- Email: [email protected]
High-Resolution Structure of a Self-Assembly-Competent Form of a Hydrophobic Peptide Captured in a Soluble β-Sheet Scaffold
doi:10.1016/j.jmb.2008.02.051
J. Mol. Biol. (2008) 378, 459–467
Available online at www.sciencedirect.com
High-Resolution Structure of a Self-Assembly-Competent Form of a Hydrophobic Peptide Captured in a Soluble β-Sheet Scaffold Koki Makabe 1 , Matthew Biancalana 1 , Shude Yan 1 , Valentina Tereshko 1 , Grzegorz Gawlak 1 , Hélène Miller-Auer 1,2 , Stephen C. Meredith 1,2 and Shohei Koide 1 ⁎ 1
Department of Biochemistry and Molecular Biology, The University of Chicago, 929 E. 57th Street, Chicago, IL 60637, USA 2
Department of Pathology, The University of Chicago, Chicago, IL 60637, USA Received 20 November 2007; received in revised form 9 February 2008; accepted 23 February 2008 Available online 4 March 2008
β-Rich self-assembly is a major structural class of polypeptides, but still little is known about its atomic structures and biophysical properties. Major impediments for structural and biophysical studies of peptide selfassemblies include their insolubility and heterogeneous composition. We have developed a model system, termed peptide self-assembly mimic (PSAM), based on the single-layer β-sheet of Borrelia outer surface protein A. PSAM allows for the capture of a defined number of self-assembly-like peptide repeats within a water-soluble protein, making structural and energetic studies possible. In this work, we extend our PSAM approach to a highly hydrophobic peptide sequence. We show that a penta-Ile peptide (Ile5), which is insoluble and forms β-rich self-assemblies in aqueous solution, can be captured within the PSAM scaffold in a form capable of selfassembly. The 1.1-Å crystal structure revealed that the Ile5 stretch forms a highly regular β-strand within this flat β-sheet. Self-assembly models built with multiple copies of the crystal structure of the Ile5 peptide segment showed no steric conflict, indicating that this conformation represents an assembly-competent form. The PSAM retained high conformational stability, suggesting that the flat β-strand of the Ile5 stretch primed for selfassembly is a low-energy conformation of the Ile5 stretch and rationalizing its high propensity for self-assembly. The ability of the PSAM to “solubilize” an otherwise insoluble peptide stretch suggests the potential of the PSAM approach to the characterization of self-assembling peptides. © 2008 Elsevier Ltd. All rights reserved.
Edited by J. Weissman
Keywords: amyloid fibril; single-layer β-sheet; solubilization; protein engineering; x-ray crystallography
Introduction It has become evident that many polypeptides can be transformed into β-rich self-assemblies.1 β-Rich self-assembly is the core structure of the so-called cross-β amyloid fibrils that are associated with *Corresponding author. E-mail address: [email protected]. Abbreviations used: PSAM, peptide self-assembly mimic; SLB, single-layer β-sheet; OspA, outer surface protein A; DMSO, dimethyl sulfoxide; AFM, atomic force microscopy; NHB, non-hydrogen bonded; HB, hydrogen bonded.
devastating human diseases. Discovery of diverse peptide sequences that can form self-assemblies has increased interest in peptide self-assembly as a means for creating nanoscale structures. Self-assembly of molecules is becoming a popular and powerful strategy to create nanomaterials, and because peptide building blocks with desired chemical composition can be readily synthesized, peptide self-assembly offers a technology platform for the production of diverse nanomaterials. Indeed, many types of selfassemblies have been produced using synthetic peptides.2,3 Therefore, increased understanding of the factors governing peptide self-assembly will have broad impacts on material sciences, biology, and medicine.
0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.
Structure of a β-Rich Self-Assembly Peptide
460 Despite their importance, still, little is known about the high-resolution structure and biophysical properties of peptide self-assemblies. Their insoluble nature and inherently heterogeneous stoichiometry make it extremely difficult to apply the standard biophysical techniques for water-soluble proteins. Solid-state NMR studies have elucidated the conformations of peptide units and modes of higher-order assemblies.4–7 Although powerful, it is a laborious method, and it is difficult to obtain high-resolution structural information. Pioneering work by the group of Eisenberg has determined the atomic structures of amyloidogenic peptides assembled into microcrystals, which has established the cross-β spine as a common structure of peptide fibrils.8,9 Although the crystallization approach is also powerful, it is limited to structural determination, and it seems that peptides are crystallized in a high-energy state.10 In addition, it may not be applicable to a broad range of amyloids beyond short peptides. To overcome these fundamental difficulties associated with characterization of self-assembling peptides, several groups have employed transplantation of a peptide segment into a water-soluble globular protein. In this approach, it is hoped that an engineered protein harboring the peptide segment remains soluble and that insights into the structure and thermodynamics of self-assembly can be gained by characterizing the engineered protein with standard biophysical techniques, such as x-ray crystallography. Stott et al. introduced Gln repeats into a flexible loop of chymotrypsin inhibitor 2.11 Although the engineered chymotrypsin inhibitor 2 oligomerized, its crystal structure revealed that the oligomerization was due to domain swapping and the Gln repeats were disordered and not forming βrich self-assembly.12 Similarly, Takano et al. replaced a C-terminal segment of ribonuclease HI with an Aβ fragment, which prevented aggregation of the Aβ fragment.13 The x-ray crystal structure of this fusion protein revealed that, although the Aβ segment had a β-sheet conformation, it did not form β-sheetmediated interactions, suggesting that the observed form lacked the capability of self-assembly. These examples illustrate the difficulty of capturing a peptide in a form primed for assembly into higherorder structures. We have developed a protein engineering strategy to overcome the fundamental challenges in highresolution characterization of peptide self-assemblies. Our approach, termed peptide self-assembly mimic (PSAM), captures a peptide segment of interest within the structural context of a flat β-sheet. A highly regular flat β-sheet is a hallmark of β-rich self-assemblies. Thus, our approach is distinct from other transplantation attempts in that it grafts a peptide of interest in an environment close to that of self-assembly. We used the single-layer β-sheet (SLB) segment of outer surface protein A (OspA) from Borrelia burgdorferi as the host for PSAM. The SLB exists as a segment linking two globular domains, which in
turn serve as “end caps” for the SLB. Although it is exposed to the solvent and thus does not contain a hydrophobic core, it is highly stable. Indeed, the presence of the globular domains would appear to inhibit the lamination of the β-sheet, regardless of its composition.14,15 The single-layer architecture eliminates complications caused by long-range interactions through a hydrophobic core that are commonly present in a water-soluble β-sheet protein. We have demonstrated that a β-hairpin sequence from the SLB forms β-rich fibrils16 and that the self-assembly of this β-hairpin segment can be captured within OspA in the form of extended SLB.17 The x-ray crystal structures of these PSAMs have enabled the establishment of structural linkage between the atomic structures of self-assembling peptides and the macroscopic morphology.14 The two globular domains sequester the “sticky” β-sheet edges, thus enabling the PSAM system to capture β-rich self-assembly within a water-soluble protein. Furthermore, unlike most β-sheets found in globular proteins,18 the SLB has a highly regular geometry and is flat, which may closely mimic the β-sheet conformation within actual peptide assemblies. Recent studies have shown that many peptide sequences self-assemble into an antiparallel β-sheet and that small sequence changes can alter the βsheet topology between parallel and antiparallel.9,19 Therefore, although our PSAM system using OspA can mimic only self-assemblies consisting of antiparallel β-sheets, its detailed structural and energetic studies would provide significant knowledge relevant to the molecular mechanism of peptide self-assembly. In our initial work to establish the PSAM strategy, we used a hydrophilic peptide sequence derived from the SLB itself. In this work, we wished to explore the potential of the PSAM to capture diverse self-assembling peptides. To this end, we introduced a penta-Ile motif in the PSAM. We show that a penta-Ile peptide forms β-rich self-assemblies and that it can be stably captured into the PSAM. The high-resolution x-ray crystal structure of the peptide captured in the PSAM reveals molecular details of βrich peptide self-assembly.
