Communication pubs.acs.org/JACS
X‑ray Crystallographic Structure of a Giant Double-Walled Peptide Nanotube Formed by a Macrocyclic β‑Sheet Containing Aβ16−22 Kevin H. Chen, Kelsey A. Corro, Stephanie P. Le, and James S. Nowick* Department of Chemistry, University of California, Irvine, Irvine, California 92697-2025, United States S Supporting Information *
connected by two δ-linked ornithine (δOrn) turn units to form a macrocycle.14 One β-strand, shown on the top in the drawing, contains Aβ16−22 with Phe19 replaced with p-iodo-phenylalanine (FI). The other β-strand, shown on the bottom in the drawing, contains the retro-peptide alignment of the same sequence, where the sequence order is reversed (EAFFVLK) and Phe19 is N-methylated.15 We term these two β-strands, respectively, the “K-E strand” and the “E-K strand”. The sequence reversal of the E-K strand places the two positively charged Lys residues and two negatively charged Glu residues at opposite ends of macrocyclic β-sheet 1.16 We incorporated p-iodo-phenylalanine to permit determination of the X-ray crystallographic phases through single-wavelength anomalous diffraction (SAD) phasing of iodine. We incorporated N-methyl-phenylalanine to block uncontrolled hydrogen bonding and prevent macrocyclic β-sheet 1 from forming amyloid fibrils.
ABSTRACT: This paper describes the supramolecular assembly of a macrocyclic β-sheet containing residues 16− 22 of the β-amyloid peptide, Aβ. X-ray crystallography reveals that the macrocyclic β-sheet assembles to form double-walled nanotubes, with an inner diameter of 7 nm and outer diameter of 11 nm. The inner wall is composed of an extended network of hydrogen-bonded dimers. The outer wall is composed of a separate extended network of β-barrel-like tetramers. These large peptide nanotubes pack into a hexagonal lattice that resembles a honeycomb. The complexity and size of the peptide nanotubes rivals some of the largest tubular biomolecular assemblies, such as GroEL and microtubules. These observations demonstrate that small amyloidogenic sequences can be used to build large nanostructures.
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esearchers have only begun to design supramolecular assemblies that rival the complexity of those found in nature.1 Microtubules, virus capsids, and other molecular machinery of biology remain beyond that which can be designed from scratch and created in the laboratory.2−4 In exploring the supramolecular assembly of a peptide derived from the βamyloid peptide, Aβ, we have discovered a supramolecular assembly that approaches these biomolecular structures in size and complexity. Here, we report the X-ray crystallographic structure of a double-walled nanotube with an inner diameter of 7 nm and an outer diameter of 11 nm that is formed by a peptide derived from Aβ. The central region of Aβ comprising residues 16−22 (KLVFFAE) provides a fascinating combination of hydrophobic and hydrophilic residues that imparts a strong propensity to assemble to form amyloid-like structures.5−10 The region consisting of residues 16−22 has a run of five hydrophobic residues (LVFFA) surrounded by polar residues of opposite charge (K and E).11 Lynn and co-workers demonstrated that the Aβ-derived heptapeptide Ac-KLVFFAE-NH2 forms ribbons comprising antiparallel β-sheets, which laminate and twist to form nanotubes, approximately 50 nm in diameter.5,6 The phenylalanine residues 19 and 20 are especially important in selfassembly; even FF dipeptides can assemble to form nanotubes.12,13 Our laboratory is also interested in understanding the self-assembly of Aβ and using it to create complex supramolecular assemblies.7−9 In the current study, we designed macrocyclic β-sheet peptide 1 to explore further the self-assembly of Aβ16−22. Macrocyclic βsheet 1 contains two heptapeptide strands derived from Aβ16−22 © 2017 American Chemical Society
