Competition between homodimerization and cholesterol binding to the C99 domain of the amyloid precursor protein

Abstract

The 99-residue transmembrane C-terminal domain (C99, also known as β-CTF) of the amyloid precursor protein (APP) is the product of the β-secretase cleavage of the full-length APP and is the substrate for γ-secretase cleavage. The latter cleavage releases the amyloid-β polypeptides that are closely associated with Alzheimer's disease. C99 is thought to form homodimers; however, the free energy in favor of dimerization has not previously been quantitated. It was also recently documented that cholesterol forms a 1:1 complex with monomeric C99 in bicelles. Here, the affinities for both homodimerization and cholesterol binding to C99 were measured in bilayered lipid vesicles using both electron paramagnetic resonance (EPR) and Förster resonance energy transfer (FRET) methods. Homodimerization and cholesterol binding were seen to be competitive processes that center on the transmembrane G₇₀₀XXXG₇₀₄XXXG₇₀₈ glycine-zipper motif and adjacent Gly709. On one hand, the observed Kd for cholesterol binding (Kd = 2.7 ± 0.3 mol %) is on the low end of the physiological cholesterol concentration range in mammalian cell membranes. On the other hand, the observed K(d) for homodimerization (K(d) = 0.47 ± 0.15 mol %) likely exceeds the physiological concentration range for C99. These results suggest that the 1:1 cholesterol/C99 complex will be more highly populated than C99 homodimers under most physiological conditions. These observations are of relevance for understanding the γ-secretase cleavage of C99.

Figure 1

Topology map of C99 protein. Not depicted is the hinge-like kink located in the transmembrane helix that occurs at G₇₀₈G₇₀₉⁽¹⁵⁾.

Figure 2

(A) and (B): CW EPR spectra of spin labeled C99 in lipid vesicles with different protein-to-lipid molar ratios (from 1:800 to 1:50). S697R1 (panel A) is the spin labeled S697C mutant, while L705R1 (panel B) is the spin labeled L705C mutant. Site 697 is located outside of the TMD, while 705 is located inside. The arrows in the top trace of each series of spectra indicates the peaks at 3464 (left) and 3474 (right) Gauss. (C) and (D): the EPR intensities at 3474 G and 3464 G from panels A and B, respectively, were plotted as a function of the total C99 concentration and fit by a homodimerization model (equation 8).

Figure 3

FRET spectra of IAEDANS-labeled C99 S730C (donor) and IANDB-labeled C99 S730 (acceptor) in lipid vesicles. (A) IAEDANS-labeled C99 (donor) was reconstituted into lipid vesicles with a protein-to-lipid molar ratio of 1:800 (black) with increasing amounts of IANDB-labeled C99 (acceptor). The acceptor IANDB-labeled C99 (acceptor) was increased relative to donor IAEDANS-labeled C99 over the following protein:protein molar ratios: 1:1 (red), 1:2 (blue), 1:4 (cyan), 1:6 (magenta), 1:8 (orange), and 1:16 (green), corresponding to total C99-to-lipid molar ratios of 1:400, 1:267, 1:160, 1:114, 1:89, and 1:47, respectively. (B) The intensity at 474 nm in each spectrum was plotted as a function of total C99 concentration and was fit by a model for homodimerization (equation 7).

Figure 4

CW EPR spectra of spin labeled C99 in lipid vesicles with different protein-to-lipid mol:mol ratios from 1:800 to 1:50. (A) Superimposed spectra (at 1:800, 1:400, 1:200, 1:100, and 1:50 C99:lipid) for G700R1, G704R1, G708R1 and G709R1. The overlays of 5 spectra for each mutant exhibit little variation within each series. (B) Superimposed spectra (same 5 ratios as above) of the G700L and G704L mutants containing a second mutation site (to Cys) for spin labeling (S697R1). As for panel A, the superimposed spectra demonstrate little variation within each series. (C) and (D): The EPR intensity at 3464 G was plotted as a function of the total C99 concentration and fit by a model for homodimerization.

Figure 5

CW EPR spectra of C99 in the presence of cholesterol. (A) CW EPR spectra of L705R1 C99 at a protein-to-lipid molar ratio of 1:100 in vesicles containing cholesterol ranging from 0 to 30 mol%. All spectra were normalized (with respect to their double integrations) to the same amount of spin label. (B) The EPR intensities at 3474 G (right arrow) and 3464 G (left arrow) from panel A were plotted as a function of total C99 concentration and then fit by a model describing competition between homodimerization of C99 and 1:1 complex formation between C99 and cholesterol (equation 15). Note that the determined “K” describes the superequilibrium (equation 12) and is not a true dissociation or association constant. (C). CW EPR spectra of S697R1 C99 in vesicles containing 20 mol% cholesterol at protein-to-lipid mol:mol ratios varying from 1:800 to 1:50. (D) Same as C except that the spin label is attached to L705C, which is located inside the membrane.

Figure 6

(A): FRET spectra of fluorescently labeled C99 S730C in lipid vesicles with a donor-to-acceptor ratio of 1:4 and a total C99-to-lipid mol:mol ratio of 1:100, containing different amounts of cholesterol: 0 mol% (black), 5 (red), 10 (orange), 15 (blue), 20 (purple), and 30 mol% (cyan). (B): The intensity of the spectrum at 474 nm was plotted as a function of the cholesterol mol% and then fit by a model (equation 17) describing competition between C99 homodimerization and 1:1 complex formation between monomeric C99 and cholesterol. (Note that 1 mol% cholesterol corresponds to 1 molecule of cholesterol for every 100 molecules of lipid and protein in the vesicles).

Figure 7

Competition between homodimerization and cholesterol binding to free C99 in lipid vesicles. The K_d values were determined in this work.