The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: ML DE. Performed the experiments: ML KFF. Analyzed the data: ML MRS. Contributed reagents/materials/analysis tools: LJ AL SAS JL JRB. Wrote the paper: ML DE. Contributed helpful discussions: AL SAS JRB.
The authors have declared that no competing interests exist.
Diagnosing and treating Alzheimer's and other diseases associated with amyloid fibers remains a great challenge despite intensive research. To aid in this effort, we present atomic structures of fiber-forming segments of proteins involved in Alzheimer's disease in complex with small molecule binders, determined by X-ray microcrystallography. The fiber-like complexes consist of pairs of β-sheets, with small molecules binding between the sheets, roughly parallel to the fiber axis. The structures suggest that apolar molecules drift along the fiber, consistent with the observation of nonspecific binding to a variety of amyloid proteins. In contrast, negatively charged orange-G binds specifically to lysine side chains of adjacent sheets. These structures provide molecular frameworks for the design of diagnostics and drugs for protein aggregation diseases.
The devastating and incurable dementia known as Alzheimer's disease affects the thinking, memory, and behavior of dozens of millions of people worldwide. Although amyloid fibers and oligomers of two proteins, tau and amyloid-β, have been identified in association with this disease, the development of diagnostics and therapeutics has proceeded to date in a near vacuum of information about their structures. Here we report the first atomic structures of small molecules bound to amyloid. These are of the dye orange-G, the natural compound curcumin, and the Alzheimer's diagnostic compound DDNP bound to amyloid-like segments of tau and amyloid-β. The structures reveal the molecular framework of small-molecule binding, within cylindrical cavities running along the β-spines of the fibers. Negatively charged orange-G wedges into a specific binding site between two sheets of the fiber, combining apolar binding with electrostatic interactions, whereas uncharged compounds slide along the cavity. We observed that different amyloid polymorphs bind different small molecules, revealing that a cocktail of compounds may be required for future amyloid therapies. The structures described here start to define the amyloid pharmacophore, opening the way to structure-based design of improved diagnostics and therapeutics.
The challenge of developing chemical interventions for Alzheimer's disease has proceeded in a virtual vacuum of information about the three-dimensional structures of the two proteins most widely accepted as being involved in the etiology. These are amyloid-beta (Aβ) and tau
More is known about the molecular structure of amyloid fibers, both those associated with Alzheimer's disease and with the numerous other amyloid conditions
The short segments forming steric zippers, when isolated from the rest of the protein, form well-ordered fibers on their own, with essentially all properties of the fibers of their full-length parent proteins
Here we use one such amyloid-forming segment from Aβ and one from tau to form co-crystals with low molecular weight compounds, with the aim of illuminating the nature of interactions of small molecules with amyloid. These complexes reveal a molecular framework which partially defines the amyloid pharmacophore, the structural features responsible for the binding of small molecules to amyloid aggregates.
