
subunit exchange owing to the similarity in the masses of 15N-labelled
and unlabelled subunits. Two biologically independent experiments were
performed (b–d).

© 2018 Springer Nature Limited. All rights reserved.

Reporting Summary
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in reporting. For further information on Nature Research policies, see Authors & Referees and the Editorial Policy Checklist.

Statistical parameters

nature research | reporting summary

Corresponding author(s): David Baker

When statistical analyses are reported, confirm that the following items are present in the relevant location (e.g. figure legend, table legend, main
text, or Methods section).
n/a Confirmed
The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement
An indication of whether measurements were taken from distinct samples or whether the same sample was measured repeatedly
The statistical test(s) used AND whether they are one- or two-sided
Only common tests should be described solely by name; describe more complex techniques in the Methods section.

A description of all covariates tested
A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons
A full description of the statistics including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND
variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals)
For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted
Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes
Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated
Clearly defined error bars
State explicitly what error bars represent (e.g. SD, SE, CI)
Our web collection on statistics for biologists may be useful.

Software and code
Policy information about availability of computer code
Data collection

Rosetta software suite was used to perform protein design calculations, Rosetta is freely available for academic users on GitHub; TMalign(Version 5/21/2016) was used to perform structural alignment.

Data analysis

Custom Python code was written to perform data analysis and can be downloaded from https://github.com/uagaug/
DeNovoHeterodimers, along with other command line scripts; Crystallographic data were analyzed with Phenix (release 1.101.1-2155)
and Coot (v0.8.7 EL); Protein models were visualized using PyMOL 2.0; ImageJ 1.48v was sued to analyze Y2H colonies; Sparky 3.115 wasused to analyze NMR spectra.

For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers
upon request. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

April 2018

1

Policy information about availability of data
All manuscripts must include a data availability statement. This statement should provide the following information, where applicable:- Accession codes, unique identifiers, or web links for publicly available datasets- A list of figures that have associated raw data
- A description of any restrictions on data availability
Coordinates and structure files have been deposited to the Protein Data Bank with accession codes: 6DMP (DHD13_XAAA), 6DKM (DHD131), 6DLC (DHD37_1:234),
6DLM (DHD127), 6DMA (DHD15 heterodimer), 6DM9 (DHD15 heterotetramer).
The native MS spectra generated during and/or analysed during the current study are available at http://files.ipd.uw.edu/pub/de_novo_heterodimers_2018/180605_native_ms_raw_spectra.tar.gz
Raw X-ray diffraction images have been deposited at proteindiffraction.org

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Data

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Life sciences

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Life sciences study design
All studies must disclose on these points even when the disclosure is negative.
Sample size

Heterodimers were designed by the Rosetta software suite and designs passing computational selection criteria were experimentally tested.
Sample size was determined by the estimated work load. In total, 158 designed proteins were tested. Sample size was considered sufficientsince no significant improvement of the success rate was observed for subsequent iterations of design/characterization.

Data exclusions

No data were excluded from the analysis.

Replication

At least two biological replicates were done for yeast-2-hybrid (Y2H) assay of individual proteins. Three technical replicates were performed
for the all-against-all Y2H assays. Three technical replicates were performed for the all-against-all native MS mixing assays. The replicatedexperiments produced similar results. CD experiments were performed once due to the expected overall stability of these proteins.

Randomization

No randomized samples allocation in this work. Because of the complexity of the designs, it is unlikely that the folding amino acids sequences
reported in this paper are due to chance rather than design calculation. Negative and positive controls were well defined and validated.

Blinding

Not relevant to the study since the experiments were well-defined by the computational design.

Reporting for specific materials, systems and methods
Materials & experimental systems

Methods

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Unique biological materials

ChIP-seq

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April 20182