Aggregation and Amyloidogenicity of the Nuclear Coactivator Binding Domain of CREB‐Binding Protein

Abstract

The nuclear coactivator binding domain (NCBD) of transcriptional co-regulator CREB-binding protein (CBP) is an example of conformationallym alleable proteins that can bind to structurally unrelated protein targets and adopt distinct folds in the respective protein complexes. Here, we show that the folding landscape of NCBD contains an alternative pathwayt hat resultsi np rotein aggregation and selfassembly into amyloid fibers. The initial steps of such protein misfolding are driven by intermolecular interactions of its N-terminal a-helix bringing multiple NCBD molecules into contact. These oligomers then undergo slow but progressive interconversion into b-sheet-containing aggregates.T o reveal the concealed aggregation potentialo fN CBD we used ac hemically synthesized mirror-image d-NCBD form. The addition of d-NCBD promoted self-assembly into amyloid precipitates presumably due to formation of thermodynamically more stable racemic b-sheet structures. The unexpecteda ggregation of NCBD needs to be taken into considerationg iven the multitude of protein-protein interactions and resulting biological functions mediated by CBP.

A crystal-structural study of Pauling–Corey rippled sheets

“…24 Our understanding of the interplay of thermodynamics and kinetics that underlie the formation of pleated vs rippled sheets remains extremely limited. Experiments performed in the laboratories of Schneider, 3,4 Nilsson, 25,26 Raskatov, 7,8 and Torbeev, 27 showed that mirror-image peptide strands may assemble into rippled sheets, but there is also evidence that some sequences may favor homochiral association. 28 The structural insights available for the MAX1/DMAX systems, 3 a short Amyloid-β (Aβ) segment, 25 and, most recently, racemic full-length Aβ40 29 were obtained from theoretical calculations constrained by a fairly limited number of experimental data.…”

The rippled β-sheet layer configuration—a novel supramolecular architecture based on predictions by Pauling and Corey

“…The rippled b-sheet layer configuration-a novel supramolecular architecture based on predictions by Pauling and Corey † Introduction Tailored proteins and peptides hold immense potential for materials development and biomedical application. Chiral (i.e., D-amino acid) substitutions may be employed to generate selfassembling peptide architectures with unique properties, [1][2][3][4][5][6] bioactive compounds with distinct activities, [7][8][9][10][11][12][13] as well as systems with enhanced crystallization behavior. [14][15][16][17][18] There is great interest in developing new peptidic systems that contain Damino acid substitutions, as this would allow systematic access to a vast structural space with unique molecular properties.…”

An Enantiomeric Fragment Pair (EFP) Approach for the Study of Cellular Uptake of Intrinsically Disordered Proteins

“…To gain insights into the Aβ-PrP C interaction, we treated wildtype and PrP C -transfected HEK293 cells with the enantiomeric pairs (Figure 1C). [12] To our surprise, while the (16-30) fragment was sufficient for stereo-differentiation, we did not observe PrP C -dependent uptake for (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). Amino acids (1-16) did not show stereo-differentiation nor PrP C dependence.…”

“…For example, Garcia et al synthesized the nuclear coactivator binding domain (NCBD) in both L-and D-forms and showed aggregation propensity enhancement upon L-NCBD and D-NCBD mixing. [25] This racemic mixture generated β-sheet structures structurally different from the enantiopure NCBDs, which under physiologically relevant neutral pH evolve into helical aggregates. In this case, the racemic mixture allowed the elucidation of an alternative aggregation pathway.…”

“…[12] Interestingly, stereo-differentiation in cellular uptake arose mostly when amino acids 16 to 30 were incorporated into the peptide sequence, as shown by flow cytometry results in Figure 1C. Segments comprising amino acids (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) and (1-30) displayed comparable levels of peptide in cells as full-length Aβ40 (data normalized to Aβ40, which showed the highest level of uptake). These results show the EFP approach is a promising tool for studying regions involved in stereospecific interactions versus regions involved in achiral interactions.…”