In our attempts to obtain complexes of small molecules with amyloid-like segments from disease-related proteins, we screened for co-crystals grown from dozens of mixtures (
The KLVFFA segment (residues 16–21) from Aβ contains apolar residues that participate in a hydrophobic spine in Aβ fibers and itself acts as an inhibitor of Aβ fibrillation
(A–B) The KLVFFA segments are packed as pairs of β-sheets forming the basic unit of the fiber, namely the steric zipper
Orange-G is bound internally to the steric zipper of KLVFFA (residues 16–21 of Aβ). It also contacts the lysine residues in the adjacent zipper. Peptide segments, forming β-sheet structures, are shown as arrows and sticks, colored by atom type with carbons in white. Orange-G carbons are in orange for one molecule and brown for the other molecule. Surface is shown for peptide atoms contacting the orange-G molecule with the orange carbons, shown as spheres. The view in (A) looks down the fiber axis. The view in (B) is perpendicular to the fiber axis. Only side chains of interacting residues are shown. The area of the fiber buried by orange-G (
Three forms of the KLVFFA segment from Aβ (A–C) and the VQIVYK segment from the tau protein (D–F) are presented. These forms serve as examples of packing polymorphism observed for amyloid fibrils
All four crystal forms of KLVFFA, including the complex with orange G, show an anti-parallel β-strand stacking in the steric zipper (Colletier et al. unpublished results and
The VQIVYK segment of tau was suggested as the minimal interaction motif for fiber formation
(A–C) The VQIVYK segments pack in parallel, in-register β-sheets (cartoon arrows) that form steric zippers (two zippers are shown in panel A). Nine layers of the fiber are depicted. VQIVYK and orange-G are shown as sticks with non-carbon atoms colored by atom type. The carbons of VQIVYK are colored white for one steric zipper and blue for the other. Two orange-G molecules (orange carbons) mediate contacts between two pairs of steric zippers; that is, orange-G is located between the protofilaments composing the fiber. In panel A, the view looks down the fiber axis. In panel B, the view is perpendicular to the fiber axis. Only the two sheets that are in contact with orange-G are shown. Backbone atoms are not shown. The unit cell dimension of the crystal along the fiber axis (4.83 Å) is indicated. The length of orange-G spans multiple unit cells of the fibril; that is, the dimensions of the small molecule and the fibril unit cell are incommensurate (see
Orange-G is bound between steric zippers of VQIVYK, i.e., internally to a bundle of protofilaments. The VQIVYK segment is located at the third repeat of the tau protein. Since there are many isoforms of tau, we will number the VQIVYK residues 1–6 for simplicity. Peptide segments, forming β-sheet structures, are shown as arrows and sticks, colored by atom type with carbons in white. Orange-G carbons are in orange. Surface is shown for peptide atoms contacting the orange-G molecule (shown as spheres). The view in (A) looks down the fiber axis. The view in (B) is perpendicular to the fiber axis. Only side chains of interacting residues are shown. The area of the fiber buried by orange-G (
Crystallization of VQIVYK alone, under identical conditions to the co-crystallization of the VQIVYK-orange-G mixture, resulted in the formation of colorless fibrous crystals (
Curcumin (
Panels A and D are micro-crystals of VQIVYK co-crystallized with DDNP and curcumin, respectively. In the structure of the complexes with DDNP (B–C) and curcumin (E–F), VQIVYK is packed in a form having a steric zipper with one β-sheet shifted in relation to the other β-sheet (cartoon arrows). The carbons of VQIVYK are colored white for one steric zipper and blue for the other. VQIVYK, DDNP, and curcumin are shown as sticks with non-carbon atoms colored by atom type. Six layers of the fiber are depicted. In panels B and E, the view looks down the fiber axis. In panels C and F, the view is perpendicular to the fiber axis. In both complexes, only the VQIVYK segment is modeled into the electron density, and in both, there is an apparent difference electron density Fo-Fc map (shown as mesh, +3σ in green and −3σ in red) located in the void formed by the shift of the steric zipper. The positive density (part of the structure that has not been modeled, green mesh) displays a continuous tube-like shape, running along the fiber axis. We attribute this density to the presence of the small molecules, yet it is too undifferentiated to fit atoms in detail. DDNP (B–C, two molecules are shown) and curcumin (E–F) (both in magenta carbons) have been computationally docked (
VQIVYK segments, forming parallel β-sheet structures, are shows as arrows and sticks, colored by atom type with carbons in white. Docked DDNP (A and B) and curcumin (C and D) are shown as spheres with carbons colored magenta. Both small molecules are bound in the void formed within two shifted steric zippers. Surface is shown for peptide atoms contacting the small molecules. The area of the fiber buried by DDNP or curcumin is 242 Å2 or 351 Å2, respectively, and is about 50% hydrophobic (contributed by the side chain of Val1 and Ile3) and 50% polar (contributed by the hydroxyl of Tyr5 and the N-termini). The view in panels A and C looks down the β-sheets (fiber axis). The view in panels B and D is perpendicular to the fiber axis, with β-strands running horizontally.