Results An oligo-Ile peptide forms β-rich self-assemblies It has been shown that certain poly-amino acids self-assemble into fibrils.20 Subsequently, amyloid aggregation propensities of amino acids have been derived.21,22 Ile has the highest aggregation propensity among aliphatic amino acids on the scale of Pawar et al.,21 and it is also among the most aggregation-prone amino acids on the scale of Rousseau et al.22 Ganesh and Jayakumar reported that short peptides containing three consecutive Ile peptides form β-rich self-assembly.23 Therefore, we chose an oligo-Ile peptide for this study. An oligo-Ile
Structure of a β-Rich Self-Assembly Peptide
peptide is very hydrophobic and distinct from the highly hydrophilic sequence of the OspA SLB; it thus represents a challenging target for transplantation into the PSAM system. A penta-Ile peptide (Ile5) was synthesized with solid-phase peptide synthesis. The purity and molecular weight of the peptide were confirmed with liquid chromatography–mass spectrometry. Because the lyophilized peptide only sparingly dissolved in an aqueous buffer, we prepared peptide solutions in two methods. First, the peptide was suspended at a 2-mg/ml concentration in an aqueous buffer with vigorous voltexing, and the supernatant of this suspension after centrifugation was used. Second, the peptide was first completely dissolved in 100% dimethyl sulfoxide (DMSO) at 20 mg/ml, and this solution was diluted 10-fold into an aqueous buffer, resulting in a solution containing 10% DMSO. Atomic force microscopy (AFM) characterization revealed needle-like objects in the peptide solution (Fig. 1b and c). Their widths ranged from ∼ 20 to ∼ 100 nm, and their lengths were a few micrometers. The needle-like objects were also found in the
461 sample in 10% DMSO (Fig. 1d), and the shape of the objects in the 10% DMSO sample was similar to that of the objects in the water sample, indicating that these objects can form from monomeric peptides in solution and that they are not an artifact from incomplete solubilization of peptide powder. The suspension supernatant exhibited a far-UV circular dichroism (CD) spectrum characteristic of β-sheet (Fig. 1a), indicating that the peptide self-assembles had high β-sheet content. The AFM objects showed no twist commonly observed for peptide self-assemblies. Both samples show x-ray diffraction maxima corresponding to 4.6- and 10-Å spacings (Fig. 1e and f), a hallmark of β-rich peptide assemblies.24 Commonly, the 4.6-Å spacing is interpreted as the spacing between β-strands within a β-sheet and the 10-Å spacing is interpreted as the spacing between two laminated β-sheets, which are the bases for the cross-β structure.24 Interestingly, the Ile5 self-assembly showed little thioflavin T binding activity. It should be noted that not all peptide fibrils bind thioflavin T.25 Taken together, their morphology, their high β-sheet content, and
Fig. 1. (a) Far-UV CD spectrum of a supernatant of homo-Ile peptide suspension in water. The CD spectrum is plotted with the ellipticity, not with the molar ellipticity, because we could not determine the concentration of the peptide solution. (b–d) AFM scan images. Onemicrometer scale bars are indicated in the images. The brightness of features increases as a function of height. (b and c) Images from the water suspension sample taken from different areas of the same sample surface. (d) Image from the 10% DMSO sample. (e and f) X-ray fiber diffraction patterns of the water (e) and 10% DMSO (f) samples. The diffraction maxima corresponding to the 4.6-and 10-Å spacings are marked with arrows.
462 the x-ray diffraction patterns suggest that the oligoIle peptide forms a cross-β self-assembly, as seen for fibrils of other peptides. Stable grafting of an oligo-Ile stretch into the PSAM Having confirmed the ability of oligo-Ile peptide to self-assemble into a β-rich structure, we proceeded to graft the Ile5 sequence within the PSAM β-sheet. We hypothesized that, if the Ile5 peptide has a high propensity to self-assemble into flat β-sheets, replacing a flat β-strand with an Ile5 stretch should not reduce the stability of the PSAM. Thus, we grafted the Ile5 sequence into the central β-strand (strand 9) of the OspA SLB (Fig. 2a). The SLB consists of three solvent-exposed strands. We chose strand 9 as the host because it is centrally located in the SLB and highly exposed to the solvent. These characteristics minimize interactions of residues in strand 9 with those in the globular domains. The five consecutive Ile residues replaced residues 120–124 (SSTEE), and we term the resulting mutant PSAM-Ile5. In spite of the introduction of a highly hydrophobic segment at highly solvent-exposed positions, PSAM-Ile5 was expressed as a soluble protein in Escherichia coli and was predominantly monomeric in aqueous solution as judged with gel-filtration chromatography (Fig. 2b). These results indicate that the PSAM scaffold effectively “solubilizes” the otherwise insoluble Ile5 peptide by sequestering it within the SLB. PSAM-Ile5 underwent a reversible unfolding reaction induced by urea (Fig. 2c). The unfolding curve was interpreted in terms of a three-state model as for
Structure of a β-Rich Self-Assembly Peptide
the wild-type OspA.15,26 In this three-state model, the first transition corresponds to unfolding of the C-terminal domain and a portion of the SLB; the second, to that of the rest of the SLB and the N-terminal domain. The overall stability of PSAMIle5 was nearly identical with that of the wild type (ΔΔGNU3M = 0.07 kcal/mol), but the first transition was destabilized (ΔΔGNI3M = − 0.75 kcal/mol) and the second transition was stabilized (ΔΔGIU3M = 0.82 kcal/mol) (ΔGNU3M, ΔGNI3M, and ΔGIU3M are the free energy difference at 3M urea between the native and unfolded states, that between the native and intermediate states, and that between the intermediate and unfolded states, respectively; ΔΔGNU3M, ΔΔGNI3M, and ΔΔGIU3M are differences in ΔGNU3M, ΔGNI3M, and ΔGIU3M between the wild type and the mutant, respectively). These compensatory changes suggest that the Ile5 replacement has altered the nature of the intermediate state. Although the detailed effects of the strand replacement on the unfolding mechanism are not clear, these results demonstrate that PSAM accommodates the strand replacement without significant stability loss. High-resolution x-ray crystal structure of PSAM-Ile5 Our crystallization efforts of PSAM-Ile5 did not yield any single crystals. To facilitate crystallization, we introduced a set of surface mutations in the globular domains that have been highly effective in crystallizing OspA and its variants.14,27 These mutations are distant from the grafted Ile5 segment and have previously been shown to cause little structural change.27 The resulting surface-engineered protein,
Fig. 2. (a) Amino acid sequence of the OspA SLB region. The line rectangles indicate residues in a β-strand whose side chains face toward the reader, and the shaded rectangles indicate those facing backward. The mutational residues are shown in rectangles. The backbone hydrogen bonds are shown as dashed lines. (b) Elution profiles of the wild type (dashed line) and PSAM-Ile5 (solid line) from a sizeexclusion column detected using absorbance at 280 nm. (c) Ureainduced unfolding of PSAM-Ile5. Normalized CD (circles) and fluorescence (cross) intensities for PSAM-Ile5 are plotted as a function of urea concentration. The profiles for the wild-type protein are also shown (CD, solid line; fluorescence, dashed line).
Structure of a β-Rich Self-Assembly Peptide
termed PSAM-Ile5-sm1, readily crystallized, and its crystal structure was determined at a resolution of 1.10 Å (Fig. 3a). The data collection and refinement statistics are summarized in Table 1. Despite the fact that Ile5 replacement in strand 9 completely disrupts the native side-chain pairings (Fig. 2a), the global fold of PSAM-Ile5-sm1 was nearly identical with the wild-type OspA containing the same set of surface mutations (Fig. 3b), with a root-mean-square deviation (RMSD) of 0.3 Å for the Cα atoms common between the wild-type and mutant structures. This structural resemblance is remarkable, because the SLB does have a level of structural plasticity. For example, a series of PSAMs with different numbers of peptide inserts exhibited a range of backbone conformations,14 and a single mutation at Phe126 located at the C-terminus of strand 9 (Fig. 2a) significantly perturbs the backbone structure, increasing the Cα RMSD to 1.15 Å.15 Thus, the small structural deviation due to the Ile5 replacement suggests that the PSAM SLB provides a structural environment that is compatible with a stable conformation of the Ile5 segment. In the crystal structure, the Ile5 segment takes on a regular β-sheet conformation with right-hand twist along the strand (Fig. 4a). In addition to the Ile5 stretch, PSAM-Ile5 contains two cross-strand Ile–Ile pairs involving the Ile5 stretch and residues in strand
463 10 (Figs. 3a and 4b). These are a non-hydrogenbonded (NHB) pair (Ile122–Ile137) and a hydrogenbonded (HB) pair (Ile123–Ile136). These cross-strand pairs provide an opportunity to see the side-chain conformations and interactions of Ile residues in the context of self-assembly, since these combinations are present in an antiparallel β-sheet structure made of homo-Ile peptides. We found distinct modes of interactions in the HB and NHB pairs (Fig. 4b). As pointed out previously,28 the Cγ atoms of the NHB Ile pair face each other with slight offset along with the strand direction. They are tightly packed and exclude solvent molecules between the side chains. In contrast, the Cγ atoms of the HB pair point to mutually opposite directions and create voids between the Cβ atoms (Fig. 4c). These voids are filled with well-defined water molecules, thus rescuing the “weak points.”27,29 This hydration pattern is conserved in the wild-type OspA-sm1 and PSAMIle5-sm1, suggesting that this is a general feature found on the surface of a flat β-sheet (Fig. 3c). Together, these results suggest that in an Ile peptide self-assembly, adjacent Ile peptides are held through the backbone hydrogen bonds, the side chains of the NHB pairs form tight interactions, and the side chains of the HB pair form suboptimal interactions that may be compensated by the main-chain hydration at the weak points.
Fig. 3. Crystal structure of PSAM-Ile5-sm1. (a) Superposition of PSAM-Ile 5 -sm1 (green) onto OspA-sm1 (gray). The mutated residues are shown in red sticks, and Ile residues in strand 10 are shown in green sticks. N- and Cterminal positions are indicated. (b) The RMSDs for the Cα atoms are plotted versus residue number. The positions of β-strands and the Cterminal helix are shown as bars. The mutation residues are indicated as a red circle. (c) Backbone hydration of the SLB. The water molecules are shown as red spheres. The mutation residues are shown as green sticks.
