Synthetic β-Barrel by Metal-Induced Folding and Assembly - Journal of

Jul 5, 2018 - The FVFV sequence, which has a latent propensity to form β-sheet ... (20). Figure 3. Structure of β-barrel 2a. (a) Side view of the cr...
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Synthetic #-barrel by metal-induced folding and assembly Motoya Yamagami, Tomohisa Sawada, and Makoto Fujita J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b04284 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 5, 2018

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Synthetic β-barrel by metal-induced folding and assembly Motoya Yamagami,† Tomohisa Sawada,*,† and Makoto Fujita*,† †

Department of Applied Chemistry, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 1138656, Japan

Supporting Information ABSTRACT: The de novo construction of repeated pro-

teins has received much attention from biologists and chemists, yet that of a β-barrel structure, one of the most well-known classes, has not been accomplished to date. Here, we report the first chemical construction of a βbarrel tertiary structure with a pore through a combination of peptide folding and metal-directed self-assembly. Coordination of zinc salts to an eight-residue peptide fragment bearing β-strand- and loop-forming sequences resulted in a β-barrel in which six-stranded cylindrical antiparallel β-sheets formed a hydrophobic pore with a specific shape.

(a)

-F-V-F-V-

(b)

O

H N

N

N H

O

H N O

-P-G-P-

O N H

O

H N

O N

O

1 ZnI2

The β-barrel is a unique protein tertiary structure in which twisted β-strands are repeated circularly and in tandem to form a large cylindrical pore.1 Inspired by the beautiful cylindrical pore structures of β-barrel proteins, researchers have synthesized synthetic mimics of β-barrels which possessed pores surrounded by β-strands.2 However, the de novo construction of more protein-like β-barrels from oligopeptides is considerably challenging3 than the formation of the other α- and α/β-folds4–11 because of the strong tendency of β-strands to aggregate into poorly-organized, insoluble fibers. To date, there are only a few reports of the de novo synthesis of β-barrels,12,13 and none of these has resulted in a precise all-β-barrel topology with an internal pore.14 However, we now report that short synthetic oligopeptide 1 undergoes a folding-and-assembly process15–18 into synthetic β-barrel 2 with sextuply repeated β-strands (Figure 1). Two organizational principles, namely peptide folding and self-assembly, work in concert in the formation of 2, and both are induced by metal coordination.

folding & assembly

+

N H

H N

N O

N

O

=

=

folding & assembly 3

(1)2(ZnI2)2 macrocycle

2a

De novo designed oligopeptide 1 was synthesized by solution-phase peptide synthesis (see the Supporting Information for details) and has an FVFVXPGP sequence [where F = phenylalanine, V = valine, X = 3-amino benzoyl (γ-amino acid residue), P = proline, G = glycine] with two pyridyl metal-binding sites at the C- and N-termini.

Figure 1. (a) Schematic representation of the bottom-up construction of an all-β-barrel. Note that the β-barrel structure in this figure does not possess the same geometry as βbarrel 2a. (b) Chemical structure of designed octapeptide ligand 1 and its Zn-triggered folding and assembly into βbarrel 2a.

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(a)

90°

(b)

(PGP loop) O

F N H

N ZnX2

O

H N O

F N H

V O

O

H N O

N H

V V

H N O

O O

N H

V

H N F

O

ZnX2 N

O N H

H N F

O

(PGP loop)