Despite the lack of differentiated electron density for curcumin and DDNP in VQIVYK, there is strong evidence for the presence of the small molecules in the crystals. The crystals show a distinctive color, whereas the control crystals (grown under identical condition without the small molecule) are colorless (
The common feature of the structures of four amyloid/small-molecule complexes is that the small molecules bind to fibers in a similar orientation, along the β-sheets, with their long axes parallel to the fiber axis. This orientation was previously proposed for the binding of thioflavin T to bovine insulin and bovine β-lactoglobulin amyloid fibrils using polarized laser confocal microscopy
Our crystal structures of small molecules bound within amyloid-like steric zippers begin to define the molecular frameworks, or pharmacophores, for the design of diagnostics and drugs for Alzheimer's and other aggregation diseases. The amyloid components in our structures are steric zippers formed by stacks of six-residue segments from Alzheimer-related proteins. Although these steric zippers cannot represent all aspects of the full-length amyloid parent proteins, they share many properties and are commonly used as models of the amyloid β-spine and of aggregation
Overall, the complexes presented here suggest the nature of two molecular frameworks for the binding of small molecules to amyloid fibers. The first molecular framework pertains to site-specific binders, such as charged compounds that form networks of interactions with sequence motifs, and is relatively well defined. The second molecular framework, far less well defined at this point, pertains to broad-spectrum binders, such as uncharged aromatic compounds that bind to tube-like cavities between β-sheets. For binding amyloid deposits in the brain, uncharged molecules could be more effective because of superior blood-brain-barrier penetrability. The same frameworks, offering cavities along β-sheets, might also exist in amyloid oligomers known to be rich in β-sheets and possibly fiber-like
The specific binding of orange-G allows definition of the chemical properties of a specific molecular framework. The prominent feature of amyloid structures is the separation of β-strands (forming a β-sheet) by ∼4.8 Å. In structures with strands packed in an antiparallel orientation, as observed for the KLVFFA fibers and for a rare mutation in Aβ that is associated with massive depositions of the mutant protein and early onset of the disease
Within our framework, an apolar aromatic spine is another essential moiety
Despite the lack of atomized electron density for the binding of curcumin and DDNP in VQIVYK fibers, the location of the binding cavity is clear. It is narrow, restricting rotation of the small molecule (
Our structures show that different small molecules bind along the β-spine of amyloid-like fibers. In case fibers contain more than a single spine, the molecules might bind to multiple sites. This is more likely for the broad-spectrum hydrophobic compounds but can also apply for charged compounds. For example, we observed orange-G to bind to two different steric zippers, of KLVFFA and VQIVYK, with the commonality of binding to lysine side chains protruding from the β-sheets.
Congo-red, a known amyloid marker, contains two sulfonic acid groups, similar to orange-G, but they are spaced ∼19 Å apart, which might account for its lack of specificity
Defining these two molecular frameworks illuminates functional attributes of specific and broad-spectrum amyloid binders. This distinction is consistent with competitive kinetic experiments demonstrating that the binding of FDDNP (the fluoridated analog of DDNP) to Aβ fibrils is displaceable by the uncharged non-steroidal anti-inflammatory naproxen, but not by the common charged dyes congo-red and thioflavin T
In the case of the complexed curcumin and DDNP structures, we hypothesize that the tube-like cavity along the β-sheets provides an adequate site for the binding of many compounds of similar properties. However, the lack of specific interactions allows the small molecule to drift along the fiber axis, leading to lower occupancy and a degree of fluidity in the structure. Extrapolating from our structures, we expect that various aromatic compounds, such as polyphenols
A subtle implication of our structures for the design of effective therapeutic treatments is the specificity they reveal of ligand binding to particular fiber polymorphs (
Four crystal structures of small molecules bound to fiber-forming segments of the two main Alzheimer's disease proteins show common features. The small molecules bind with their long axes parallel to the fiber axis. The structures reveal a sequence-specific binder which forms salt links with side-chains of the steric zipper spines of the fibers and non-specific binders which lie in cylindrical cavities formed at the edges of several steric zippers. Small-molecule binding is specific to particular steric-zipper polymorphs, suggesting that effective Alzheimer's diagnostics and therapeutics may have to be mixtures of various compounds to bind to all polymorphs present. The complexes presented here suggest routes for structure-based design of combinations of compounds that can bind to a spectrum of polymorphic aggregates, to be used as markers of fibers and as inhibitors of aggregation.