Structure of a β-Rich Self-Assembly Peptide
464 Table 1. Data collection and refinement statistics for PSAM-Ile5-sm1 P21
Space group Data collection statistics Cell parameters a b c β Beamline Wavelength (Å) Resolution (Å) Completeness (%) I/σ(I) Rmergea Average redundancy Refinement statistics Resolution range (Å) Reflections used (free) R-factorb Rfreec RMSDs Bonds (Å) Angles (°) No. of protein residues No. of water molecules Average B-factor (Å2) Ramachandran plot statistics (%) Most favored regions Additionally allowed regions Generally allowed regions
33.26 54.71 66.50 99.91 APS 23ID 0.9795 50–1.1 (1.14–1.10) 97.8 (86.2) 19.25 (2.88) 0.053 (0.318) 3.3 (2.3) 20.0–1.1 88,420 (4669) 0.155 0.171 0.008 1.281 246 378 9.82 91.9 8.1 0.0
Data in parentheses are for the highest-resolution shell. a Rmerge = ∑hkl∑i|I(hkl)i − 〈I(hkl)〉|/∑hkl∑i〈I(hkl)i〉 over i observations of a reflection hkl. b R-factor = ∑||Fobs|−|Fcalc||/∑|Fobs|. c Rfree is R with 5% of reflections sequestered before refinement.
Atomic models of the Ile5 self-assembly To examine if the Ile5 structure in PSAM-Ile5-sm1 reflects the structure of an Ile5 peptide within the βrich self-assemblies, we constructed an atomic model of a self-assembly from the x-ray crystal structure of the Ile5 segment. In this model, multiple copies of the peptide structure were placed side by side such that a continuous β-sheet is formed (Fig. 4d). We used the backbone structures of an insulin peptide that forms antiparallel β-sheet assembly [Protein Data Bank (PDB) ID 2OMQ]9 as the template for placing the Ile5 structures. It is important to emphasize that the template was used only to define the spacing and that we did not alter the conformation of the Ile5 segment in this modeling. The resulting β-sheet structure of the model has no steric clash, indicating that the Ile5 conformation in PSAM-Ile5-sm1 can self-assemble without conformational adjustment. Thus, this model may represent the basic unit for the selfassembly of the Ile5 peptide. Although further lamination of the β-sheet units is required to account for the dimensions of the self-assembly seen in the AFM images, this model helps connect the atomic structure and the macroscopic morphology of peptide self-assembly.
Discussion We have shown that an Ile5 peptide forms a β-rich self-assembly and that it can be captured within a soluble PSAM in a form that is clearly compatible with self-assembly. To our knowledge, this is the first example of the high-resolution structure of a homo-oligomer of an amino acid in an assemblycompetent form. PSAM-Ile5 was highly stable, and it retained the flat β-sheet seen in other PSAMs, strongly suggesting that the highly regular flat βstrand is an energetically favorable conformation of the Ile5 peptide. Our results with PSAM-Ile5 are in stark contrast to previous results of grafting studies of self-assembling peptides. Often, soluble host proteins are recruited into amyloids and/or aggregates.30,31 In other cases where the grafting location was carefully chosen so that the protein solubility was maintained, the grafted peptide was found to be in a conformation incapable of self-assembly. 12,13 The main difference between our host and the others is that ours captures a peptide of interest within a welldefined flat β-sheet, while others capture a peptide within a flexible segment. Lamination of β-sheet does not occur, presumably because this is prevented by the globular domains. The flat β-sheet is the native conformation of the host SLB. Thus, the formation of a flat β-strand conformation of the grafted peptide does not promote a nonnative conformation of the PSAM host that might cause denaturation and/or misfolding. Because the flat βsheet is a favorable conformation of self-assembling peptides, grafting of a peptide segment with a high self-assembly propensity stabilizes the native, flat structure of the PSAM scaffold. β-Sheet assembly requires the formation of a backbone hydrogen-bond network across β-strands. In general, an exposed β-sheet edge is poised to interact with another β-sheet edge, and in natural proteins, multitudes of negative elements are installed at an exposed β-sheet edge to prevent aberrant intermolecular interactions. 18 Thus, it is unlikely that a grafted peptide segment finds a highly regular β-sheet edge in the host protein to which it can dock. Accordingly, it is unlikely that simple grafting of a self-assembling peptide unit in a natural host protein would promote the formation of a self-assembly-mimicking conformation (e.g., β-sheet) of the peptide. Furthermore, simple grafting provides no clear mechanism for preventing uncontrolled self-assembly of a grafted peptide. Our PSAM approach converts peptide self-assembly into protein folding, allowing for high-resolution characterization of structural features in peptide self-assemblies using well-established tools. While the PSAM scaffold promotes the β-sheet formation of the grafted sequence, the scaffold has enough structural plasticity such that the grafted peptide can adapt its own favorable conformation. The work presented here demonstrates that the PSAM is applicable to diverse peptide sequences, including extremely hydrophobic ones such as Ile5, suggesting
Structure of a β-Rich Self-Assembly Peptide
465
Fig. 4. Construction of the βassembly models. (a) Ile5 structure in PSAM-Ile5-sm1. (b) HB pair and NHB pair of Ile residues in the crystal structure. The mutated residues are shown as red sticks, and Ile residues in strand 10 are shown as green sticks. Backbone hydrogen bonds are shown as a blue dashed line. (c) Interstrand Ile pairing in the crystal structure. Ile residues are shown in Corey–Pauling–Koltun representation. The left panel shows the NHB pair (shown in red), whereas the right panel shows the HB pair (shown in red). Water moleculepositioned “weak points” are shown in cyan. (d) Models of SLB assembly. Antiparallel β-sheet model is created based on the amyloidforming peptide crystal structure.
that sequences from natural fibril-forming proteins can also be captured in the PSAM. We speculate that, with the ability of the PSAM to accommodate larger segments of self-assembly mimics,14 this strategy will complement existing ones, such as solidstate NMR spectroscopy and microcrystallography, to shed new light on the molecular structure and biophysical properties of peptide self-assemblies.
been described previously.26 The gene for the PSAM-Ile5 was subcloned in the equivalent region of the OspA surface mutant for crystallization (“OspAsm1”)27 using SpeI and PstI restriction enzyme sites, resulting in 12 surface mutations in total. The mutant was expressed and purified as described before.27 Analytical size-exclusion chromatography was performed using a Superdex 75 HR 10/30 column (GE Healthcare) in 50 mM Tris–HCl, pH 8.0, containing 200 mM NaCl at room temperature.
Materials and Methods
CD spectroscopy
Peptide preparation The Ile5 peptide was synthesized by solid-phase peptide synthesis using standard Fmoc (9-fluoroenylmethyloxycarbonyl) chemistry on a Rink MBHA resin. Its N-terminus was unblocked, and its C-terminus was amidated. The product was cleaved from the resin using trifluoroacetic acid/triisopropylsilane/H2O = 95:2.5:2.5 (v:v:v) and dissolved in 50% acetic acid and purified using reversedphase HPLC. Protein preparation Methods for site-directed mutagenesis, urea denaturation experiments, and analysis of denaturation data have
CD measurements were carried out using an Aviv 202 spectropolarimeter (Aviv Associates, Lakewood, NJ). The spectrum was taken at 25 °C in a 1-mm pathlength cuvette with a 1-nm step size, a 3-nm bandwidth, and a 1-s averaging time. AFM Samples were imaged after incubation for ∼1 month at room temperature. Samples were deposited onto a freshly cleaved mica surface, left on mica for 30 s, and then rinsed with deionized water. The mica surface with the adsorbed peptide was then dried with nitrogen gas. The images shown were obtained by scanning the mica surface in air by AFM (Multimode, Digital Instruments, Santa Barbara, CA)
466 with a spring constant of 2 N/m and a tip radius of curvature of b 10 nm (AC240TS-C2, Olympus). AFM scans were taken at 512 × 512 pixels of resolution and produced topographic images of the samples, in which the parameters of the brightness of features were as follows: tapping frequency, ∼70 kHz; RMS amplitude before engagement, 1 V; integral and proportional gains, 0.2–0.6 and 0.3–1.0, respectively; set point, 0.8 V; and scanning speed, 1 Hz. X-ray fiber diffraction The same batch of Ile5 samples that were used in the AFM experiments was placed in 1-mm diameter quartz capillary and vacuum dried. Diffraction data were collected at the APS (Advanced Photon Source) synchrotron radiation 23ID beamline facility of the Argonne National Laboratory and examined using Mosflm software.32 Crystallization and structure determination The crystals were grown in 30% PEG (polyethylene glycol) 400, 0.1 M Tris–HCl, pH 8.0, using the hangingdrop vapor-diffusion method. The x-ray diffraction data were collected at the APS 23ID beamline. Crystal data and data collection statistics are summarized in Table 1. The x-ray diffraction data were processed with HKL2000.33 The structure was determined by molecular replacement with the program MOLREP in CCP4.32 The OspA-sm1 structure (PDB ID 2G8C) was used as the search model. CNS1.134 and Refmac535 were used for the structural refinement, and final positional and anisotropic temperature factor refinements were performed using Refmac5. Model building was carried out using the Coot program.36 The self-assembly model was built by superposing the five Cα positions of the Ile5 structure of PSAM-Ile5 onto the Cα positions of the crystal structure of the insulin peptide. Molecular graphics were generated using PyMOL†. PDB accession code The coordinates have been deposited in the PDB with entry code 2I5V.
