Simon Sharpe

Structure of an Intermediate State in Protein Folding and Aggregation

Protein Tipping Point Amyloid fibrils are insoluble protein aggregates that play a role in various degenerative diseases. Recent experiments have provided insight into fibrillar structures; however, the mechanisms of aggregation remain unclear. Neudecker et al. (p. 362; see the Perspective by Eliezer) report the structure of a transient folding intermediate in a protein SH3 domain known to undergo aggregation. The intermediate is stabilized by non-native interactions and exposes an aggregation-prone β strand. Thus, for this protein, folding from the intermediate state will compete with aggregation. A folding intermediate of a protein SH3 domain is prone to aggregation, which competes with native folding. Protein-folding intermediates have been implicated in amyloid fibril formation involved in neurodegenerative disorders. However, the structural mechanisms by which intermediates initiate fibrillar aggregation have remained largely elusive. To gain insight, we used relaxation dispersion nuclear magnetic resonance spectroscopy to determine the structure of a low-populated, on-pathway folding intermediate of the A39V/N53P/V55L (A, Ala; V, Val; N, Asn; P, Pro; L, Leu) Fyn SH3 domain. The carboxyl terminus remains disordered in this intermediate, thereby exposing the aggregation-prone amino-terminal β strand. Accordingly, mutants lacking the carboxyl terminus and thus mimicking the intermediate fail to safeguard the folding route and spontaneously form fibrillar aggregates. The structure provides a detailed characterization of the non-native interactions stabilizing an aggregation-prone intermediate under native conditions and insight into how such an intermediate can derail folding and initiate fibrillation.

Core structure of amyloid fibrils formed by residues 106-126 of the human prion protein.

Peptides comprising residues 106-126 of the human prion protein (PrP) exhibit many features of the full-length protein. PrP(106-126) induces apoptosis in neurons, forms fibrillar aggregates, and can mediate the conversion of native cellular PrP (PrP(C)) to the scrapie form (PrP(Sc)). Despite a wide range of biochemical and biophysical studies on this peptide, including investigation of its propensity for aggregation, interactions with cell membranes, and PrP-like toxicity, the structure of amyloid fibrils formed by PrP(106-126) remains poorly defined. In this study we use solid-state nuclear magnetic resonance to define the secondary and quaternary structure of PrP(106-126) fibrils. Our results reveal that PrP(106-126) forms in-register parallel beta sheets, stacked in an antiparallel fashion within the mature fibril. The close intermolecular contacts observed in the fibril core provide a rational for the sequence-dependent behavior of PrP(106-126), and provide a basis for further investigation of its biological properties.

Oligomerization state and supramolecular structure of the HIV-1 Vpu protein transmembrane segment in phospholipid bilayers

HIV‐1 Vpu is an 81‐residue protein with a single N‐terminal transmembrane (TM) helical segment that is involved in the release of new virions from host cell membranes. Vpu and its TM segment form ion channels in phospholipid bilayers, presumably by oligomerization of TM helices into a pore‐like structure. We describe measurements that provide new constraints on the oligomerization state and supramolecular structure of residues 1–40 of Vpu (Vpu1–40), including analytical ultracentrifugation measurements to investigate oligomerization in detergent micelles, photo‐induced crosslinking experiments to investigate oligomerization in bilayers, and solid‐state nuclear magnetic resonance measurements to obtain constraints on intermolecular contacts between and orientations of TM helices in bilayers. From these data, we develop molecular models for Vpu TM oligomers. The data indicate that a variety of oligomers coexist in phospholipid bilayers, so that a unique supramolecular structure can not be defined. Nonetheless, since oligomers of various sizes have similar intermolecular contacts and orientations, molecular models developed from our data are most likely representative of Vpu TM oligomers that exist in host cell membranes.

Structural Properties and Dynamic Behavior of Nonfibrillar Oligomers Formed by PrP(106−126)

The formation of nonfibrillar oligomers has been proposed as a common element of the aggregation pathway of proteins and peptides associated with neurodegenerative diseases such as Alzheimers and Creutzfeldt-Jakob disease. While fibrillar structures have long been considered indicators of diseases linked with the accumulation of amyloid plaques, it has more recently been proposed that amyloid oligomers are in fact the cytotoxic form. Here we describe the local structure and dynamics of stable oligomers formed by a peptide comprising residues 106-126 of the human prion protein (PrP). Structural constraints from solid-state NMR reveal quaternary packing interactions within the hydrophobic core, similar to those previously reported for amyloid fibrils formed by this peptide, and consistent with structural studies of oligomers formed by the Alzheimers beta-amyloid peptide. However, a hydration-dependent increase in disorder is observed for nonfibrillar oligomers of PrP(106-126). In solution NMR spectra we observe narrow (1)H and (13)C resonances corresponding to a monomer in exchange with the approximately 30 nm diameter nonfibrillar oligomers, giving additional information on the molecular structure of these species. Taken together, our data support a model in which the local structure of the oligomers contains the basic elements of amyloid fibrils, but with long-range disorder and local mobility that distinguishes these assemblies from the fibrillar form of PrP(106-126). These characteristics may provide a basis for the differing biological activities of amyloid fibrils and oligomers.