Peptide segments (custom synthesis) were purchased from CS Bio. Orange-G and curcumin were purchased from Sigma-Aldrich. DDNP was synthesized as described in
All crystals were grown at 18°C via hanging-drop vapor diffusion. All crystals appeared within 1 wk, except the negative control crystals of VQIVYK+DDNP that took 8 mo to grow.
The drop was a mixture of 10 mM VQIVYK and 1 mM orange-G in water, and reservoir solution (0.1 M zinc acetate dehydrate, 18% polyethylene glycol 3350). The structure was solved to 1.8 Å resolution and contained one segment, one orange-G, two water molecules, two zinc atoms, and one acetate molecule in the asymmetric unit.
The drop was a mixture of 6 mM VQIVYK and 1 mM DDNP in 60% ethanol, and reservoir solution (0.52 M potassium sodium tartrate, 0.065 M HEPES-Na pH 7.5, 35% glycerol). The structure was solved to 1.2 Å resolution and contained one segment and three water molecules in the asymmetric unit.
The drop was a mixture of 6 mM VQIVYK and 1 mM DDNP in 60% ethanol, and reservoir solution (1.2 M DL-malic acid pH 7.0, 0.1 M BIS-TRIS propane pH 7.0). The structure was solved to 1.65 Å resolution and contained one segment, and three water molecules in the asymmetric unit.
The drop was a mixture of 6 mM VQIVYK in 60% ethanol and reservoir solution (0.52 M potassium sodium tartrate, 0.065 M HEPES-Na pH 7.5, 35% glycerol). The structure was solved to 1.2 Å resolution and contained one segment, and one water molecule in the asymmetric unit.
The drop was a mixture of 10 mM VQIVYK and 1 mM curcumin in 80% dimethyl sulfoxide (DMSO), and reservoir solution (0.1 M Tris.HCl pH 8.5, 70% (v/v) MPD (2-methyl-2,4-pentanediol)). The structure was solved to 1.3 Å resolution and contained one segment, and two water molecules in the asymmetric unit.
The drop was a mixture of 10 mM VQIVYK in 80% DMSO and reservoir solution (0.1 M Tris.HCl pH 8.5, 70% (v/v) MPD (2-methyl-2,4-pentanediol)). The structure was solved to 1.3 Å resolution and contained one segment, and one water molecule in the asymmetric unit.
The drop was a mixture of 10 mM KLVFFA and 1 mM orange-G in water, and reservoir solution (10% w/v polyethylene glycol 1,500, 30% v/v glycerol). Another drop was a mixture of 5 mM KLVFFA and 1 mM orange-G in water, and reservoir solution (30% w/v polyethylene glycol 1,500, 20% v/v glycerol). The structure was solved to 1.8 Å resolution and contained four segments, two orange-G molecules, and 11 water molecules in the asymmetric unit.
The drop was a mixture of 10 mM KLVFFA in water, and reservoir solution (10% w/v polyethylene glycol 1,500, 30% v/v glycerol). Another drop was a mixture of 5 mM KLVFFA in water, and reservoir solution (30% w/v polyethylene glycol 1,500, 20% v/v glycerol). The structure was solved to 2.1 Å resolution and contained one segment and three water molecules in the asymmetric unit.