Macrocyclic β-sheet 1 grew hexagonal pointed prism shaped crystals suitable for X-ray crystallography from a 10 mg/mL solution with 0.1 M bis-Tris at pH 5.5, 0.2 M ammonium acetate, and 45% v/v 2-methyl-2,4-pentanediol. X-ray diffraction data were collected with a copper anode source and processed with the PHENIX software suite. The X-ray crystallographic assembly of macrocyclic β-sheet 1 was solved and refined in space group P6122. The resolution of the final structural model is 2.1 Å. The X-ray crystallographic structure of macrocyclic β-sheet 1 reveals a unique hierarchical supramolecular assembly. Macrocyclic β-sheet 1 folds to form β-hairpin monomers. Six monomers make up the X-ray crystallographic asymmetric unit. The asymmetric units assemble in a honeycomb-like fashion to form the crystal lattice. The crystal lattice reveals a network of tightly packed, double-walled peptide nanotubes. Two of the six monomers are relatively flat; four are more twisted (Figure 1 and S1). Each β-hairpin has two surfaces. One surface displays the polar residues Lys16 and Glu22, as well as the hydrophobic residues Val18 and Phe20. The other surface displays the hydrophobic residues Leu17 and Ala21, as well as the unnatural Received: April 17, 2017 Published: June 9, 2017 8102
DOI: 10.1021/jacs.7b03890 J. Am. Chem. Soc. 2017, 139, 8102−8105
Communication
Journal of the American Chemical Society
Figure 1. X-ray crystallographic structure of the β-hairpin monomers that comprise the dimer (A) and tetramer (B) formed by macrocyclic βsheet 1.
amino acids p-iodo-Phe19 and N-methyl-Phe19. We term these surfaces, respectively, the “major face” and the “minor face” (Figure 2). The flatter β-hairpins make up dimers; the more twisted β-hairpins make up tetramers. Each monomer of the dimer comes from two adjacent asymmetric units. The dimers and tetramers pack through hydrophobic interactions between their minor faces, most notably among the side chains of p-iodoPhe19 and N-methyl-Phe19. The dimer resembles a four-stranded antiparallel β-sheet (Figure 2A). The K-E strands of the two macrocyclic β-sheets make up the dimerization interface and are fully aligned through nine intermolecular hydrogen bonds. Two salt bridges between the side chains of Lys16 and Glu22 on either end of the β-strands further stabilize the dimer. The tetramer resembles a β-barrel-like assembly with a continuously hydrogen-bonded network that connects all 16 βstrands of the monomers (Figure 2B). It is not a traditional βbarrel in which there is extensive hydrogen bonding between the β-strands of each monomer that results in an upright, cylindrical structure. Instead, the tetramer is substantially flatter than a canonical β-barrel and has only nine intermolecular hydrogen bonds among the four component monomers. There are only three unique pairings among the nine hydrogen bonds. At two of the junctions between monomers, δOrn1 and Leu17 of the K-E strand hydrogen bond. At the other two junctions between monomers, Leu17 and N-methyl-Phe19 of the E-K strand hydrogen bond. Hydrophobic interactions stabilize the tetramer. The eight side chains of Leu17 pack tightly together to form a hydrophobic core (Figure 2C). The formation of this leucine core appears to promote the twisting of the four β-hairpins in the tetramer. Aromatic interactions between Phe20 on the major face further stabilize the tetramer. Macrocyclic β-sheet 1 forms a honeycomb-like lattice composed of double-walled peptide nanotubes (Figure 3). Each nanotube is surrounded by six other identical nanotubes. Aromatic interactions among N-methyl-Phe19 and p-iodo-Phe19
Figure 2. Hydrogen-bonded dimer (A) formed by macrocyclic β-sheet 1. The β-barrel-like tetramer: side view (B) and top view (C).
on the minor face stabilize each junction in the lattice where three nanotubes meet (Figure S2). A small triangular cavity, about 2 nm across, sits at the center of each junction. Each nanotube consists of an inner wall and an outer wall. The inner wall consists of the dimers (cyan), and the outer wall consists of the tetramers (green). The diameters of the inner and outer walls are 7 and 11 nm, respectively (Figure 4A). The thickness of the nanotube wall is approximately 2 nm. The nanotube is largely hollow and runs the length of the crystal lattice (Figure 4B). 8103
DOI: 10.1021/jacs.7b03890 J. Am. Chem. Soc. 2017, 139, 8102−8105
Communication
Journal of the American Chemical Society
Figure 3. Honeycomb-like crystal lattice form by macrocyclic β-sheet 1.