Acknowledgements This work was supported in part by the National Institutes of Health through grant R01-GM72688 and the National Science Foundation through grant CMMI-0709079. M. B. was supported by the National Institutes of Health (grant T90-DK070076) and the Paul K. Richter and Evalyn E. Cobb Richter Memorial Fund. Use of the APS was supported by the Office of Science Basic Energy Sciences Program of the U.S. Department of Energy under contract no. W-31-109ENG-38. GM/CA CAT (APS 23ID) has been supported in whole or in part with federal funds from the National Cancer Institute (Y1-CO-1020) and the National Institute of General Medical Science (Y1† http://www.pymol.org
Structure of a β-Rich Self-Assembly Peptide
GM-1104). We thank Dr. Justin Jureller of the University of Chicago NanoBio Facility for his assistance with AFM.
References 1. Dobson, C. M. (2003). Protein folding and disease: a view from the first Horizon Symposium. Nat. Rev. Drug Discov. 2, 154–160. 2. Rajagopal, K. & Schneider, J. P. (2004). Self-assembling peptides and proteins for nanotechnological applications. Curr. Opin. Struct. Biol. 14, 480–486. 3. Fairman, R. & Akerfeldt, K. S. (2005). Peptides as novel smart materials. Curr. Opin. Struct. Biol. 15, 453–463. 4. Jaroniec, C. P., MacPhee, C. E., Bajaj, V. S., McMahon, M. T., Dobson, C. M. & Griffin, R. G. (2004). Highresolution molecular structure of a peptide in an amyloid fibril determined by magic angle spinning NMR spectroscopy. Proc. Natl Acad. Sci. USA, 101, 711–716. 5. Luhrs, T., Ritter, C., Adrian, M., Riek-Loher, D., Bohrmann, B., Dobeli, H. et al. (2005). 3D structure of Alzheimer's amyloid-β(1–42) fibrils. Proc. Natl Acad. Sci. USA, 102, 17342–17347. 6. Petkova, A. T., Yau, W. M. & Tycko, R. (2006). Experimental constraints on quaternary structure in Alzheimer's beta-amyloid fibrils. Biochemistry, 45, 498–512. 7. Tycko, R. (2004). Progress towards a molecular-level structural understanding of amyloid fibrils. Curr. Opin. Struct. Biol. 14, 96–103. 8. Nelson, R., Sawaya, M. R., Balbirnie, M., Madsen, A. O., Riekel, C., Grothe, R. & Eisenberg, D. (2005). Structure of the cross-beta spine of amyloid-like fibrils. Nature, 435, 773–778. 9. Sawaya, M. R., Sambashivan, S., Nelson, R., Ivanova, M. I., Sievers, S. A., Apostol, M. I. et al. (2007). Atomic structures of amyloid cross-beta spines reveal varied steric zippers. Nature, 447, 453–457. 10. Esposito, L., Pedone, C. & Vitagliano, L. (2006). Molecular dynamics analyses of cross-β-spine steric zipper models: β-sheet twisting and aggregation. Proc. Natl Acad. Sci. USA, 103, 11533–11538. 11. Stott, K., Blackburn, J. M., Butler, P. J. & Perutz, M. (1995). Incorporation of glutamine repeats makes protein oligomerize: implications for neurodegenerative diseases. Proc. Natl Acad. Sci. USA, 92, 6509–6513. 12. Chen, Y. W., Stott, K. & Perutz, M. F. (1999). Crystal structure of a dimeric chymotrypsin inhibitor 2 mutant containing an inserted glutamine repeat. Proc. Natl Acad. Sci. USA, 96, 1257–1261. 13. Takano, K., Endo, S., Mukaiyama, A., Chon, H., Matsumura, H., Koga, Y. & Kanaya, S. (2006). Structure of amyloid beta fragments in aqueous environments. FEBS J. 273, 150–158. 14. Makabe, K., McElheny, D., Tereshko, V., Hilyard, A., Gawlak, G., Yan, S. et al. (2006). Atomic structures of peptide self-assembly mimics. Proc. Natl Acad. Sci. USA, 103, 17753–17758. 15. Yan, S., Gawlak, G., Makabe, K., Tereshko, V., Koide, A. & Koide, S. (2007). Hydrophobic surface burial is the major stability determinant of a flat, single-layer beta-sheet. J. Mol. Biol. 368, 230–243. 16. Ohnishi, S., Koide, A. & Koide, S. (2000). Solution conformation and amyloid-like fibril formation of a polar peptide derived from a β-hairpin in the OspA single-layer β-sheet. J. Mol. Biol. 301, 477–489. 17. Koide, S., Huang, X., Link, K., Koide, A., Bu, Z. & Engelman, D. M. (2000). Design of single-layer betasheets without a hydrophobic core. Nature, 403, 456–460.
Structure of a β-Rich Self-Assembly Peptide 18. Richardson, J. S. & Richardson, D. C. (2002). Natural beta-sheet proteins use negative design to avoid edgeto-edge aggregation. Proc. Natl Acad. Sci. USA, 99, 2754–2759. 19. Gordon, D. J., Balbach, J. J., Tycko, R. & Meredith, S. C. (2004). Increasing the amphiphilicity of an amyloidogenic peptide changes the beta-sheet structure in the fibrils from antiparallel to parallel. Biophys. J. 86, 428–434. 20. Fandrich, M. & Dobson, C. M. (2002). The behaviour of polyamino acids reveals an inverse side chain effect in amyloid structure formation. EMBO J. 21, 5682–5690. 21. Pawar, A. P., Dubay, K. F., Zurdo, J., Chiti, F., Vendruscolo, M. & Dobson, C. M. (2005). Prediction of “aggregation-prone” and “aggregation-susceptible” regions in proteins associated with neurodegenerative diseases. J. Mol. Biol. 350, 379–392. 22. Rousseau, F., Schymkowitz, J. & Serrano, L. (2006). Protein aggregation and amyloidosis: confusion of the kinds? Curr. Opin. Struct. Biol. 16, 118–126. 23. Ganesh, S. & Jayakumar, R. (2003). Structural transitions involved in a novel amyloid-like beta-sheet assemblage of tripeptide derivatives. Biopolymers, 70, 336–345. 24. Sunde, M., Serpell, L. C., Bartlam, M., Fraser, P. E., Pepys, M. B. & Blake, C. C. (1997). Common core structure of amyloid fibrils by synchrotron X-ray diffraction. J. Mol. Biol. 273, 729–739. 25. Ivanova, M. I., Thompson, M. J. & Eisenberg, D. (2006). A systematic screen of β2-microglobulin and insulin for amyloid-like segments. Proc. Natl Acad. Sci. USA, 103, 4079–4082. 26. Yan, S., Gawlak, G., Smith, J., Silver, L., Koide, A. & Koide, S. (2004). Conformational heterogeneity of an equilibrium folding intermediate quantified and mapped by scanning mutagenesis. J. Mol. Biol. 338, 811–825. 27. Makabe, K., Tereshko, V., Gawlak, G., Yan, S. & Koide,
467
28.
29. 30.
31.
32. 33. 34.
35.
36.
S. (2006). Atomic-resolution crystal structure of Borrelia burgdorferi outer surface protein A via surface engineering. Protein Sci. 15, 1907–1914. Wouters, M. A. & Curmi, P. M. (1995). An analysis of side chain interactions and pair correlations within antiparallel beta-sheets: the differences between backbone hydrogen-bonded and non-hydrogenbonded residue pairs. Proteins: Struct., Funct., Genet. 22, 119–131. Finkelstein, A. V. & Nakamura, H. (1993). Weak points of antiparallel beta-sheets. How are they filled up in globular proteins? Protein Eng. 6, 367–372. Esteras-Chopo, A., Serrano, L. & Lopez de la Paz, M. (2005). The amyloid stretch hypothesis: recruiting proteins toward the dark side. Proc. Natl Acad. Sci. USA, 102, 16672–16677. Kim, W. & Hecht, M. H. (2006). Generic hydrophobic residues are sufficient to promote aggregation of the Alzheimer's Aβ42 peptide. Proc. Natl Acad. Sci. USA, 103, 15824–15829. Collaborative Computational Project (1994). The CCP4 Suite: programs for protein crystallography. Acta Crystallogr., Sect. D: Biol. Crystallogr. 50, 760–763. Otwinowski, Z. & Minor, W. (1997). Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W. et al. (1998). Crystallography & NMR System: a new software suite for macromolecular structure determination. Acta Crystallogr., Sect. D: Biol. Crystallogr. 54, 905–921. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. (1997). Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr., Sect. D: Biol. Crystallogr. 53, 240–255. Emsley, P. & Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 60, 2126–2132.