X-ray diffraction data were collected at beamline 24-ID-E of the Advanced Photon Source (APS), Argonne National Laboratory; wavelength of data collection was 0.9792 Å. Data were collected at 100 K. Molecular replacement solutions for all segments were obtained using the program Phaser
Three-dimensional (3-D) structures of the small molecules were generated using Corina (Molecular Networks;
The area buried of the small molecules within the fiber structure was calculated using Areaimol
Binding energy and corresponding dissociation constant of one orange-G molecule to the KLVFFA fiber were estimated from the apolar surface area (contributed by carbon atoms) that is covered by the interaction and was calculated using Areaimol
Liquid chromatography tandem mass spectrometry (LC-MSMS) was used to measure the molar ratios of the peptide segments and the small molecules within the crystals. Authentic samples of the peptides and each of the small molecules were used to prepare standard response curves. Crystals from each of the four mixtures of peptides and small compounds were individually picked (using a sharpened glass capillary) and re-dissolved in 5%–10% acetonitrile. The samples were divided into two aliquots, one for the peptide analyses and the other for the small molecule analyses, and the amount of each component in the samples was interpolated using the standard curves.
Peptide standards (dry powder of VQIVYK and KLVFFA) were dissolved in water and prepared in concentrations ranging from 0.05 µM to 0.01 mM in 0.1% TFA. Aliquots of the standards and the re-dissolved crystals were separately injected (50 µL) onto a polymeric reverse phase column (PLRP/S, 2×150 mm, 5 µm, 300 Å; Varian) equilibrated in Buffer A (0.1% formic acid in water) and eluted (0.25 mL/min) with an increasing concentration of Buffer B (0.1% formic acid in acetonitrile). The effluent from the column was directed to an Ionspray source attached to a triple quadrupole mass spectrometer (Perkin Elmer/Sciex API III+) operating under previously optimized positive ion mode conditions. Data were collected in the positive ion multiple reaction monitoring (MRM) mode in which the intensity of specific parent→fragment ion transitions were recorded (VQIVYK, m/z 749.5→341.3, 749.5→409.4, 749.5→440.3, 749.5→522.5; KLVFFA, 724.4→84, 724.4→488.3, 362.7→84, 362.7→120.1).
Similar procedures were used for the analyses of the small molecules. Orange-G was dissolved in water and diluted with 10% ammonium acetate to concentrations ranging from 2 nM to 20 µM. Solutions of the standard and the re-dissolved crystals were separately injected (50 µL) onto a silica based reverse phase column (Supelco Ascentis Express C18, 150×2.1 mm, 2.7 µm) equilibrated in Buffer A (10 mM ammonium acetate) and eluted (0.2 mL/min) with an increasing concentration of Buffer B (acetonitrile/Isopropanol 1∶1 containing 10 mM ammonium acetate). The negative ion MRM transitions were m/z 407.1→302.1 and 407.1→222.1.
DDNP was dissolved in 95% ethanol and diluted with 10% ammonium acetate to concentrations ranging from 2 nM to 20 µM. Solutions of the standards and the re-dissolved crystals (further diluted with acetonitrile:methanol:water:acetic-acid (41∶23∶36∶1, v/v/v/v) to ensure dissolution) were separately injected (50 µL) onto a silica based reverse phase column (Supelco Ascentis Express C18, 150×2.1 mm, 2.7 µm) equilibrated in Buffer A (10 mM ammonium acetate) and eluted (0.2 mL/min) with an increasing concentration of Buffer B (acetonitrile/Isopropanol 1∶1 containing 10 mM ammonium acetate). The positive ion MRM transition was: DDNP − m/z 262.1→247.1.
Curcumin was dissolved and diluted in acetonitrile:methanol:water:acetic-acid (41∶23∶36∶1, v/v/v/v) to concentrations ranging from 2 nM to 2 µM. Aliquots of the standards and the re-dissolved crystals (further diluted with acetonitrile:methanol:water:acetic-acid (41∶23∶36∶1, v/v/v/v) to ensure dissolution) were injected (100 µL) onto a silica based reverse phase column (Waters Symmetry Shield RP18 5 µM, 3.9×150 mm) equilibrated in Buffer A (10 mM ammonium acetate) and eluted (0.5 mL/min) with an increasing concentration of Buffer B (acetonitrile/Isopropanol 1∶1 containing 10 mM ammonium acetate). The negative ion MRM transitions were m/z 367.1→173.1, 367.1→149.
