Nucleation of α-Synuclein Amyloid Fibrils Induced by Cross-Interaction with ß-Hairpin Peptides Derived from Immunoglobulin Light Chains.

Heterologous interactions between different amyloid-forming proteins, also called cross-interactions, may have a critical impact on disease-related amyloid formation. ß-hairpin conformers of amyloid-forming proteins have been shown to affect homologous interactions in the amyloid self-assembly process. Here, we applied two ß-hairpin-forming peptides derived from immunoglobulin light chains as models to test how heterologous ß-hairpins modulate the fibril formation of Parkinson's disease-associated protein α-synuclein (αSyn). The peptides SMAhp and LENhp comprise ß-strands C and C' of the κ4 antibodies SMA and LEN, which are associated with light chain amyloidosis and multiple myeloma, respectively. SMAhp and LENhp bind with high affinity to the ß-hairpin-binding protein ß-wrapin AS10 according to isothermal titration calorimetry and NMR spectroscopy. The addition of SMAhp and LENhp affects the kinetics of αSyn aggregation monitored by Thioflavin T (ThT) fluorescence, with the effect depending on assay conditions, salt concentration, and the applied ß-hairpin peptide. In the absence of agitation, substoichiometric concentrations of the hairpin peptides strongly reduce the lag time of αSyn aggregation, suggesting that they support the nucleation of αSyn amyloid fibrils. The effect is also observed for the aggregation of αSyn fragments lacking the N-terminus or the C-terminus, indicating that the promotion of nucleation involves the interaction of hairpin peptides with the hydrophobic non-amyloid-ß component (NAC) region.

Pyroglutamate-Modified Amyloid-ß(3-42) Shows α-Helical Intermediates before Amyloid Formation.

Pyroglutamate-modified amyloid-ß (pEAß) has been described as a relevant Aß species in Alzheimer's-disease-affected brains, with pEAß (3-42) as a dominant isoform. Aß (1-40) and Aß (1-42) have been well characterized under various solution conditions, including aqueous solutions containing trifluoroethanol (TFE). To characterize structural properties of pEAß (3-42) possibly underlying its drastically increased aggregation propensity compared to Aß (1-42), we started our studies in various TFE-water mixtures and found striking differences between the two Aß species. Soluble pEAß (3-42) has an increased tendency to form ß-sheet-rich structures compared to Aß (1-42), as indicated by circular dichroism spectroscopy data. Kinetic assays monitored by thioflavin-T show drastically accelerated aggregation leading to large fibrils visualized by electron microscopy of pEAß (3-42) in contrast to Aß (1-42). NMR spectroscopy was performed for backbone and side-chain chemical-shift assignments of monomeric pEAß (3-42) in 40% TFE solution. Although the difference between pEAß (3-42) and Aß (1-42) is purely N-terminal, it has a significant impact on the chemical environment of >20% of the total amino acid residues, as revealed by their NMR chemical-shift differences. Freshly dissolved pEAß (3-42) contains two α-helical regions connected by a flexible linker, whereas the N-terminus remains unstructured. We found that these α-helices act as a transient intermediate to ß-sheet and fibril formation of pEAß (3-42).

The Nedd4-1 WW Domain Recognizes the PY Motif Peptide through Coupled Folding and Binding Equilibria.