intra-cycle H-bonds inter-cycle H-bonds

Figure 2. Structure of the (1)2(ZnI2)2 macrocyclic unit in 2a. (a) Cartoon representations of the macrocyclic unit from different viewpoints. (b) Chemical structure of the (1)2(ZnI2)2 macrocyclic unit (the PGP loop moieties have been omitted for clarity). The FVFV sequence has a latent propensity to form βsheet secondary structures, and the PGP sequence, which forms a loop as shown in our previous work,16 are linked through the X spacer residue for separation of the two moieties. 1H NMR measurement of 1 showed the existence of multiple conformers, which indicates the conformational flexibility of 1 in the solution state (Figures S9 and S11). Folding and assembly of peptide ligand 1 on coordination to ZnI2 was then conducted as follows: a chloroform solution of ligand 1 (13 mM, 150 µL), a buffer solution (chloroform/ethanol = 1:1 (v/v), 150 µL), and an ethanol solution of ZnI2 (13 mM, 150 µL) were layered in a capped microtube and allowed to stand for 2 weeks in an incubator at 20 °C. Peptide–ZnI2 complex 2a was formed as single crystals and isolated in good yield (69%). Elemental analysis revealed that the product had (1)•(ZnI2) stoichiometry. A single crystal of 2a was subjected to an X-ray diffraction study. Data collection was carried out at a synchrotron facility (SPring-8) and the crystal structure was refined in the P41212 space group. The asymmetric unit includes one and a half (1)2(ZnI2)2 macrocycles, which each exhibit an internal antiparallel β-sheet and are conformationally almost identical. Three (1)2(ZnI2)2 macrocycles are circularly assembled through intermolecular antiparallel β-sheet formation into all-β-barrel cylindrical structure

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[(1)2(ZnI2)2]3 (2a) (Figure 1b). To the best of our knowledge, this is the first example of the de novo construction of an all-β-barrel topology with a large pore. The conformation of ligand 1 in 2a (Figure 2a) was analyzed using plots of dihedral angles ɸ (N–Cα torsion) and ψ (Cα–Ccarbonyl torsion) (Ramachandran plot19). All of the amide bonds were confirmed to be in the trans configuration, and as expected, the FVFV moiety is folded into a βstrand (ɸ: −75°~−140°; ψ: 126°~146°) (Figure S15). However, the PGP sequence adopts an extended S-shaped conformation rather than the expected Ω-shaped conformation observed in our previous study16 (Figures S16 and S18). The (1)2(ZnI2)2 macrocycle component has pseudo-C2 symmetry (Figure 2), in which two β-strands with opposite N→C direction form an antiparallel β-sheet. The aromatic γ-amino acid residue (X) is also involved in this intra-cycle β-sheet formation; four hydrogen bonds are observed within the macrocycle with N···O=C distances between the X residue and the facing F residue of 2.8–2.9 Å (Figure 2b, red). In addition to this intra-cycle β-sheet formation, intercycle antiparallel β-sheet formation [N···O=C distances of ~3.0 Å (Figure 2b, green)] results in the circular assembly of three macrocyclic units into the pseudo-D3 symmetry [(1)2(ZnI2)2]3 β-barrel structure of 2a (Figures 3a and 4a). β-Barrel 2a is characterized as a (6, 12) β-barrel—the structural features of β-barrels are defined by their (n, S) values, where n indicates the number of strands and S indicates the shear number20,21 (Figure 3b)—and the slope of the strands with respect to the barrel axis is 56°, fully consistent with the theoretical value for this β-barrel.20 (a)

(b)

= Phe

barrel axis 56° strand axis

S

=1

= Val =

H N

O

2

Figure 3. Structure of β-barrel 2a. (a) Side view of the crystal structure of 2a. β-Strand regions are highlighted in green. (b) Geometrical analysis. The arrows with five circles on them represent amino acid residues forming βstrands. The orange dotted guidelines indicate the domain of a (1)2(ZnI2)2 macrocyclic unit.

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(a)

(a)

(b)

Figure 5. Comparison of the two major types of PGP loops. (a) Ω-shaped loop found in our previous work16 (top) and overlaid coordinates of PGP loops with the same conformation found in natural protein structures reported in the PDB (bottom). (b) S-shaped loop found in 2a (top) and overlaid coordinates from the PDB (bottom). For details, see Figure S17.

(b)

Figure 4. Top view of β-barrel 2a. (a) Cartoon representation, with each (1)2(ZnI2)2 macrocyclic unit in a different color. (b) Interior V residues and exterior F residues colored gold and purple, respectively. Complexation of 1 with ZnBr2 and ZnCl2 gave almost identical β-barrels [(1)2(ZnBr2)2]3 (2b) and [(1)2(ZnCl2)2]3 (2c) (in 68% and 55% yields, respectively). These structures were also characterized by X-ray crystallographic analysis, which showed that the structures of 2a–2c are almost superimposable (Figure S14). Notably, all of the isopropyl side chains of the 12 valine residues are located inside barrel 2a, which results in a highly hydrophobic pore (Figure 4b, gold). The crosssectional area of the interior space along the barrel axis was estimated to be approximately 30 Å2, which means that the barrel is potentially capable of accommodating or channeling small molecules. In contrast, all of the phenylalanine side chains are located outside of the barrel (Figure 4b, purple). No particular inter-barrel interactions are observed because all of the hydrogen-bonding sites of the FVFV sequence are used for the formation of the anti-parallel βsheets. The aromatic X residue seems to play a pivotal role, not only participating in the β-sheet formation, but also introducing curvature into the β-strands to prevent polymerization into β-sheet fibrils.