Structures of KLVFFA complexed with orange-G and VQIVYK complexed with orange-G are deposited in the Protein Data Bank (PDB) with accession codes 3OVJ and 3OVL, respectively (
Chemical structures of the small molecule binders.
(TIF)
The crystal structure of the KLVFFA segment from Aβ complexed with orange-G shows extensive interactions between two orange-G molecules and the fiber. The KLVFFA segments are packed as pairs of β-sheets with orange-G bound internally to the steric zipper. The asymmetric unit of the crystal contains four peptide segments, two orange-G molecules, and 11 water molecules. Here, four layers of β-strands and two steric zippers are shown. KLVFFA and orange-G are shown as sticks with non-carbon atoms colored by atom type. The anti-parallel strands (cartoon arrows) are alternately colored white and blue, with the adjacent steric zipper colored in darker hues. The two orange-G molecules in the asymmetric unit, with carbons in orange and brown, display similar interactions with the fiber. In panel A, the view looks down the fiber axis. In panels B–C, the view is perpendicular to the fiber axis. The sulfonic acid groups of orange-G form salt links (pink lines) with five lysine residues, four protruding from facing β-sheets of the steric zipper and one from the adjacent zipper (only the latter is presented as pink lines in panel A). Only side chains of residues participating in salt links are shown. One of the sheets from the adjacent pair and one orange-G molecule are removed for clarity.
(TIF)
Crystals of the KLVFFA segment from Aβ and of the VQIVYK segment from the tau protein grown with and without orange-G. (A–B) Micro-crystals of the KLVFFA segment of Aβ grown under identical conditions (
(TIF)
Crystal structures used as controls for the complexes of the VQIVYK segment from the tau protein with DDNP and curcumin. (A) Micro-crystals of VQIVYK co-crystallized with DDNP; the structure is shown in panel C. (B) Micro-crystals of VQIVYK crystallized under identical conditions to the crystals in panel A, lacking DDNP (
(TIF)
Electron density maps and simulated annealing composite omit maps of the KLVFFA segment from Aβ complexed with orange-G. The KLVFFA segments and orange-G molecules are shown as sticks with non-carbon atoms colored by atom type. The β-sheets are formed via stacks of anti-parallel strands, alternately colored with carbons in white and in blue. The carbons of the orange-G molecules are colored orange. Water molecules are shown as aqua spheres. The view here is perpendicular to the fiber axis. (A–C) The electron density 2Fo-Fc map is shown as grey mesh (1.3σ). The difference electron density Fo-Fc map is shown as mesh (+3σ in green and −3σ in red). (D–F) The simulated annealing composite omit 2Fo-Fc map (10% omitted) is shown as grey mesh (1.3σ). Panels B–C and D–E focus on the two orange-G molecules in the asymmetric unit.
(TIF)
Electron density map of the VQIVYK segment from the tau protein complexed with orange-G. The VQIVYK segment and orange-G are shown as sticks with carbon atoms colored grey and orange, respectively, and non-carbon atoms colored by atom type. The view in panel A looks down the fiber axis. The view in panel B is perpendicular to the fiber axis and focuses on orange-G. The electron density 2Fo-Fc map is shown as grey mesh (1.3σ). The difference electron density Fo-Fc map is shown as mesh (+3σ in green and −3σ in red).
(TIF)
Screening for co-crystals from mixtures of amyloid-like segments with small molecules.
(PDF)
Data collection and refinement statistics (molecular replacement).
(PDF)
(I) Using computational docking for structure determination. (II) Incommensurate structures.
(PDF)
We thank Daniel H. Anderson and Nicholas K. Sauter for discussions and assistance with the evaluation of the incommensurate structures. We thank the anonymous referee who informed us of the paper by Schütz et al., also in the review process. J.R.B. gratefully acknowledges the support of the Elizabeth and Thomas Plott Chair Endowment in Gerontology. This work is based upon research conducted at the Northeastern Collaborative Access Team beam lines of the Advanced Photon Source. We are grateful for the beamtime and to the staff for help during data collection.
amyloid-beta
nuclear magnetic resonance