The four WW domains of human Nedd4-1 (neuronal precursor cell expressed developmentally downregulated gene 4-1) interact with the PPxY (PY) motifs of the human epithelial Na(+) channel (hENaC) subunits, with the third WW domain (WW3*) showing the highest affinity. We have shown previously that the α-hENaC PY motif binding interface of WW3* undergoes conformational exchange on the millisecond time scale, indicating that conformational sampling plays a role in peptide recognition. To further understand this role, the structure and dynamics of hNedd4-1 WW3* were investigated. The nuclear Overhauser effect-derived structure of apo-WW3* resembles the domain in complex with the α-hENaC peptide, although particular side chain conformations change upon peptide binding, which was further investigated by molecular dynamics simulations. Model-free analysis of the (15)N nuclear magnetic resonance spin relaxation data showed that the apo and peptide-bound states of WW3* have similar backbone picosecond to nanosecond time scale dynamics. However, apo-WW3* exhibits pronounced chemical exchange on the millisecond time scale that is quenched upon peptide binding. (1)HN and (15)N Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments at various temperatures revealed that apo-WW3* exists in an equilibrium between the natively folded peptide binding-competent state and a random coil-like denatured state. The thermodynamics of the folding equilibrium was determined by fitting a thermal denaturation profile monitored by circular dichroism spectroscopy in combination with the CPMG data, leading to the conclusion that the unfolded state is populated to ∼ 20% at 37 °C. These results show that the binding of the hNedd4-1 WW3* domain to α-hENaC is coupled to the folding equilibrium.

Conformational Polymorphism in Autophagy-Related Protein GATE-16.

Autophagy is a fundamental homeostatic process in eukaryotic organisms, fulfilling essential roles in development and adaptation to stress. Among other factors, formation of autophagosomes critically depends on proteins of the Atg8 (autophagy-related protein 8) family, which are reversibly conjugated to membrane lipids. We have applied X-ray crystallography, nuclear magnetic resonance spectroscopy, and molecular dynamics simulations to study the conformational dynamics of Atg8-type proteins, using GATE-16 (Golgi-associated ATPase enhancer of 16 kDa), also known as GABARAPL2, as a model system. This combination of complementary approaches provides new insight into a structural transition centered on the C-terminus, which is crucial for the biological activity of these proteins.

Alternative conformations of the Tau repeat domain in complex with an engineered binding protein.

The aggregation of Tau into paired helical filaments is involved in the pathogenesis of several neurodegenerative diseases, including Alzheimer disease. The aggregation reaction is characterized by conformational conversion of the repeat domain, which partially adopts a cross-ß-structure in the resulting amyloid-like fibrils. Here, we report the selection and characterization of an engineered binding protein, ß-wrapin TP4, targeting the Tau repeat domain. TP4 was obtained by phage display using the four-repeat Tau construct K18ΔK280 as a target. TP4 binds K18ΔK280 as well as the longest isoform of human Tau, hTau40, with nanomolar affinity. NMR spectroscopy identified two alternative TP4-binding sites in the four-repeat domain, with each including two hexapeptide motifs with high ß-sheet propensity. Both binding sites contain the aggregation-determining PHF6 hexapeptide within repeat 3. In addition, one binding site includes the PHF6* hexapeptide within repeat 2, whereas the other includes the corresponding hexapeptide Tau(337-342) within repeat 4, denoted PHF6**. Comparison of TP4-binding with Tau aggregation reveals that the same regions of Tau are involved in both processes. TP4 inhibits Tau aggregation at substoichiometric concentration, demonstrating that it interferes with aggregation nucleation. This study provides residue-level insight into the interaction of Tau with an aggregation inhibitor and highlights the structural flexibility of Tau.

A ß-hairpin-binding protein for three different disease-related amyloidogenic proteins.

Amyloidogenic proteins share a propensity to convert to the ß-structure-rich amyloid state that is associated with the progression of several protein-misfolding disorders. Here we show that a single engineered ß-hairpin-binding protein, the ß-wrapin AS10, binds monomers of three different amyloidogenic proteins, that is, amyloid-ß peptide, α-synuclein, and islet amyloid polypeptide, with sub-micromolar affinity. AS10 binding inhibits the aggregation and toxicity of all three proteins. The results demonstrate common conformational preferences and related binding sites in a subset of the amyloidogenic proteins. These commonalities enable the generation of multispecific monomer-binding agents.

Contact between the ß1 and ß2 Segments of α-Synuclein that Inhibits Amyloid Formation.

