Neuronal nAChRs are a diverse family of pentameric ion channels with wide distribution throughout cells of the nervous and immune systems. However, the role of specific subtypes in normal and pathological states remains poorly understood due to the lack of selective probes. Here, we used a binding assay based on acetylcholine-binding protein (AChBP), a homolog of the nicotinic acetylcholine ligand-binding domain, to discover a novel α-conotoxin (α-TxIA) in the venom of Conus textile. α-TxIA bound with high affinity to AChBPs from different species and selectively targeted the α3β2 nAChR subtype. A co-crystal structure of Ac-AChBP with the enhanced potency analog TxIA(A10L), revealed a 20° backbone tilt compared to other AChBP–conotoxin complexes. This reorientation was coordinated by a key salt bridge formed between Arg5 (TxIA) and Asp195 (Ac-AChBP). Mutagenesis studies, biochemical assays and electrophysiological recordings directly correlated the interactions observed in the co-crystal structure to binding affinity at AChBP and different nAChR subtypes. Together, these results establish a new pharmacophore for the design of novel subtype-selective ligands with therapeutic potential in nAChR-related diseases.
Previous studies suggest that the toxic soluble-oligomeric form of different amyloid proteins share a common backbone conformation, but the amorphous nature of this oligomer prevents its structural characterization by experiment. Based on molecular dynamics simulations we proposed that toxic intermediates of different amyloid proteins adopt a common, nonstandard secondary structure, called α-sheet. Here we report the experimental characterization of peptides designed to be complementary to the α-sheet conformation observed in the simulations. We demonstrate inhibition of aggregation in two different amyloid systems, β-amyloid peptide (Aβ) and transthyretin, by these designed α-sheet peptides. When immobilized the α-sheet designs preferentially bind species from solutions enriched in the toxic conformer compared with non-aggregated, nontoxic species or mature fibrils. The designs display characteristic spectroscopic signatures distinguishing them from conventional secondary structures, supporting α-sheet as a structure involved in the toxic oligomer stage of amyloid formation and paving the way for novel therapeutics and diagnostics.DOI:
http://dx.doi.org/10.7554/eLife.01681.001
There is now substantial evidence that soluble oligomers are primary toxic agents in amyloid diseases. Development of an antibody recognizing the toxic soluble oligomeric forms of different and unrelated amyloid species suggests a common conformational intermediate during amyloidogenesis. We previously observed common occurrence of a novel secondary structure element, which we call α-sheet, in molecular dynamics simulations of various amyloidogenic proteins, and we hypothesized that the toxic conformer is comprised of α-sheet structure. As such, α-sheet may represent a conformational signature of the misfolded intermediates of amyloidogenesis and a potential unique binding target for peptide inhibitors. Recently, we reported the design and characterization of a novel hairpin peptide (α1 or AP90) that adopts stable α-sheet structure and inhibits the aggregation of the β-Amyloid Peptide Aβ42, and transthyretin. AP90 is a 23-residue hairpin peptide featuring alternating D- and L- amino acids with favorable conformational propensities for α-sheet formation, and a designed turn. For this study, we reverse engineered AP90 to identify which of its design features is most responsible for conferring α-sheet stability and inhibitory activity. We present experimental characterization (CD and FTIR) of 7 peptides designed to accomplish this. In addition, we measured their ability to inhibit aggregation in three unrelated amyloid species: Aβ42, transthyretin, and human islet amylin polypeptide. We found that a hairpin peptide featuring alternating L- and D-amino acids, independent of sequence, is sufficient for conferring α-sheet structure and inhibition of aggregation. Additionally, we show a correlation between α-sheet structural stability and inhibitory activity.
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