Solid-State NMR characterization of autofluorescent fibrils formed by the elastin-derived peptide GVGVAGVG.

The characterization of the molecular structure and physical properties of self-assembling peptides is an important aspect of optimizing their utility as scaffolds for biomaterials and other applications. Here we report the formation of autofluorescent fibrils by an octapeptide (GVGVAGVG) derived via a single amino acid substitution in one of the hydrophobic repeat elements of human elastin. This is the shortest and most well-defined peptide so far reported to exhibit intrinsic fluorescence in the absence of a discrete fluorophore. Structural characterization by FTIR and solid-state NMR reveals a predominantly β-sheet conformation for the peptide in the fibrils, which are likely assembled in an amyloid-like cross-β structure. Investigation of dynamics and the effects of hydration on the peptide are consistent with a rigid, water excluded structure, which has implications for the likely mechanism of intrinsic fibril fluorescence.

The Mechanism of Membrane Disruption by Cytotoxic Amyloid Oligomers Formed by Prion Protein(106–126) Is Dependent on Bilayer Composition

Background: Nonfibrillar amyloid oligomers are cytotoxic and may act through physical disruption of cell membranes. Results: Cytotoxic oligomers of the amyloid peptide PrP(106–126) disrupt membranes through distinct mechanisms, depending on lipid composition. Conclusion: Cytotoxicity of PrP(106–126) oligomers can occur through at least two different physical processes. Significance: Mechanisms for the membrane disruption of amyloid oligomers are proposed, providing new insight into their cytotoxicity. The formation of fibrillar aggregates has long been associated with neurodegenerative disorders such as Alzheimer and Parkinson diseases. Although fibrils are still considered important to the pathology of these disorders, it is now widely understood that smaller amyloid oligomers are the toxic entities along the misfolding pathway. One characteristic shared by the majority of amyloid oligomers is the ability to disrupt membranes, a commonality proposed to be responsible for their toxicity, although the mechanisms linking this to cell death are poorly understood. Here, we describe the physical basis for the cytotoxicity of oligomers formed by the prion protein (PrP)-derived amyloid peptide PrP(106–126). We show that oligomers of this peptide kill several mammalian cells lines, as well as mouse cerebellar organotypic cultures, and we also show that they exhibit antimicrobial activity. Physical perturbation of model membranes mimicking bacterial or mammalian cells was investigated using atomic force microscopy, polarized total internal reflection fluorescence microscopy, and NMR spectroscopy. Disruption of anionic membranes proceeds through a carpet or detergent model as proposed for other antimicrobial peptides. By contrast, when added to zwitterionic membranes containing cholesterol-rich ordered domains, PrP(106–126) oligomers induce a loss of domain separation and decreased membrane disorder. Loss of raft-like domains may lead to activation of apoptotic pathways, resulting in cell death. This work sheds new light on the physical mechanisms of amyloid cytotoxicity and is the first to clearly show membrane type-specific modes of action for a cytotoxic peptide.

Morphology and Secondary Structure of Stable β-Oligomers Formed by Amyloid Peptide PrP(106―126)

The formation of nonfibrillar oligomers has been proposed to be a common element of the aggregation pathway of amyloid peptides. Here we describe the first detailed investigation of the morphology and secondary structure of stable oligomers formed by a peptide comprising residues 106-126 of the human prion protein (PrP). These oligomers have an apparent hydrodynamic radius of approximately 30 nm and are more membrane-active than monomeric or fibrillar PrP(106-126). Circular dichroism and solid state NMR data support formation of an extended beta-strand by the hydrophobic core of PrP(106-126), while negative thioflavin-T binding implies an absence of cross-beta structure in nonfibrillar oligomers.