Conversion of the intrinsically disordered protein α-synuclein (α-syn) into amyloid aggregates is a key process in Parkinson's disease. The sequence region 35-59 contains ß-strand segments ß1 and ß2 of α-syn amyloid fibril models and most disease-related mutations. ß1 and ß2 frequently engage in transient interactions in monomeric α-syn. The consequences of ß1-ß2 contacts are evaluated by disulfide engineering, biophysical techniques, and cell viability assays. The double-cysteine mutant α-synCC, with a disulfide linking ß1 and ß2, is aggregation-incompetent and inhibits aggregation and toxicity of wild-type α-syn. We show that α-syn delays the aggregation of amyloid-ß peptide and islet amyloid polypeptide involved in Alzheimer's disease and type 2 diabetes, an effect enhanced in the α-synCC mutant. Tertiary interactions in the ß1-ß2 region of α-syn interfere with the nucleation of amyloid formation, suggesting promotion of such interactions as a potential therapeutic approach.

Interaction of Bcl-2 with the autophagy-related GABAA receptor-associated protein (GABARAP): biophysical characterization and functional implications.

Apoptosis and autophagy are fundamental homeostatic processes in eukaryotic organisms fulfilling essential roles in development and adaptation. Recently, the anti-apoptotic factor Bcl-2 has been reported to also inhibit autophagy, thus establishing a potential link between these pathways, but the mechanistic details are only beginning to emerge. Here we show that Bcl-2 directly binds to the phagophore-associated protein GABARAP. NMR experiments revealed that the interaction critically depends on a three-residue segment (EWD) of Bcl-2 adjacent to the BH4 region, which is anchored to one of the two hydrophobic pockets on the GABARAP molecule. This is at variance with the majority of GABARAP interaction partners identified previously, which occupy both hydrophobic pockets simultaneously. Bcl-2 affinity could also be detected for GEC1, but not for other mammalian Atg8 homologs. Finally, we provide evidence that overexpression of Bcl-2 inhibits lipidation of GABARAP, a key step in autophagosome formation, possibly via competition with the lipid conjugation machinery. These results support the regulatory role of Bcl-2 in autophagy and define GABARAP as a novel interaction partner involved in this intricate connection.

Hepatitis C virus NS5A is able to competitively displace c-Myc from the Bin1 SH3 domain in vitro.

We studied the interaction of the SH3 domain of Bin1 with a 15-mer peptide of HCV NS5A and show its potency to competitively displace a 15-mer human c-Myc fragment, which is a physiological ligand of Bin1, using NMR spectroscopy. Fluorescence spectroscopy and ITC were employed to determine the affinity of Bin1 SH3 to NS5A(347-361), yielding a submicromolar affinity to NS5A. Our study compares the binding dynamics and affinities of the relevant regions for binding of c-Myc and NS5A to Bin1 SH3. The result gives further insights into the potential role of NS5A in Bin1-mediated apoptosis.

Structural insights into conformational changes of a cyclic nucleotide-binding domain in solution from Mesorhizobium loti K1 channel.

Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels, are activated by binding of ligands to a domain (CNBD) located on the cytoplasmic side of the channel. The underlying mechanisms are not well understood. To elucidate the gating mechanism, structures of both the ligand-free and -bound CNBD are required. Several crystal structures of the CNBD from HCN2 and a bacterial CNG channel (MloK1) have been solved. However, for HCN2, the cAMP-free and -bound state did not reveal substantial structural rearrangements. For MloK1, structural information for the cAMP-free state has only been gained from mutant CNBDs. Moreover, in the crystal, the CNBD molecules form an interface between dimers, proposed to be important for allosteric channel gating. Here, we have determined the solution structure by NMR spectroscopy of the cAMP-free wild-type CNBD of MloK1. A comparison of the solution structure of cAMP-free and -bound states reveals large conformational rearrangement on ligand binding. The two structures provide insights on a unique set of conformational events that accompany gating within the ligand-binding site.

