The Amyloid Precursor Protein C-Terminal Fragment C100 Occurs in Monomeric and Dimeric Stable Conformations and Binds γ-Secretase Modulators

The amyloid-beta (A beta) peptide is contained within the C-terminal fragment (beta-CTF) of the amyloid precursor protein (APP) and is intimately linked to Alzheimer's disease. In vivo, A beta is generated by sequential cleavage of beta-CTF within the gamma-secretase module. To investigate gamma-secretase function, in vitro assays are in widespread use which require a recombinant beta-CTF substrate expressed in bacteria and purified from inclusion bodies, termed C100. So far, little is known about the conformation of C100 under different conditions of purification and refolding. Since C100 dimerization influences the efficiency and specificity of gamma-secretase cleavage, it is also of great interest to determine the secondary structure and the oligomeric state of the synthetic substrate as well as the binding properties of small molecules named gamma-secretase modulators (GSMs) which we could previously show to modulate APP transmembrane sequence interactions [Richter et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 14597-14602]. Here, we use circular dichroism and continuous-wave electron spin resonance measurements to show that C100 purified in a buffer containing SDS at micelle-forming concentrations adopts a highly stable alpha-helical conformation, in which it shows little tendency to aggregate or to form higher oligomers than dimers. By surface plasmon resonance analysis and molecular modeling we show that the GSM sulindac sulfide binds to C100 and has a preference for C100 dimers.