J. Mol. Biol. (2008) 378, 459–467
Available online at www.sciencedirect.com
High-Resolution Structure of a Self-Assembly-Competent Form of a Hydrophobic Peptide Captured in a Soluble β-Sheet Scaffold Koki Makabe 1 , Matthew Biancalana 1 , Shude Yan 1 , Valentina Tereshko 1 , Grzegorz Gawlak 1 , Hélène Miller-Auer 1,2 , Stephen C. Meredith 1,2 and Shohei Koide 1 ⁎ 1
Department of Biochemistry and Molecular Biology, The University of Chicago, 929 E. 57th Street, Chicago, IL 60637, USA 2
Department of Pathology, The University of Chicago, Chicago, IL 60637, USA Received 20 November 2007; received in revised form 9 February 2008; accepted 23 February 2008 Available online 4 March 2008
β-Rich self-assembly is a major structural class of polypeptides, but still little is known about its atomic structures and biophysical properties. Major impediments for structural and biophysical studies of peptide selfassemblies include their insolubility and heterogeneous composition. We have developed a model system, termed peptide self-assembly mimic (PSAM), based on the single-layer β-sheet of Borrelia outer surface protein A. PSAM allows for the capture of a defined number of self-assembly-like peptide repeats within a water-soluble protein, making structural and energetic studies possible. In this work, we extend our PSAM approach to a highly hydrophobic peptide sequence. We show that a penta-Ile peptide (Ile5), which is insoluble and forms β-rich self-assemblies in aqueous solution, can be captured within the PSAM scaffold in a form capable of selfassembly. The 1.1-Å crystal structure revealed that the Ile5 stretch forms a highly regular β-strand within this flat β-sheet. Self-assembly models built with multiple copies of the crystal structure of the Ile5 peptide segment showed no steric conflict, indicating that this conformation represents an assembly-competent form. The PSAM retained high conformational stability, suggesting that the flat β-strand of the Ile5 stretch primed for selfassembly is a low-energy conformation of the Ile5 stretch and rationalizing its high propensity for self-assembly. The ability of the PSAM to “solubilize” an otherwise insoluble peptide stretch suggests the potential of the PSAM approach to the characterization of self-assembling peptides. © 2008 Elsevier Ltd. All rights reserved.
Edited by J. Weissman
Keywords: amyloid fibril; single-layer β-sheet; solubilization; protein engineering; x-ray crystallography
Introduction It has become evident that many polypeptides can be transformed into β-rich self-assemblies.1 β-Rich self-assembly is the core structure of the so-called cross-β amyloid fibrils that are associated with *Corresponding author. E-mail address: [email protected]. Abbreviations used: PSAM, peptide self-assembly mimic; SLB, single-layer β-sheet; OspA, outer surface protein A; DMSO, dimethyl sulfoxide; AFM, atomic force microscopy; NHB, non-hydrogen bonded; HB, hydrogen bonded.
devastating human diseases. Discovery of diverse peptide sequences that can form self-assemblies has increased interest in peptide self-assembly as a means for creating nanoscale structures. Self-assembly of molecules is becoming a popular and powerful strategy to create nanomaterials, and because peptide building blocks with desired chemical composition can be readily synthesized, peptide self-assembly offers a technology platform for the production of diverse nanomaterials. Indeed, many types of selfassemblies have been produced using synthetic peptides.2,3 Therefore, increased understanding of the factors governing peptide self-assembly will have broad impacts on material sciences, biology, and medicine.
0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.
Structure of a β-Rich Self-Assembly Peptide
460 Despite their importance, still, little is known about the high-resolution structure and biophysical properties of peptide self-assemblies. Their insoluble nature and inherently heterogeneous stoichiometry make it extremely difficult to apply the standard biophysical techniques for water-soluble proteins. Solid-state NMR studies have elucidated the conformations of peptide units and modes of higher-order assemblies.4–7 Although powerful, it is a laborious method, and it is difficult to obtain high-resolution structural information. Pioneering work by the group of Eisenberg has determined the atomic structures of amyloidogenic peptides assembled into microcrystals, which has established the cross-β spine as a common structure of peptide fibrils.8,9 Although the crystallization approach is also powerful, it is limited to structural determination, and it seems that peptides are crystallized in a high-energy state.10 In addition, it may not be applicable to a broad range of amyloids beyond short peptides. To overcome these fundamental difficulties associated with characterization of self-assembling peptides, several groups have employed transplantation of a peptide segment into a water-soluble globular protein. In this approach, it is hoped that an engineered protein harboring the peptide segment remains soluble and that insights into the structure and thermodynamics of self-assembly can be gained by characterizing the engineered protein with standard biophysical techniques, such as x-ray crystallography. Stott et al. introduced Gln repeats into a flexible loop of chymotrypsin inhibitor 2.11 Although the engineered chymotrypsin inhibitor 2 oligomerized, its crystal structure revealed that the oligomerization was due to domain swapping and the Gln repeats were disordered and not forming βrich self-assembly.12 Similarly, Takano et al. replaced a C-terminal segment of ribonuclease HI with an Aβ fragment, which prevented aggregation of the Aβ fragment.13 The x-ray crystal structure of this fusion protein revealed that, although the Aβ segment had a β-sheet conformation, it did not form β-sheetmediated interactions, suggesting that the observed form lacked the capability of self-assembly. These examples illustrate the difficulty of capturing a peptide in a form primed for assembly into higherorder structures. We have developed a protein engineering strategy to overcome the fundamental challenges in highresolution characterization of peptide self-assemblies. Our approach, termed peptide self-assembly mimic (PSAM), captures a peptide segment of interest within the structural context of a flat β-sheet. A highly regular flat β-sheet is a hallmark of β-rich self-assemblies. Thus, our approach is distinct from other transplantation attempts in that it grafts a peptide of interest in an environment close to that of self-assembly. We used the single-layer β-sheet (SLB) segment of outer surface protein A (OspA) from Borrelia burgdorferi as the host for PSAM. The SLB exists as a segment linking two globular domains, which in
turn serve as “end caps” for the SLB. Although it is exposed to the solvent and thus does not contain a hydrophobic core, it is highly stable. Indeed, the presence of the globular domains would appear to inhibit the lamination of the β-sheet, regardless of its composition.14,15 The single-layer architecture eliminates complications caused by long-range interactions through a hydrophobic core that are commonly present in a water-soluble β-sheet protein. We have demonstrated that a β-hairpin sequence from the SLB forms β-rich fibrils16 and that the self-assembly of this β-hairpin segment can be captured within OspA in the form of extended SLB.17 The x-ray crystal structures of these PSAMs have enabled the establishment of structural linkage between the atomic structures of self-assembling peptides and the macroscopic morphology.14 The two globular domains sequester the “sticky” β-sheet edges, thus enabling the PSAM system to capture β-rich self-assembly within a water-soluble protein. Furthermore, unlike most β-sheets found in globular proteins,18 the SLB has a highly regular geometry and is flat, which may closely mimic the β-sheet conformation within actual peptide assemblies. Recent studies have shown that many peptide sequences self-assemble into an antiparallel β-sheet and that small sequence changes can alter the βsheet topology between parallel and antiparallel.9,19 Therefore, although our PSAM system using OspA can mimic only self-assemblies consisting of antiparallel β-sheets, its detailed structural and energetic studies would provide significant knowledge relevant to the molecular mechanism of peptide self-assembly. In our initial work to establish the PSAM strategy, we used a hydrophilic peptide sequence derived from the SLB itself. In this work, we wished to explore the potential of the PSAM to capture diverse self-assembling peptides. To this end, we introduced a penta-Ile motif in the PSAM. We show that a penta-Ile peptide forms β-rich self-assemblies and that it can be stably captured into the PSAM. The high-resolution x-ray crystal structure of the peptide captured in the PSAM reveals molecular details of βrich peptide self-assembly.
