Page 112 - Science

RESEARCH

PROTEIN DESIGN with Rosetta. To specify the orientation of the
designs (11) in the membrane when expressed
Accurate computational design of in cells, at the designed lipid-water boundary
on the extracellular/periplasmic side, we incor-
porated a ring of amphipathic aromatic resi-
multipass transmembrane proteins dues and, at the lipid-water boundary on the
cytoplasmic side, a ring of positively charged
residues (Figs. 1A and 2A). Between these two
3
Peilong Lu, 1,2 Duyoung Min, Frank DiMaio, 1,2 Kathy Y. Wei, 1,2 * Michael D. Vahey, 4 rings, the surface residues are exposed to the
Scott E. Boyken, 1,2 Zibo Chen, 1,2 Jorge A. Fallas, 1,2 George Ueda, 1,2 William Sheffler, 1,2 hydrophobic membrane environment; these po-
5
3
Vikram Khipple Mulligan, 1,2 Wenqing Xu, James U. Bowie, David Baker 1,2,6 † sitions in Rosetta sequence design calculations
were restricted to hydrophobic amino acids
The computational design of transmembrane proteins with more than one membrane- (supplementary materials). Consistent with the
spanning region remains a major challenge. We report the design of transmembrane design, TMHMM predicts that the dimer de-
monomers, homodimers, trimers, and tetramers with 76 to 215 residue subunits containing signs contain 2 TMs and the single-chain design
two to four membrane-spanning regions and up to 860 total residues that adopt the (scTMHC2) contains 4 TMs (fig. S1). On average,
target oligomerization state in detergent solution. The designed proteins localize to the for each residue ~68% of the side-chain surface
plasma membrane in bacteria and in mammalian cells, and magnetic tweezer unfolding area is buried in the design models, which could
experiments in the membrane indicate that they are very stable. Crystal structures provide substantial van der Waals stabiliza-
of the designed dimer and tetramer—a rocket-shaped structure with a wide cytoplasmic tion (12).
base that funnels into eight transmembrane helices—are very close to the design Synthetic genes encoding the designs were
models. Our results pave the way for the design of multispan membrane proteins with obtained and the proteins expressed in E. coli
new functions. and mammalian cells. The dimer design with
the shortest hydrophobic span (15 residues;
TMHC2_S) was poorly behaved in both E. coli Downloaded from
n recent years, it has become possible to de A major challenge for membrane protein and mammalian cells, but the dimer designs
novo design, with high accuracy, soluble pro- design stems from the similarity of the mem- with longer spans—TMHC2, TMHC2_E, and
tein structures ranging from short con- brane environment to protein hydrophobic cores. TMHC2_L—localized to the cell membrane when
strained peptides to megadalton protein In the design of soluble proteins, the secondary expressed in human embryonic kidney (HEK)
I cages (1). There have also been advances in structure and overall topology can be specified 293T cells (Fig. 1B) and in E. coli. The designed
membrane protein design, as illustrated by an by the pattern of hydrophobic and hydrophilic proteins were purified by extracting the E. coli
elegant zinc-transporting transmembrane pep- residues, with the former inside the protein and membrane fraction with detergent, followed
tide tetramer named Rocker (2)and an engi- the latter outside, facing solvent. This core de- by nickel–nitrilotriacetic acid (NTA) chroma-
neered ion-conducting oligomer based on the sign principle cannot be used for membrane tography and size exclusion chromatography http://science.sciencemag.org/
C-terminal transmembrane segment (TMs) of proteins because the apolar environment of the (SEC) with a yield of ~2 mg/L (fig. S2, A and B).
