Stabilization and structural analysis of a membrane-associated hIAPP aggregation intermediate

Abstract

Membrane-assisted amyloid formation is implicated in human diseases, and many of the aggregating species accelerate amyloid formation and induce cell death. While structures of membrane-associated intermediates would provide tremendous insights into the pathology and aid in the design of compounds to potentially treat the diseases, it has not been feasible to overcome the challenges posed by the cell membrane. Here, we use NMR experimental constraints to solve the structure of a type-2 diabetes related human islet amyloid polypeptide intermediate stabilized in nanodiscs. ROSETTA and MD simulations resulted in a unique β-strand structure distinct from the conventional amyloid β-hairpin and revealed that the nucleating NFGAIL region remains flexible and accessible within this isolated intermediate, suggesting a mechanism by which membrane-associated aggregation may be propagated. The ability of nanodiscs to trap amyloid intermediates as demonstrated could become one of the most powerful approaches to dissect the complicated misfolding pathways of protein aggregation.

A Split‐Intein‐Based Method for the Efficient Production of Circularized Nanodiscs for Structural Studies of Membrane Proteins

“…In addition, nanodiscs are suited to the study of membrane‐attached proteins and complexes with membrane proteins in a native environment . Furthermore, the long‐term stability of nanodiscs is also essential for biochemical assays, for example, for probing amyloid binding to lipid surfaces . To improve the homogeneity of nanodiscs for structural applications, circular MSP (cMSP) proteins have been produced by using sortase A mediated protein ligation, However, this additional enzymatic reaction needs to be optimized for each batch of enzyme, and ligation yields can vary considerably.…”

Semen‐derived amyloidogenic peptides—Key players of HIV infection

“…In addition, determining the high‐resolution structure of SEVI in lipid bilayers composed of varying membrane composition, and in viral or host cell membrane composition, could provide more physiologically‐relevant structure that would valuable for the development of potential drugs. Although this is a difficult task due to the challenges posed by the lipid membrane for high‐resolution structural studies, the recently developed nanodiscs can be used to accomplish this task . In particular, the recently developed polymers that vary in charge and can form lipid nanodiscs would be valuable to trap the intermediate structures of SEVI in different membrane compositions .…”

“…Although this is a difficult task due to the challenges posed by the lipid membrane for highresolution structural studies, the recently developed nanodiscs can be used to accomplish this task. 42,43 In particular, the recently developed polymers that vary in charge and can form lipid nanodiscs would be valuable to trap the intermediate structures of SEVI in different membrane compositions. 44 In addition, the use of "styrene-free" methacrylate polymer nanodiscs would enable high-throughput screening of conditions by ThT fluorescence and CD experiments as demonstrated recently.…”

Picturing the Membrane‐assisted Choreography of Cytochrome P450 with Lipid Nanodiscs

“…[99] As a matter of fact, given the heterogeneity of the P450's metabolon in terms of substrate catalysis, we anticipate that lipid-protein interactions could also play a role in the catalytic sorting. In fact, several other biological processes are governed by specific lipid-protein interactions, including amyloid aggregation, [103] the insertion of pore-forming peptides, [104][105] and virus entry, [106] among others. [12,100] The formation of electron transfer binary and ternary complexes with CPR and cytb 5 could .…”