Results An oligo-Ile peptide forms β-rich self-assemblies It has been shown that certain poly-amino acids self-assemble into fibrils.20 Subsequently, amyloid aggregation propensities of amino acids have been derived.21,22 Ile has the highest aggregation propensity among aliphatic amino acids on the scale of Pawar et al.,21 and it is also among the most aggregation-prone amino acids on the scale of Rousseau et al.22 Ganesh and Jayakumar reported that short peptides containing three consecutive Ile peptides form β-rich self-assembly.23 Therefore, we chose an oligo-Ile peptide for this study. An oligo-Ile
Structure of a β-Rich Self-Assembly Peptide
peptide is very hydrophobic and distinct from the highly hydrophilic sequence of the OspA SLB; it thus represents a challenging target for transplantation into the PSAM system. A penta-Ile peptide (Ile5) was synthesized with solid-phase peptide synthesis. The purity and molecular weight of the peptide were confirmed with liquid chromatography–mass spectrometry. Because the lyophilized peptide only sparingly dissolved in an aqueous buffer, we prepared peptide solutions in two methods. First, the peptide was suspended at a 2-mg/ml concentration in an aqueous buffer with vigorous voltexing, and the supernatant of this suspension after centrifugation was used. Second, the peptide was first completely dissolved in 100% dimethyl sulfoxide (DMSO) at 20 mg/ml, and this solution was diluted 10-fold into an aqueous buffer, resulting in a solution containing 10% DMSO. Atomic force microscopy (AFM) characterization revealed needle-like objects in the peptide solution (Fig. 1b and c). Their widths ranged from ∼ 20 to ∼ 100 nm, and their lengths were a few micrometers. The needle-like objects were also found in the
461 sample in 10% DMSO (Fig. 1d), and the shape of the objects in the 10% DMSO sample was similar to that of the objects in the water sample, indicating that these objects can form from monomeric peptides in solution and that they are not an artifact from incomplete solubilization of peptide powder. The suspension supernatant exhibited a far-UV circular dichroism (CD) spectrum characteristic of β-sheet (Fig. 1a), indicating that the peptide self-assembles had high β-sheet content. The AFM objects showed no twist commonly observed for peptide self-assemblies. Both samples show x-ray diffraction maxima corresponding to 4.6- and 10-Å spacings (Fig. 1e and f), a hallmark of β-rich peptide assemblies.24 Commonly, the 4.6-Å spacing is interpreted as the spacing between β-strands within a β-sheet and the 10-Å spacing is interpreted as the spacing between two laminated β-sheets, which are the bases for the cross-β structure.24 Interestingly, the Ile5 self-assembly showed little thioflavin T binding activity. It should be noted that not all peptide fibrils bind thioflavin T.25 Taken together, their morphology, their high β-sheet content, and
Fig. 1. (a) Far-UV CD spectrum of a supernatant of homo-Ile peptide suspension in water. The CD spectrum is plotted with the ellipticity, not with the molar ellipticity, because we could not determine the concentration of the peptide solution. (b–d) AFM scan images. Onemicrometer scale bars are indicated in the images. The brightness of features increases as a function of height. (b and c) Images from the water suspension sample taken from different areas of the same sample surface. (d) Image from the 10% DMSO sample. (e and f) X-ray fiber diffraction patterns of the water (e) and 10% DMSO (f) samples. The diffraction maxima corresponding to the 4.6-and 10-Å spacings are marked with arrows.
462 the x-ray diffraction patterns suggest that the oligoIle peptide forms a cross-β self-assembly, as seen for fibrils of other peptides. Stable grafting of an oligo-Ile stretch into the PSAM Having confirmed the ability of oligo-Ile peptide to self-assemble into a β-rich structure, we proceeded to graft the Ile5 sequence within the PSAM β-sheet. We hypothesized that, if the Ile5 peptide has a high propensity to self-assemble into flat β-sheets, replacing a flat β-strand with an Ile5 stretch should not reduce the stability of the PSAM. Thus, we grafted the Ile5 sequence into the central β-strand (strand 9) of the OspA SLB (Fig. 2a). The SLB consists of three solvent-exposed strands. We chose strand 9 as the host because it is centrally located in the SLB and highly exposed to the solvent. These characteristics minimize interactions of residues in strand 9 with those in the globular domains. The five consecutive Ile residues replaced residues 120–124 (SSTEE), and we term the resulting mutant PSAM-Ile5. In spite of the introduction of a highly hydrophobic segment at highly solvent-exposed positions, PSAM-Ile5 was expressed as a soluble protein in Escherichia coli and was predominantly monomeric in aqueous solution as judged with gel-filtration chromatography (Fig. 2b). These results indicate that the PSAM scaffold effectively “solubilizes” the otherwise insoluble Ile5 peptide by sequestering it within the SLB. PSAM-Ile5 underwent a reversible unfolding reaction induced by urea (Fig. 2c). The unfolding curve was interpreted in terms of a three-state model as for
Structure of a β-Rich Self-Assembly Peptide
the wild-type OspA.15,26 In this three-state model, the first transition corresponds to unfolding of the C-terminal domain and a portion of the SLB; the second, to that of the rest of the SLB and the N-terminal domain. The overall stability of PSAMIle5 was nearly identical with that of the wild type (ΔΔGNU3M = 0.07 kcal/mol), but the first transition was destabilized (ΔΔGNI3M = − 0.75 kcal/mol) and the second transition was stabilized (ΔΔGIU3M = 0.82 kcal/mol) (ΔGNU3M, ΔGNI3M, and ΔGIU3M are the free energy difference at 3M urea between the native and unfolded states, that between the native and intermediate states, and that between the intermediate and unfolded states, respectively; ΔΔGNU3M, ΔΔGNI3M, and ΔΔGIU3M are differences in ΔGNU3M, ΔGNI3M, and ΔGIU3M between the wild type and the mutant, respectively). These compensatory changes suggest that the Ile5 replacement has altered the nature of the intermediate state. Although the detailed effects of the strand replacement on the unfolding mechanism are not clear, these results demonstrate that PSAM accommodates the strand replacement without significant stability loss. High-resolution x-ray crystal structure of PSAM-Ile5 Our crystallization efforts of PSAM-Ile5 did not yield any single crystals. To facilitate crystallization, we introduced a set of surface mutations in the globular domains that have been highly effective in crystallizing OspA and its variants.14,27 These mutations are distant from the grafted Ile5 segment and have previously been shown to cause little structural change.27 The resulting surface-engineered protein,
Fig. 2. (a) Amino acid sequence of the OspA SLB region. The line rectangles indicate residues in a β-strand whose side chains face toward the reader, and the shaded rectangles indicate those facing backward. The mutational residues are shown in rectangles. The backbone hydrogen bonds are shown as dashed lines. (b) Elution profiles of the wild type (dashed line) and PSAM-Ile5 (solid line) from a sizeexclusion column detected using absorbance at 280 nm. (c) Ureainduced unfolding of PSAM-Ile5. Normalized CD (circles) and fluorescence (cross) intensities for PSAM-Ile5 are plotted as a function of urea concentration. The profiles for the wild-type protein are also shown (CD, solid line; fluorescence, dashed line).
Structure of a β-Rich Self-Assembly Peptide
termed PSAM-Ile5-sm1, readily crystallized, and its crystal structure was determined at a resolution of 1.10 Å (Fig. 3a). The data collection and refinement statistics are summarized in Table 1. Despite the fact that Ile5 replacement in strand 9 completely disrupts the native side-chain pairings (Fig. 2a), the global fold of PSAM-Ile5-sm1 was nearly identical with the wild-type OspA containing the same set of surface mutations (Fig. 3b), with a root-mean-square deviation (RMSD) of 0.3 Å for the Cα atoms common between the wild-type and mutant structures. This structural resemblance is remarkable, because the SLB does have a level of structural plasticity. For example, a series of PSAMs with different numbers of peptide inserts exhibited a range of backbone conformations,14 and a single mutation at Phe126 located at the C-terminus of strand 9 (Fig. 2a) significantly perturbs the backbone structure, increasing the Cα RMSD to 1.15 Å.15 Thus, the small structural deviation due to the Ile5 replacement suggests that the PSAM SLB provides a structural environment that is compatible with a stable conformation of the Ile5 segment. In the crystal structure, the Ile5 segment takes on a regular β-sheet conformation with right-hand twist along the strand (Fig. 4a). In addition to the Ile5 stretch, PSAM-Ile5 contains two cross-strand Ile–Ile pairs involving the Ile5 stretch and residues in strand
463 10 (Figs. 3a and 4b). These are a non-hydrogenbonded (NHB) pair (Ile122–Ile137) and a hydrogenbonded (HB) pair (Ile123–Ile136). These cross-strand pairs provide an opportunity to see the side-chain conformations and interactions of Ile residues in the context of self-assembly, since these combinations are present in an antiparallel β-sheet structure made of homo-Ile peptides. We found distinct modes of interactions in the HB and NHB pairs (Fig. 4b). As pointed out previously,28 the Cγ atoms of the NHB Ile pair face each other with slight offset along with the strand direction. They are tightly packed and exclude solvent molecules between the side chains. In contrast, the Cγ atoms of the HB pair point to mutually opposite directions and create voids between the Cβ atoms (Fig. 4c). These voids are filled with well-defined water molecules, thus rescuing the “weak points.”27,29 This hydration pattern is conserved in the wild-type OspA-sm1 and PSAMIle5-sm1, suggesting that this is a general feature found on the surface of a flat β-sheet (Fig. 3c). Together, these results suggest that in an Ile peptide self-assembly, adjacent Ile peptides are held through the backbone hydrogen bonds, the side chains of the NHB pairs form tight interactions, and the side chains of the HB pair form suboptimal interactions that may be compensated by the main-chain hydration at the weak points.
Fig. 3. Crystal structure of PSAM-Ile5-sm1. (a) Superposition of PSAM-Ile 5 -sm1 (green) onto OspA-sm1 (gray). The mutated residues are shown in red sticks, and Ile residues in strand 10 are shown in green sticks. N- and Cterminal positions are indicated. (b) The RMSDs for the Cα atoms are plotted versus residue number. The positions of β-strands and the Cterminal helix are shown as bars. The mutation residues are indicated as a red circle. (c) Backbone hydration of the SLB. The water molecules are shown as red spheres. The mutation residues are shown as green sticks.