Direct observation of structure and dynamics during phase separation of an elastomeric protein

Significance An increasing number of proteins have been shown to undergo liquid–liquid phase separation in response to changes in their environment, resulting in formation of a dense protein-rich phase (coacervate), and plays an important role in several systems regulating the growth and development of cells and tissues. Determining the effects of phase separation on protein structure and dynamics is critical for understanding how it modulates protein function. However, structural studies have been limited by the intrinsic disorder and decreased mobility of coacervated proteins. We report direct observation of protein structure and dynamics during the phase transition of an elastomeric protein. Despite large changes in dynamics, coacervation has little effect on protein structure, such that intrinsic disorder is retained. Despite its growing importance in biology and in biomaterials development, liquid–liquid phase separation of proteins remains poorly understood. In particular, the molecular mechanisms underlying simple coacervation of proteins, such as the extracellular matrix protein elastin, have not been reported. Coacervation of the elastin monomer, tropoelastin, in response to heat and salt is a critical step in the assembly of elastic fibers in vivo, preceding chemical cross-linking. Elastin-like polypeptides (ELPs) derived from the tropoelastin sequence have been shown to undergo a similar phase separation, allowing formation of biomaterials that closely mimic the material properties of native elastin. We have used NMR spectroscopy to obtain site-specific structure and dynamics of a self-assembling elastin-like polypeptide along its entire self-assembly pathway, from monomer through coacervation and into a cross-linked elastic material. Our data reveal that elastin-like hydrophobic domains are composed of transient β-turns in a highly dynamic and disordered chain, and that this disorder is retained both after phase separation and in elastic materials. Cross-linking domains are also highly disordered in monomeric and coacervated ELP3 and form stable helices only after chemical cross-linking. Detailed structural analysis combined with dynamic measurements from NMR relaxation and diffusion data provides direct evidence for an entropy-driven mechanism of simple coacervation of a protein in which transient and nonspecific intermolecular hydrophobic contacts are formed by disordered chains, whereas bulk water and salt are excluded.

Dynamic Equilibria between Monomeric and Oligomeric Misfolded States of the Mammalian Prion Protein Measured by 19F NMR

The assembly of misfolded proteins is a critical step in the pathogenesis of amyloid and prion diseases, although the molecular mechanisms underlying this phenomenon are not completely understood. Here, we use (19)F NMR spectroscopy to examine the thermodynamic driving forces surrounding formation of β-sheet-rich oligomers early in the misfolding and aggregation pathway of the mammalian prion protein. We show that initial assembly of a small octameric intermediate is entropically driven, while further assembly to putative prefibrillar aggregates is driven by a favorable change in enthalpy. Kinetic data suggest that formation of the β-octamer represents a rate-limiting step in the assembly of prion aggregates. A disease-related mutation (F198S) known to destabilize the native state of PrP was also found to stabilize the β-octamer, suggesting that it can influence susceptibility to prion disease through two distinct mechanisms. This study provides new insight into the misfolding pathway leading to critical oligomers of the prion protein and suggests a physical basis for increased assembly of the F198S mutant.

Conformational Transitions of the Cross-linking Domains of Elastin during Self-assembly

Background: Elastin is a polymeric protein providing extensibility and elastic recoil to tissues. Results: Cross-linking domain structure shifts from random coil to β-strand to α-helix during assembly of elastin matrix. Conclusion: Cross-linking domains have a previously unappreciated structural lability during assembly, which is highly susceptible to mutations of lysine residues. Significance: Identification of conformational transitions in cross-linking domains of elastin during self-assembly is essential for understanding the mechanisms of formation of the elastic matrix. Elastin is the intrinsically disordered polymeric protein imparting the exceptional properties of extension and elastic recoil to the extracellular matrix of most vertebrates. The monomeric precursor of elastin, tropoelastin, as well as polypeptides containing smaller subsets of the tropoelastin sequence, can self-assemble through a colloidal phase separation process called coacervation. Present understanding suggests that self-assembly is promoted by association of hydrophobic domains contained within the tropoelastin sequence, whereas polymerization is achieved by covalent joining of lysine side chains within distinct alanine-rich, α-helical cross-linking domains. In this study, model elastin polypeptides were used to determine the structure of cross-linking domains during the assembly process and the effect of sequence alterations in these domains on assembly and structure. CD temperature melts indicated that partial α-helical structure in cross-linking domains at lower temperatures was absent at physiological temperature. Solid-state NMR demonstrated that β-strand structure of the cross-linking domains dominated in the coacervate state, although α-helix was predominant after subsequent cross-linking of lysine side chains with genipin. Mutation of lysine residues to hydrophobic amino acids, tyrosine or alanine, leads to increased propensity for β-structure and the formation of amyloid-like fibrils, characterized by thioflavin-T binding and transmission electron microscopy. These findings indicate that cross-linking domains are structurally labile during assembly, adapting to changes in their environment and aggregated state. Furthermore, the sequence of cross-linking domains has a dramatic effect on self-assembly properties of elastin-like polypeptides, and the presence of lysine residues in these domains may serve to prevent inappropriate ordered aggregation.