the Escherichia coli polysaccharide transporter hydrocarbon core of the lipid bilayer requires The designed proteins TMHC2, TMHC2_E, and
Wza (3). Both are single membrane–spanning that outward-facing residues in the membrane TMHC2_L eluted as single peaks in SEC, and
synthesized peptides with fewer than 36 resi- also be nonpolar. Buried hydrogen bonds be- in analytical ultracentrifugation (AUC) experi-
dues. It has also been possible to design and tween polar side chains have been demonstra- ments in detergent solution, the proteins sedi-
confirm the transmembrane topology of multi- ted to play an important role in the association mented as dimers, which is consistent with the
pass membrane proteins by using simple se- of helical peptides within the membrane, over- design models (Fig. 1C and fig. S3). For the
quence hydrophobicity and charge-based models coming the degeneracy in the nonpolar inter- single-chain scTMHC2, the major species in SEC on March 1, 2018
(4), but the extent to which the transmembrane actions (5–7). was the monomer, with a small side peak that
helicespack witheachother is notclear.Design We reasoned that a recently developed meth- was readily removed by purification (fig. S2B).
of structurally defined multipass membrane pro- od for designing buried hydrogen bond net- Circular dichroism (CD) measurements showed
teins has remained a major challenge because works (8) could allow specification of the packing that the designs were a-helical and highly ther-
of the difficulty in specifying structure within interactions of transmembrane helices in multi- mal stable; the CD spectra at 95°C were similar
the membrane and in experimentally determin- pass transmembrane proteins. We first explored to those at 25°C (Figs. 1D and 2B). TOXCAT-
ing membrane protein structures generally; crys- the design of helical transmembrane proteins b−lactamase (TbL) assays (13), which couple
tal structures of the full designed oligomeric with four TMs—dimers of 76- to 104-residue E. coli survival to oligomerization and proper
states of Rocker- and the Wza-derived channel hairpins or a single chain design of 156 residues— orientation of fused antibiotic resistance mark-
have not yet been determined, and to date, there with hydrophobic spanning regions ranging ers on the N and C termini, suggest that the N
are no crystal structures of de novo–designed multi- from 21 to 35 Å (Figs. 1A and 2A), repurposing and C termini of TMHC2 are in the cytoplasm,
pass membrane proteins. the Ser- and Gln-containing hydrogen bond as in the design models (fig. S4).
networks in a designed soluble four-helix dimer We more quantitatively characterized the fold-
with C2 symmetry [2L4HC2_23; Protein Data ing stability of scTMHC2 using single-molecule
1 Department of Biochemistry, University of Washington,
2
Seattle, WA 98195, USA. Institute for Protein Design, Bank (PDB) ID 5J0K] (8) to provide structural forced unfolding experiments (Fig. 2) (14, 15).
University of Washington, Seattle, WA 98195, USA. specificity. Four-helix bundles of different lengths The designed protein reconstituted in a bicelle
3 Department of Chemistry and Biochemistry, UCLA-DOE with backbone geometries capable of host- was covalently attached to a magnetic bead and
Institute, Molecular Biology Institute, University of California, ing these networks were produced by using a glass surface through its N and C termini
4
Los Angeles (UCLA), CA 90095, USA. Department of parametric generating equations (9), residues (Fig. 2A and fig. S5). The distance between the
Bioengineering and Biophysics Group, University of California,
5
Berkeley, Berkeley, CA 94720, USA. Department of Biological comprising the hydrogen bond networks and bead and the surface was determined as a
Structure, University of Washington, Seattle, WA 98195, neighboring packing residues were introduced, function of the applied mechanical tension. In
6
USA. Howard Hughes Medical Institute, University of and the remainder of the sequence was opti- unfolding experiments with the force slowly
Washington, Seattle, WA 98195, USA. mized by using Rosetta Monte Carlo (10) design increasing (~0.5 pN/s), unfolding transitions
*Present address: Department of Bioengineering, University of
California, Berkeley, CA 94720, USA. calculations to obtain low-energy sequences. were observed at ~18 pN and, upon force de-
†Corresponding author. Email: dabaker@u.washington.edu Connecting loops between the helices were built ramping, refolding transitions were observed
Lu et al., Science 359, 1042–1046 (2018) 2 March 2018 1of 5