A Hexameric Peptide Barrel as Building Block of Amyloid‐β Protofibrils

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Most, if not all, peptide-and protein-based hydrogels formed by self-assembly can be characterized as kinetically trapped 3D networks of fibrils. The propensity of disease-associated amyloidforming peptides and proteins to assemble into polymorphic fibrils suggests that cross-β fibrils comprising hydrogels may also be polymorphic. We use solid-state NMR to determine the molecular and supramolecular structure of MAX1, a de novo designed gelforming peptide, in its fibrillar state. We find that MAX1 adopts a β-hairpin conformation and self-assembles with high fidelity into a double-layered cross-β structure. Hairpins assemble with an in-register Syn orientation within each β-sheet layer and with an Anti orientation between layers. Surprisingly, although the MAX1 fibril network is kinetically trapped, solid-state NMR data show that fibrils within this network are monomorphic and most likely represent the thermodynamic ground state. Intermolecular interactions not available in alternative structural arrangements apparently dictate this monomorphic behavior.M AX1 is a 20-residue peptide designed de novo to fold into an amphiphilic β-hairpin that self-assembles to form a fibrillar network within a self-supporting hydrogel (1). The MAX1 gel exhibits shear thin-recovery rheological behavior (2), is cytocompatible toward mammalian cells, yet is inherently antimicrobial (3) and thus has applications in tissue engineering and drug delivery. In addition to exploring the utility of the gel, we seek to understand the mechanism of gelation, the macroscale morphology of its fibrillar network, and the underlying molecular structure of its fibrils.MAX1 contains two segments of alternating lysine and valine residues, connected by a four-residue turn-forming segment. When initially dissolved in water, electrostatic repulsions among protonated lysine sidechains lead to an ensemble of monomeric random coil conformations (1). Peptide folding and self-assembly, leading to gelation (Fig. 1), can be triggered by attenuating electrostatic repulsions, by adjusting the solution pH and/or ionic strength. Increasing the solution temperature also drives hydrophobic collapse of valine sidechains, further favoring MAX1 assembly. According to circular dichroism (1), cryo-transmission electron microscopy (TEM) (4), small-angle neutron scattering (5), and dynamic light scattering coupled with rheological measurements (4), soon after the triggering event, peptides assemble into branched clusters of β-sheet-rich, semiflexible nanofibrils throughout the solution. Individual clusters contain dangling fibril ends that grow and interpenetrate neighboring clusters as the network evolves. Multiple particle tracking microrheology shows that the time at which the fibril network percolates the entire sample volume, defining the gel point, is less than 1 min at 1% (wt/vol) peptide (6). In this mechanism of gelation, the growing fibrils become kinetically trapped in the evolving network as they percolate the sample volume. Fibrils do not precipitate, but rather ...

Section: Discussionmentioning

confidence: 99%

Molecular structure of monomorphic peptide fibrils within a kinetically trapped hydrogel network

Nagy-Smith

Moore

Schneider

et al. 2015

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“…Several lines of evidence link the amyloid β peptide (Aβ) and its self-assembly reaction to AD (5-7). Monomeric Aβ peptides are unstructured in solution, but oligomeric (8,9) and fibrillar (10)(11)(12)(13) aggregates contain a high proportion of β-sheet structure. Monomers and fibrils appear to be relatively harmless, whereas at least some oligomeric species have significant toxicity and are likely to be linked to neurodegeneration (14,15).…”

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confidence: 99%

Phage display and kinetic selection of antibodies that specifically inhibit amyloid self-replication