Structure of a β-Rich Self-Assembly Peptide
464 Table 1. Data collection and refinement statistics for PSAM-Ile5-sm1 P21
Space group Data collection statistics Cell parameters a b c β Beamline Wavelength (Å) Resolution (Å) Completeness (%) I/σ(I) Rmergea Average redundancy Refinement statistics Resolution range (Å) Reflections used (free) R-factorb Rfreec RMSDs Bonds (Å) Angles (°) No. of protein residues No. of water molecules Average B-factor (Å2) Ramachandran plot statistics (%) Most favored regions Additionally allowed regions Generally allowed regions
33.26 54.71 66.50 99.91 APS 23ID 0.9795 50–1.1 (1.14–1.10) 97.8 (86.2) 19.25 (2.88) 0.053 (0.318) 3.3 (2.3) 20.0–1.1 88,420 (4669) 0.155 0.171 0.008 1.281 246 378 9.82 91.9 8.1 0.0
Data in parentheses are for the highest-resolution shell. a Rmerge = ∑hkl∑i|I(hkl)i − 〈I(hkl)〉|/∑hkl∑i〈I(hkl)i〉 over i observations of a reflection hkl. b R-factor = ∑||Fobs|−|Fcalc||/∑|Fobs|. c Rfree is R with 5% of reflections sequestered before refinement.
Atomic models of the Ile5 self-assembly To examine if the Ile5 structure in PSAM-Ile5-sm1 reflects the structure of an Ile5 peptide within the βrich self-assemblies, we constructed an atomic model of a self-assembly from the x-ray crystal structure of the Ile5 segment. In this model, multiple copies of the peptide structure were placed side by side such that a continuous β-sheet is formed (Fig. 4d). We used the backbone structures of an insulin peptide that forms antiparallel β-sheet assembly [Protein Data Bank (PDB) ID 2OMQ]9 as the template for placing the Ile5 structures. It is important to emphasize that the template was used only to define the spacing and that we did not alter the conformation of the Ile5 segment in this modeling. The resulting β-sheet structure of the model has no steric clash, indicating that the Ile5 conformation in PSAM-Ile5-sm1 can self-assemble without conformational adjustment. Thus, this model may represent the basic unit for the selfassembly of the Ile5 peptide. Although further lamination of the β-sheet units is required to account for the dimensions of the self-assembly seen in the AFM images, this model helps connect the atomic structure and the macroscopic morphology of peptide self-assembly.
Discussion We have shown that an Ile5 peptide forms a β-rich self-assembly and that it can be captured within a soluble PSAM in a form that is clearly compatible with self-assembly. To our knowledge, this is the first example of the high-resolution structure of a homo-oligomer of an amino acid in an assemblycompetent form. PSAM-Ile5 was highly stable, and it retained the flat β-sheet seen in other PSAMs, strongly suggesting that the highly regular flat βstrand is an energetically favorable conformation of the Ile5 peptide. Our results with PSAM-Ile5 are in stark contrast to previous results of grafting studies of self-assembling peptides. Often, soluble host proteins are recruited into amyloids and/or aggregates.30,31 In other cases where the grafting location was carefully chosen so that the protein solubility was maintained, the grafted peptide was found to be in a conformation incapable of self-assembly. 12,13 The main difference between our host and the others is that ours captures a peptide of interest within a welldefined flat β-sheet, while others capture a peptide within a flexible segment. Lamination of β-sheet does not occur, presumably because this is prevented by the globular domains. The flat β-sheet is the native conformation of the host SLB. Thus, the formation of a flat β-strand conformation of the grafted peptide does not promote a nonnative conformation of the PSAM host that might cause denaturation and/or misfolding. Because the flat βsheet is a favorable conformation of self-assembling peptides, grafting of a peptide segment with a high self-assembly propensity stabilizes the native, flat structure of the PSAM scaffold. β-Sheet assembly requires the formation of a backbone hydrogen-bond network across β-strands. In general, an exposed β-sheet edge is poised to interact with another β-sheet edge, and in natural proteins, multitudes of negative elements are installed at an exposed β-sheet edge to prevent aberrant intermolecular interactions. 18 Thus, it is unlikely that a grafted peptide segment finds a highly regular β-sheet edge in the host protein to which it can dock. Accordingly, it is unlikely that simple grafting of a self-assembling peptide unit in a natural host protein would promote the formation of a self-assembly-mimicking conformation (e.g., β-sheet) of the peptide. Furthermore, simple grafting provides no clear mechanism for preventing uncontrolled self-assembly of a grafted peptide. Our PSAM approach converts peptide self-assembly into protein folding, allowing for high-resolution characterization of structural features in peptide self-assemblies using well-established tools. While the PSAM scaffold promotes the β-sheet formation of the grafted sequence, the scaffold has enough structural plasticity such that the grafted peptide can adapt its own favorable conformation. The work presented here demonstrates that the PSAM is applicable to diverse peptide sequences, including extremely hydrophobic ones such as Ile5, suggesting
Structure of a β-Rich Self-Assembly Peptide
465
Fig. 4. Construction of the βassembly models. (a) Ile5 structure in PSAM-Ile5-sm1. (b) HB pair and NHB pair of Ile residues in the crystal structure. The mutated residues are shown as red sticks, and Ile residues in strand 10 are shown as green sticks. Backbone hydrogen bonds are shown as a blue dashed line. (c) Interstrand Ile pairing in the crystal structure. Ile residues are shown in Corey–Pauling–Koltun representation. The left panel shows the NHB pair (shown in red), whereas the right panel shows the HB pair (shown in red). Water moleculepositioned “weak points” are shown in cyan. (d) Models of SLB assembly. Antiparallel β-sheet model is created based on the amyloidforming peptide crystal structure.
that sequences from natural fibril-forming proteins can also be captured in the PSAM. We speculate that, with the ability of the PSAM to accommodate larger segments of self-assembly mimics,14 this strategy will complement existing ones, such as solidstate NMR spectroscopy and microcrystallography, to shed new light on the molecular structure and biophysical properties of peptide self-assemblies.
been described previously.26 The gene for the PSAM-Ile5 was subcloned in the equivalent region of the OspA surface mutant for crystallization (“OspAsm1”)27 using SpeI and PstI restriction enzyme sites, resulting in 12 surface mutations in total. The mutant was expressed and purified as described before.27 Analytical size-exclusion chromatography was performed using a Superdex 75 HR 10/30 column (GE Healthcare) in 50 mM Tris–HCl, pH 8.0, containing 200 mM NaCl at room temperature.
Materials and Methods
CD spectroscopy
Peptide preparation The Ile5 peptide was synthesized by solid-phase peptide synthesis using standard Fmoc (9-fluoroenylmethyloxycarbonyl) chemistry on a Rink MBHA resin. Its N-terminus was unblocked, and its C-terminus was amidated. The product was cleaved from the resin using trifluoroacetic acid/triisopropylsilane/H2O = 95:2.5:2.5 (v:v:v) and dissolved in 50% acetic acid and purified using reversedphase HPLC. Protein preparation Methods for site-directed mutagenesis, urea denaturation experiments, and analysis of denaturation data have
CD measurements were carried out using an Aviv 202 spectropolarimeter (Aviv Associates, Lakewood, NJ). The spectrum was taken at 25 °C in a 1-mm pathlength cuvette with a 1-nm step size, a 3-nm bandwidth, and a 1-s averaging time. AFM Samples were imaged after incubation for ∼1 month at room temperature. Samples were deposited onto a freshly cleaved mica surface, left on mica for 30 s, and then rinsed with deionized water. The mica surface with the adsorbed peptide was then dried with nitrogen gas. The images shown were obtained by scanning the mica surface in air by AFM (Multimode, Digital Instruments, Santa Barbara, CA)
466 with a spring constant of 2 N/m and a tip radius of curvature of b 10 nm (AC240TS-C2, Olympus). AFM scans were taken at 512 × 512 pixels of resolution and produced topographic images of the samples, in which the parameters of the brightness of features were as follows: tapping frequency, ∼70 kHz; RMS amplitude before engagement, 1 V; integral and proportional gains, 0.2–0.6 and 0.3–1.0, respectively; set point, 0.8 V; and scanning speed, 1 Hz. X-ray fiber diffraction The same batch of Ile5 samples that were used in the AFM experiments was placed in 1-mm diameter quartz capillary and vacuum dried. Diffraction data were collected at the APS (Advanced Photon Source) synchrotron radiation 23ID beamline facility of the Argonne National Laboratory and examined using Mosflm software.32 Crystallization and structure determination The crystals were grown in 30% PEG (polyethylene glycol) 400, 0.1 M Tris–HCl, pH 8.0, using the hangingdrop vapor-diffusion method. The x-ray diffraction data were collected at the APS 23ID beamline. Crystal data and data collection statistics are summarized in Table 1. The x-ray diffraction data were processed with HKL2000.33 The structure was determined by molecular replacement with the program MOLREP in CCP4.32 The OspA-sm1 structure (PDB ID 2G8C) was used as the search model. CNS1.134 and Refmac535 were used for the structural refinement, and final positional and anisotropic temperature factor refinements were performed using Refmac5. Model building was carried out using the Coot program.36 The self-assembly model was built by superposing the five Cα positions of the Ile5 structure of PSAM-Ile5 onto the Cα positions of the crystal structure of the insulin peptide. Molecular graphics were generated using PyMOL†. PDB accession code The coordinates have been deposited in the PDB with entry code 2I5V.