The conformation of the PGP region also deserves consideration. In our previous study using a PGP ligand,16 the same PGP sequence adopted a compact Ω-shaped loop through complexation, which induced a highly entwined structure. However, in 2, the extended S-shaped loop seems to induce the formation of the (1)2(ZnI2)2 closed βsheet array and also stabilizes this structure. These S- and Ω-shaped loops should thus be considered not as random conformations but as dominant folding patterns in protein structures. A search of the PDB (Protein Data Bank) revealed that, among approximately 4000 examples, the conformational preference of the PGP sequence was the Ωshaped loop in about 47% of cases, and the S-shaped loop in about 43% (see the SI for details). We suggest that the folding of the PGP region into an S-shaped loop plays an important role in the formation of the stable (1)2(ZnI2)2 macrocyclic β-sheet and further induces its trimerization into the β-barrel structure. In conclusion, we have succeeded in the bottom-up chemical construction of a precise β-barrel structure with a large pore. The metal coordination to a peptide fragment with only eight residues shown in this study is quite simple, but has proven to be sufficient for the formation of such a β-barrel structure. We note that the obtained structures here have interior spaces, whereas the previous rare examples of β-barrel structures were obtained from amyloidbased short peptides,12,13 in which the strong β-sheets aggregation hindered the pore formation. We thus emphasize that elaborate, naturally occurring peptide tertiary or quaternary structures can be finely mimicked through the design of a concerted folding (intra-strand organization) and assembly (inter-strand organization) process, referred to as the general strategy of folding and assembly. These two organizational processes have been studied individually, but their combination and concertation have previously

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only been achieved by nature. Solubilization and elucidation of the solution state behaviors of the constructed βbarrels are our next challenge in due course.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Crystallographic data of 2a–2c are available from the Cambridge Structural Database under refcode 1832733– 1832735 (CIF). Full synthetic details, including preparative procedures and spectroscopic data for characterization of compounds (PDF).

AUTHOR INFORMATION Corresponding Authors

*[email protected] *[email protected] Notes

The authors declare no competing interests.

ACKNOWLEDGMENT This work was supported by Grants-in-Aid for Specially Promoted Research (24000009) for MF and Young Scientists (A) (JP15H05481) for TS. MY acknowledges a JSPS Research Fellowship for Young Scientists. The synchrotron X-ray crystallography was performed at the BL38B1 beamline at SPring-8 (2017B0120).