Sequestration of a ß-hairpin for control of α-synuclein aggregation.

The misfolding and aggregation of the protein α-synuclein (α-syn), which results in the formation of amyloid fibrils, is involved in the pathogenesis of Parkinson's disease and other synucleinopathies. The emergence of amyloid toxicity is associated with the formation of partially folded aggregation intermediates. Here, we engineered a class of binding proteins termed ß-wrapins (ß-wrap proteins) with affinity for α-synuclein (α-syn). The NMR structure of an α-syn:ß-wrapin complex reveals a ß-hairpin of α-syn comprising the sequence region α-syn(37-54). The ß-wrapin inhibits α-syn aggregation and toxicity at substoichiometric concentrations, demonstrating that it interferes with the nucleation of aggregation.

Sequence-based identification of amyloidogenic ß-hairpins reveals a prostatic acid phosphatase fragment promoting semen amyloid formation.

ß-Structure-rich amyloid fibrils are hallmarks of several diseases, including Alzheimer's (AD), Parkinson's (PD), and type 2 diabetes (T2D). While amyloid fibrils typically consist of parallel ß-sheets, the anti-parallel ß-hairpin is a structural motif accessible to amyloidogenic proteins in their monomeric and oligomeric states. Here, to investigate implications of ß-hairpins in amyloid formation, potential ß-hairpin-forming amyloidogenic segments in the human proteome were predicted based on sequence similarity with ß-hairpins previously observed in Aß, α-synuclein, and islet amyloid polypeptide, amyloidogenic proteins associated with AD, PD, and T2D, respectively. These three ß-hairpins, established upon binding to the engineered binding protein ß-wrapin AS10, are characterized by proximity of two sequence segments rich in hydrophobic and aromatic amino acids, with high ß-aggregation scores according to the TANGO algorithm. Using these criteria, 2505 potential ß-hairpin-forming amyloidogenic segments in 2098 human proteins were identified. Characterization of a test set of eight protein segments showed that seven assembled into Thioflavin T-positive aggregates and four formed ß-hairpins in complex with AS10 according to NMR. One of those is a segment of prostatic acid phosphatase (PAP) comprising amino acids 185-208. PAP is naturally cleaved into fragments, including PAP(248-286) which forms functional amyloid in semen. We find that PAP(185-208) strongly decreases the protein concentrations required for fibril formation of PAP(248-286) and of another semen amyloid peptide, SEM1(86-107), indicating that it promotes nucleation of semen amyloids. In conclusion, ß-hairpin-forming amyloidogenic protein segments could be identified in the human proteome with potential roles in functional or disease-related amyloid formation.

Structural snapshot of cyclic nucleotide binding domains from cyclic nucleotide-sensitive ion channels.

Cyclic nucleotide-binding domains (CNBDs) that are present in various channel proteins play crucial roles in signal amplification cascades. Although atomic resolution structures of some of those CNBDs are available, the detailed mechanism by which they confer cyclic nucleotide-binding to the ion channel pore remains poorly understood. In this review, we describe structural insights about cyclic nucleotide-binding-induced conformational changes in CNBDs and their potential coupling with channel gating.

Structural analysis of the pyroglutamate-modified isoform of the Alzheimer's disease-related amyloid-ß using NMR spectroscopy.