Munke

Persson

Weiffert

et al. 2017

The aggregation of the amyloid β peptide (Aβ) into amyloid fibrils is a defining characteristic of Alzheimer's disease. Because of the complexity of this aggregation process, effective therapeutic inhibitors will need to target the specific microscopic steps that lead to the production of neurotoxic species. We introduce a strategy for generating fibril-specific antibodies that selectively suppress fibril-dependent secondary nucleation of the 42-residue form of Aβ (Aβ42). We target this step because it has been shown to produce the majority of neurotoxic species during aggregation of Aβ42. Starting from large phage display libraries of single-chain antibody fragments (scFvs), the three-stage approach that we describe includes (i) selection of scFvs with high affinity for Aβ42 fibrils after removal of scFvs that bind Aβ42 in its monomeric form; (ii) ranking, by surface plasmon resonance affinity measurements, of the resulting candidate scFvs that bind to the Aβ42 fibrils; and (iii) kinetic screening and analysis to find the scFvs that inhibit selectively the fibril-catalyzed secondary nucleation process in Aβ42 aggregation. By applying this approach, we have identified four scFvs that inhibit specifically the fibril-dependent secondary nucleation process. Our method also makes it possible to discard antibodies that inhibit elongation, an important factor because the suppression of elongation does not target directly the production of toxic oligomers and may even lead to its increase. On the basis of our results, we suggest that the method described here could form the basis for rationally designed immunotherapy strategies to combat Alzheimer's and related neurodegenerative diseases.Alzheimer | antibody | inhibitor | drug development | self-assembly

“…To enable molecular studies of such states, we have developed a chemical approach whereby a high-energy state of apo AdK was arrested with a covalent disulfide bond. The relevance of cross-linking proteins through disulfide bonding (16)(17)(18) has been shown, e.g., by inhibition of fibril nucleation and elongation of α-synuclein, amyloid-β peptide, and islet amyloid polypeptide (19). The disulfide-linked AdK variant could be purified to homogeneity in the arrested high-energy state enabeling subsequent comprehensive biophysical characterization of the structure, dynamics, and activity of this functionally crucial conformation.…”

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confidence: 99%

Structural basis for ligand binding to an enzyme by a conformational selection pathway

Kovermann

Grundström

Sauer‐Eriksson

et al. 2017

Proteins can bind target molecules through either induced fit or conformational selection pathways. In the conformational selection model, a protein samples a scarcely populated high-energy state that resembles a target-bound conformation. In enzymatic catalysis, such high-energy states have been identified as crucial entities for activity and the dynamic interconversion between ground states and high-energy states can constitute the ratelimiting step for catalytic turnover. The transient nature of these states has precluded direct observation of their properties. Here, we present a molecular description of a high-energy enzyme state in a conformational selection pathway by an experimental strategy centered on NMR spectroscopy, protein engineering, and X-ray crystallography. Through the introduction of a disulfide bond, we succeeded in arresting the enzyme adenylate kinase in a closed high-energy conformation that is on-pathway for catalysis. A 1.9-Å X-ray structure of the arrested enzyme in complex with a transition state analog shows that catalytic sidechains are properly aligned for catalysis. We discovered that the structural sampling of the substrate free enzyme corresponds to the complete amplitude that is associated with formation of the closed and catalytically active state. In addition, we found that the trapped high-energy state displayed improved ligand binding affinity, compared with the wild-type enzyme, demonstrating that substrate binding to the high-energy state is not occluded by steric hindrance. Finally, we show that quenching of fast time scale motions observed upon ligand binding to adenylate kinase is dominated by enzyme-substrate interactions and not by intramolecular interactions resulting from the conformational change.enzymatic catalysis | ligand binding | structural biology | adenylate kinase S ubstantial proportions of cellular processes depend on chemical reactions that in aqueous solution often are several orders of magnitude too slow to support biological life (1). This difference between "chemical" and "biological" time scales is bridged by acceleration of the rates of chemical reactions by enzymes (2). The catalytic power of an enzyme depends on a significant reduction of the free energy barrier for the chemical reaction (2). Several factors collectively contribute to an enzyme's efficiency as catalysts, including balanced substrate-binding affinity to ensure selectivity but at the same time avoid kinetic traps, optimal alignment of substrates, activation of a substrate's functional groups, and dehydration of active sites. All of these factors to some degree depend on conformational dynamics of the enzyme (3), where dynamics is defined as the time-dependent displacement of atomic coordinates. Structural excursions from enzymatic ground states to high-energy states have been observed with NMR spectroscopy, and in a few cases, were shown to be rate limiting for catalytic turnover (3-7). Furthermore, it has been proposed that the conformational dynamics required for substrate bindin...