REFERENCES

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(1) Cowan, S. W.; Schirmer, T.; Rummel, G.; Steiert, M.; Ghosh, R.; Pauptit, R. A.; Jansonius, J. N.; Rosenbusch, J. P. Nature 1992, 358, 727. (2) Sakai, N.; Mareda, J.; Matile, S. Acc. Chem. Res. 2008, 41, 1354. (3) Blaber, M.; Lee, J. Curr. Opin. Struct. Biol. 2012, 22, 442. (4) Regan, L.; DeGrado, W. F. Science 1988, 241, 976. (5) Harbury, P. B.; Plecs, J. J.; Tidor, B.; Alber, T.; Kim, P. S. Science 1998, 282, 1462. (6) Bryson, J. W.; Betz, S. F.; Lu, H. S.; Suich, D. J.; Zhou, H. X.; O’Neil, K. T.; DeGrado, W. F.; Science 1995, 270, 935. (7) Zaccai, N. R.; Chi, B.; Thomson, A. R.; Boyle, A. L.; Bartlett, G. J.; Bruning, M.; Linden, N.; Sessions, R. B.; Booth, P. J.; Brady, R. L.; Woolfson, D. N. Nat. Chem. Biol. 2011, 7, 935. (8) Hodges, R. S.; Saund, A. K.; Chong, P. C. S.; St.-Pierre, S. A.; Reid, R. E. J. Biol. Chem. 1981, 256, 1214. (9) Huang, P.-S.; Feldmeier, K.; Parmeggiani, F.; Velasco, D. A. F.; Höcker, B.; Baker, D. Nat. Chem. Biol. 2016, 12, 29. (10) Rämisch, S.; Weininger, U.; Martinsson, J.; Akke, M.; André, I. Proc. Natl. Acad. Sci. USA 2014, 111, 17875. (11) Doyle, L.; Hallinan, J.; Bolduc, J.; Parmeggiani, F.; Baker, D.; Stoddard, B. L.; Bradley, P. Nature 2015, 528, 585. (12) Laganowsky, A.; Liu, C.; Sawaya, M. R.; Whitelegge, J. P.; Park, J.; Zhao, M.; Pensalfini, A.; Soriaga, A. B.; Landau, M.; Teng, P. K.; Cascio, D.; Glabe, C.; Eisenberg, D. Science 2012, 335, 1228. (13) Liu, C.; Zhao, M.; Jiang, L.; Cheng, P.-N.; Park, J.; Sawaya, M. R.; Pensalfini, A.; Gou, D.; Berk, A. J.; Glabe, C. G.; Nowick, J.; Eisenberg, D. Proc. Natl. Acad. Sci. USA 2012, 109, 20913. (14) Ljubetič, A.; Gradišar, H.; Jerala, R.; Curr. Opin. Struct. Biol. 2017, 40, 65. (15) Sawada, T.; Matsumoto, A.; Fujita, M.; Angew. Chem. Int. Ed. 2014, 53, 7228. (16) Sawada, T.; Yamagami, M.; Ohara, K.; Yamaguchi, K.; Fujita, M. Angew. Chem. Int. Ed. 2016, 55, 4519. (17) Sawada, T.; Inomata, Y.; Yamagami, M.; Fujita, M. Chem. Lett. 2017, 46, 1119. (18) Sawada, T.; Yamagami, M.; Akinaga, S.; Miyaji, T.; Fujita, M. Chem. Asian J. 2017, 12, 1715. (19) Ramachandran, G. N.; Ramakrishnan, C.; Sasisekharan, V. J. Mol. Biol. 1963, 7, 95. (20) Murzin, A. G.; Lesk, A. M.; Chothia, C. J. Mol. Biol. 1994, 236, 1369. (21) Murzin, A. G.; Lesk, A. M.; Chothia, C. J. Mol. Biol. 1994, 236, 1382.

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Figure 1. (a) Schematic representation of the bottom-up construction of an all-β-barrel. Note that the βbarrel structure in this figure does not possess the same geometry as β-barrel 2a. (b) Chemical structure of designed octapeptide ligand 1 and its Zn-triggered folding and assembly into β-barrel 2a. 213x368mm (300 x 300 DPI)

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Figure 2. Structure of the (1)2(ZnI2)2 macrocyclic unit in 2a. (a) Cartoon representations of the macrocyclic unit from different viewpoints. (b) Chemical structure of the (1)2(ZnI2)2 macrocyclic unit (the PGP loop moieties have been omitted for clarity). 217x326mm (300 x 300 DPI)

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Figure 3. Structure of β-barrel 2a. (a) Side view of the crystal structure of 2a. β-Strand regions are highlighted in green. (b) Geometrical analysis. The arrows with five circles on them represent amino acid residues forming β-strands. The orange dotted guidelines indicate the domain of a (1)2(ZnI2)2 macrocyclic unit. 279x290mm (300 x 300 DPI)

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Figure 4. Top view of β-barrel 2a. (a) Cartoon representation, with each (1)2(ZnI2)2 macrocyclic unit in a different color. (b) Interior V residues and exterior F residues colored gold and purple, respectively. 134x236mm (300 x 300 DPI)

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Figure 5. Comparison of the two major types of PGP loops. (a) Ω-shaped loop found in our previous work16 (top) and overlaid coordinates of PGP loops with the same conformation found in natural protein structures reported in the PDB (bottom). (b) S-shaped loop found in 2a (top) and overlaid coordinates from the PDB (bottom). For details, see Figure S17. 172x113mm (300 x 300 DPI)

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