The aggregation of the Aß plays a fundamental role in the pathology of AD. Recently, N-terminally modified Aß species, pE-Aß, have been described as major constituents of Aß deposits in the brains of AD patients. pE-Aß has an increased aggregation propensity and shows increased toxicity compared with Aß1-40 and Aß1-42. In the present work, high-resolution NMR spectroscopy was performed to study pE-Aß3-40 in aqueous TFE-containing solution. Two-dimensional TOCSY and NOESY experiments were performed. On the basis of NOE and chemical shift data, pE-Aß3-40 was shown to contain two helical regions formed by residues 14-22 and 30-36. This is similar as previously described for Aß1-40. However, the secondary chemical shift data indicate decreased helical propensity in pE-Aß3-40 when compared with Aß1-40 under exactly the same conditions. This is in agreement with the observation that pE-Aß3-40 shows a drastically increased tendency to form ß-sheet-rich structures under more physiologic conditions. Structural studies of pE-Aß are crucial for better understanding the structural basis of amyloid fibril formation in the brain during development of AD, especially because an increasing number of reports indicate a decisive role of pE-Aß for the pathogenesis of AD.

Alpha-Synuclein-Specific Naturally Occurring Antibodies Inhibit Aggregation In Vitro and In Vivo.

Parkinson's disease (PD) is associated with motor and non-motor symptoms and characterized by aggregates of alpha-synuclein (αSyn). Naturally occurring antibodies (nAbs) are part of the innate immune system, produced without prior contact to their specific antigen, and polyreactive. The abundance of nAbs against αSyn is altered in patients with PD. In this work, we biophysically characterized nAbs against αSyn (nAbs-αSyn) and determined their biological effects. nAbs-αSyn were isolated from commercial intravenous immunoglobulins using column affinity purification. Biophysical properties were characterized using a battery of established in vitro assays. Biological effects were characterized in HEK293T cells transiently transfected with fluorescently tagged αSyn. Specific binding of nAbs-αSyn to monomeric αSyn was demonstrated by Dot blot, ELISA, and Surface Plasmon Resonance. nAbs-αSyn did not affect viability of HEK293T cells as reported by Cell Titer Blue and LDH Assays. nAbs-αSyn inhibited fibrillation of αSyn reported by the Thioflavin T aggregation assay. Altered fibril formation was confirmed with atomic force microscopy. In cells transfected with EGFP-tagged αSyn we observed reduced formation of aggresomes, perinuclear accumulations of αSyn aggregates. The results demonstrate that serum of healthy individuals contains nAbs that specifically bind αSyn and inhibit aggregation of αSyn in vitro. The addition of nAbs-αSyn to cultured cells affects intracellular αSyn aggregates. These findings help understanding the role of the innate immune systems for the pathogenesis of PD and suggest that systemic αSyn binding agents could potentially affect neuronal αSyn pathology.

Solution structure of the Mesorhizobium loti K1 channel cyclic nucleotide-binding domain in complex with cAMP.

Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels, are crucial in neuronal excitability and signal transduction of sensory cells. HCN and CNG channels are activated by binding of cyclic nucleotides to their intracellular cyclic nucleotide-binding domain (CNBD). However, the mechanism by which the binding of cyclic nucleotides opens these channels is not well understood. Here, we report the solution structure of the isolated CNBD of a cyclic nucleotide-sensitive K(+) channel from Mesorhizobium loti. The protein consists of a wide anti-parallel beta-roll topped by a helical bundle comprising five alpha-helices and a short 3(10)-helix. In contrast to the dimeric arrangement ('dimer-of-dimers') in the crystal structure, the solution structure clearly shows a monomeric fold. The monomeric structure of the CNBD supports the hypothesis that the CNBDs transmit the binding signal to the channel pore independently of each other.

Solution structure of Atg8 reveals conformational polymorphism of the N-terminal domain.

During autophagy a crescent shaped like membrane is formed, which engulfs the material that is to be degraded. This membrane grows further until its edges fuse to form the double membrane covered autophagosome. Atg8 is a protein, which is required for this initial step of autophagy. Therefore, a multistage conjugation process of newly synthesized Atg8 to phosphatidylethanolamine is of critical importance. Here we present the high resolution structure of unprocessed Atg8 determined by nuclear magnetic resonance spectroscopy. Its C-terminal subdomain shows a well-defined ubiquitin-like fold with slightly elevated mobility in the pico- to nanosecond timescale as determined by heteronuclear NOE data. In comparison to unprocessed Atg8, cleaved Atg8(G116) shows a decreased mobility behaviour. The N-terminal domain adopts different conformations within the micro- to millisecond timescale. The possible biological relevance of the differences in dynamic behaviours between both subdomains as well as between the cleaved and uncleaved forms is discussed.