The inner wall is composed of an extended network of dimers (Figure 5A). Each dimer hydrogen bonds with two other dimers through the E-K strand. The dimers are shifted out of alignment by four residues toward the N-termini to accommodate the Nmethyl groups. Each dimer forms four hydrogen bonds with each neighboring dimer, with Leu17 hydrogen bonding with Leu17 and N-methyl-Phe19 hydrogen bonding with δOrn1 in the respective E-K strands. The outer wall is composed of an extended network of tetramers (Figure 5B). Each tetramer hydrogen bonds with four other tetramers through the K-E strands. The tetramers are shifted out of alignment by four residues toward the C-termini, with p-iodo-Phe19 hydrogen bonding with Ala21 in the respective K-E strands. The Aβ16−22 sequence, displayed forward and backward in macrocyclic β-sheet 1, imparts a rich architecture of supramolecular assembly. By adopting two conformations, one flat and one twisted, macrocyclic β-sheet 1 is able to form the inner and outer walls of the giant double-walled peptide nanotube. At 11 nm in diameter, the nanotube is almost as large as the largest homogeneous tubular biomolecular assemblies, such as GroEL (13 nm), tobacco mosaic virus (18 nm), and microtubules (24 nm). Although macrocyclic β-sheet 1 contains only 16 residues, it is able to form assemblies comparable in size to proteins containing hundreds of residues. This finding demonstrates the potential of using small amyloidogenic sequences to build large nanostructures. The structure of the double-walled nanotube is an emergent property that cannot currently be predicted from the Aβ16−22 sequence.17 These monodisperse structures may eventually serve as templates in which to form nanowires or other materials.18−21 Different amyloidogenic sequences may yield other unusual nanostructures, with wide-ranging potential applications. Although prediction of these higher order supramolecular assemblies remains largely out of reach, the β-amyloid peptide provides fertile ground for discovering new structures.
Figure 4. Hollow peptide nanotube formed by macrocyclic β-sheet 1: top view (A) and side view (B).
Figure 5. Inner wall of the peptide nanotube is composed of an extended network of dimers (A). The outer wall of the peptide nanotube is composed of an extended network of tetramers (B).
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DOI: 10.1021/jacs.7b03890 J. Am. Chem. Soc. 2017, 139, 8102−8105
Communication
Journal of the American Chemical Society
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(17) Lu, K.; Guo, L.; Mehta, A. K.; Childers, W. S.; Dublin, S. N.; Skanthakumar, S.; Conticello, V. P.; Thiyagarajan, P.; Apkarian, R. P.; Lynn, D. G. Chem. Commun. 2007, 2729−2731. (18) Reches, M.; Gazit, E. Science 2003, 300, 625−627. (19) Cherny, I.; Gazit, E. Angew. Chem., Int. Ed. 2008, 47, 4062−4069. (20) Hamley, I. W. Angew. Chem., Int. Ed. 2014, 53, 6866−6881. (21) Sun, J.; Jiang, X.; Lund, R.; Downing, K. H.; Balsara, N. P.; Zuckermann, R. N. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 3954−3959.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b03890. Crystallographic data for macrocyclic β-sheet 1 (PDB) Crystallographic data for macrocyclic β-sheet 1 (CIF) Procedures for the synthesis and crystallization of macrocyclic β-sheet 1; details of X-ray diffraction data collection, processing, and refinement (PDF)
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AUTHOR INFORMATION
Corresponding Author
*
[email protected] ORCID
James S. Nowick: 0000-0002-2273-1029 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank NSF (CHE-1507840) for funding. Crystallographic coordinates of macrocyclic β-sheet 1 were deposited into the Protein Data Bank with PDB code 5VF1.
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REFERENCES
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DOI: 10.1021/jacs.7b03890 J. Am. Chem. Soc. 2017, 139, 8102−8105