Acknowledgements This work was supported in part by the National Institutes of Health through grant R01-GM72688 and the National Science Foundation through grant CMMI-0709079. M. B. was supported by the National Institutes of Health (grant T90-DK070076) and the Paul K. Richter and Evalyn E. Cobb Richter Memorial Fund. Use of the APS was supported by the Office of Science Basic Energy Sciences Program of the U.S. Department of Energy under contract no. W-31-109ENG-38. GM/CA CAT (APS 23ID) has been supported in whole or in part with federal funds from the National Cancer Institute (Y1-CO-1020) and the National Institute of General Medical Science (Y1† http://www.pymol.org
Structure of a β-Rich Self-Assembly Peptide
GM-1104). We thank Dr. Justin Jureller of the University of Chicago NanoBio Facility for his assistance with AFM.
References 1. Dobson, C. M. (2003). Protein folding and disease: a view from the first Horizon Symposium. Nat. Rev. Drug Discov. 2, 154–160. 2. Rajagopal, K. & Schneider, J. P. (2004). Self-assembling peptides and proteins for nanotechnological applications. Curr. Opin. Struct. Biol. 14, 480–486. 3. Fairman, R. & Akerfeldt, K. S. (2005). Peptides as novel smart materials. Curr. Opin. Struct. Biol. 15, 453–463. 4. Jaroniec, C. P., MacPhee, C. E., Bajaj, V. S., McMahon, M. T., Dobson, C. M. & Griffin, R. G. (2004). Highresolution molecular structure of a peptide in an amyloid fibril determined by magic angle spinning NMR spectroscopy. Proc. Natl Acad. Sci. USA, 101, 711–716. 5. Luhrs, T., Ritter, C., Adrian, M., Riek-Loher, D., Bohrmann, B., Dobeli, H. et al. (2005). 3D structure of Alzheimer's amyloid-β(1–42) fibrils. Proc. Natl Acad. Sci. USA, 102, 17342–17347. 6. Petkova, A. T., Yau, W. M. & Tycko, R. (2006). Experimental constraints on quaternary structure in Alzheimer's beta-amyloid fibrils. Biochemistry, 45, 498–512. 7. Tycko, R. (2004). Progress towards a molecular-level structural understanding of amyloid fibrils. Curr. Opin. Struct. Biol. 14, 96–103. 8. Nelson, R., Sawaya, M. R., Balbirnie, M., Madsen, A. O., Riekel, C., Grothe, R. & Eisenberg, D. (2005). Structure of the cross-beta spine of amyloid-like fibrils. Nature, 435, 773–778. 9. Sawaya, M. R., Sambashivan, S., Nelson, R., Ivanova, M. I., Sievers, S. A., Apostol, M. I. et al. (2007). Atomic structures of amyloid cross-beta spines reveal varied steric zippers. Nature, 447, 453–457. 10. Esposito, L., Pedone, C. & Vitagliano, L. (2006). Molecular dynamics analyses of cross-β-spine steric zipper models: β-sheet twisting and aggregation. Proc. Natl Acad. Sci. USA, 103, 11533–11538. 11. Stott, K., Blackburn, J. M., Butler, P. J. & Perutz, M. (1995). Incorporation of glutamine repeats makes protein oligomerize: implications for neurodegenerative diseases. Proc. Natl Acad. Sci. USA, 92, 6509–6513. 12. Chen, Y. W., Stott, K. & Perutz, M. F. (1999). Crystal structure of a dimeric chymotrypsin inhibitor 2 mutant containing an inserted glutamine repeat. Proc. Natl Acad. Sci. USA, 96, 1257–1261. 13. Takano, K., Endo, S., Mukaiyama, A., Chon, H., Matsumura, H., Koga, Y. & Kanaya, S. (2006). Structure of amyloid beta fragments in aqueous environments. FEBS J. 273, 150–158. 14. Makabe, K., McElheny, D., Tereshko, V., Hilyard, A., Gawlak, G., Yan, S. et al. (2006). Atomic structures of peptide self-assembly mimics. Proc. Natl Acad. Sci. USA, 103, 17753–17758. 15. Yan, S., Gawlak, G., Makabe, K., Tereshko, V., Koide, A. & Koide, S. (2007). Hydrophobic surface burial is the major stability determinant of a flat, single-layer beta-sheet. J. Mol. Biol. 368, 230–243. 16. Ohnishi, S., Koide, A. & Koide, S. (2000). Solution conformation and amyloid-like fibril formation of a polar peptide derived from a β-hairpin in the OspA single-layer β-sheet. J. Mol. Biol. 301, 477–489. 17. Koide, S., Huang, X., Link, K., Koide, A., Bu, Z. & Engelman, D. M. (2000). Design of single-layer betasheets without a hydrophobic core. Nature, 403, 456–460.
Structure of a β-Rich Self-Assembly Peptide 18. Richardson, J. S. & Richardson, D. C. (2002). Natural beta-sheet proteins use negative design to avoid edgeto-edge aggregation. Proc. Natl Acad. Sci. USA, 99, 2754–2759. 19. Gordon, D. J., Balbach, J. J., Tycko, R. & Meredith, S. C. (2004). Increasing the amphiphilicity of an amyloidogenic peptide changes the beta-sheet structure in the fibrils from antiparallel to parallel. Biophys. J. 86, 428–434. 20. Fandrich, M. & Dobson, C. M. (2002). The behaviour of polyamino acids reveals an inverse side chain effect in amyloid structure formation. EMBO J. 21, 5682–5690. 21. Pawar, A. P., Dubay, K. F., Zurdo, J., Chiti, F., Vendruscolo, M. & Dobson, C. M. (2005). Prediction of “aggregation-prone” and “aggregation-susceptible” regions in proteins associated with neurodegenerative diseases. J. Mol. Biol. 350, 379–392. 22. Rousseau, F., Schymkowitz, J. & Serrano, L. (2006). Protein aggregation and amyloidosis: confusion of the kinds? Curr. Opin. Struct. Biol. 16, 118–126. 23. Ganesh, S. & Jayakumar, R. (2003). Structural transitions involved in a novel amyloid-like beta-sheet assemblage of tripeptide derivatives. Biopolymers, 70, 336–345. 24. Sunde, M., Serpell, L. C., Bartlam, M., Fraser, P. E., Pepys, M. B. & Blake, C. C. (1997). Common core structure of amyloid fibrils by synchrotron X-ray diffraction. J. Mol. Biol. 273, 729–739. 25. Ivanova, M. I., Thompson, M. J. & Eisenberg, D. (2006). A systematic screen of β2-microglobulin and insulin for amyloid-like segments. Proc. Natl Acad. Sci. USA, 103, 4079–4082. 26. Yan, S., Gawlak, G., Smith, J., Silver, L., Koide, A. & Koide, S. (2004). Conformational heterogeneity of an equilibrium folding intermediate quantified and mapped by scanning mutagenesis. J. Mol. Biol. 338, 811–825. 27. Makabe, K., Tereshko, V., Gawlak, G., Yan, S. & Koide,
467
28.
29. 30.
31.
32. 33. 34.
35.
36.
S. (2006). Atomic-resolution crystal structure of Borrelia burgdorferi outer surface protein A via surface engineering. Protein Sci. 15, 1907–1914. Wouters, M. A. & Curmi, P. M. (1995). An analysis of side chain interactions and pair correlations within antiparallel beta-sheets: the differences between backbone hydrogen-bonded and non-hydrogenbonded residue pairs. Proteins: Struct., Funct., Genet. 22, 119–131. Finkelstein, A. V. & Nakamura, H. (1993). Weak points of antiparallel beta-sheets. How are they filled up in globular proteins? Protein Eng. 6, 367–372. Esteras-Chopo, A., Serrano, L. & Lopez de la Paz, M. (2005). The amyloid stretch hypothesis: recruiting proteins toward the dark side. Proc. Natl Acad. Sci. USA, 102, 16672–16677. Kim, W. & Hecht, M. H. (2006). Generic hydrophobic residues are sufficient to promote aggregation of the Alzheimer's Aβ42 peptide. Proc. Natl Acad. Sci. USA, 103, 15824–15829. Collaborative Computational Project (1994). The CCP4 Suite: programs for protein crystallography. Acta Crystallogr., Sect. D: Biol. Crystallogr. 50, 760–763. Otwinowski, Z. & Minor, W. (1997). Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W. et al. (1998). Crystallography & NMR System: a new software suite for macromolecular structure determination. Acta Crystallogr., Sect. D: Biol. Crystallogr. 54, 905–921. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. (1997). Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr., Sect. D: Biol. Crystallogr. 53, 240–255. Emsley, P. & Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 60, 2126–2132.