Helix formation in arrestin accompanies recognition of photoactivated rhodopsin.

Binding of arrestin to photoactivated phosphorylated rhodopsin terminates the amplification of visual signals in photoreceptor cells. Currently, there is no crystal structure of a rhodopsin-arrestin complex available, although structures of unbound rhodopsin and arrestin have been determined. High-affinity receptor binding is dependent on distinct arrestin sites responsible for recognition of rhodopsin activation and phosphorylation. The loop connecting beta-strands V and VI in rod arrestin has been implicated in the recognition of active rhodopsin. We report the structure of receptor-bound arrestin peptide Arr(67-77) mimicking this loop based on solution NMR data. The peptide binds photoactivated rhodopsin in the unphosphorylated and phosphorylated form with similar affinities and stabilizes the metarhodopsin II photointermediate. A largely alpha-helical conformation of the receptor-bound peptide is observed.

Structure of the SLy1 SAM homodimer reveals a new interface for SAM domain self-association.

Sterile alpha motif (SAM) domains are protein interaction modules that are involved in a diverse range of biological functions such as transcriptional and translational regulation, cellular signalling, and regulation of developmental processes. SH3 domain-containing protein expressed in lymphocytes 1 (SLy1) is involved in immune regulation and contains a SAM domain of unknown function. In this report, the structure of the SLy1 SAM domain was solved and revealed that this SAM domain forms a symmetric homodimer through a novel interface. The interface consists primarily of the two long C-terminal helices, α5 and α5', of the domains packing against each other. The dimerization is characterized by a dissociation constant in the lower micromolar range. A SLy1 SAM domain construct with an extended N-terminus containing five additional amino acids of the SLy1 sequence further increases the stability of the homodimer, making the SLy1 SAM dimer two orders of magnitude more stable than previously studied SAM homodimers, suggesting that the SLy1 SAM dimerization is of functional significance. The SLy1 SAM homodimer contains an exposed mid-loop surface on each monomer, which may provide a scaffold for mediating interactions with other SAM domain-containing proteins via a typical mid-loop-end-helix interface.

Solution structure of a Hck SH3 domain ligand complex reveals novel interaction modes.

We studied the interaction of hematopoietic cell kinase SH3 domain (HckSH3) with an artificial 12-residue proline-rich peptide PD1 (HSKYPLPPLPSL) identified as high affinity ligand (K(D)=0.2 muM). PD1 shows an unusual ligand sequence for SH3 binding in type I orientation because it lacks the typical basic anchor residue at position P(-3), but instead has a tyrosine residue at this position. A basic lysine residue, however, is present at position P(-4). The solution structure of the HckSH3:PD1 complex, which is the first HckSH3 complex structure available, clearly reveals that the P(-3) tyrosine residue of PD1 does not take the position of the typical anchor residue but rather forms additional van der Waals interactions with the HckSH3 RT loop. Instead, lysine at position P(-4) of PD1 substitutes the function of the P(-3) anchor residue. This finding expands the well known ligand consensus sequence +xxPpxP by +xxxPpxP. Thus, software tools like iSPOT fail to identify PD1 as a high-affinity HckSH3 ligand so far. In addition, a short antiparallel beta-sheet in the RT loop of HckSH3 is observed upon PD1 binding. The structure of the HckSH3:PD1 complex reveals novel features of SH3 ligand binding and yields new insights into the structural basics of SH3-ligand interactions. Consequences for computational prediction tools adressing SH3-ligand interactions as well as the biological relevance of our findings are discussed.