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. 2012 Jun 1;336(6085):1168-71.
doi: 10.1126/science.1219988.

The Amyloid Precursor Protein Has a Flexible Transmembrane Domain and Binds Cholesterol

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Free PMC article

The Amyloid Precursor Protein Has a Flexible Transmembrane Domain and Binds Cholesterol

Paul J Barrett et al. Science. .
Free PMC article

Abstract

C99 is the transmembrane carboxyl-terminal domain of the amyloid precursor protein that is cleaved by γ-secretase to release the amyloid-β polypeptides, which are associated with Alzheimer's disease. Nuclear magnetic resonance and electron paramagnetic resonance spectroscopy show that the extracellular amino terminus of C99 includes a surface-embedded "N-helix" followed by a short "N-loop" connecting to the transmembrane domain (TMD). The TMD is a flexibly curved α helix, making it well suited for processive cleavage by γ-secretase. Titration of C99 reveals a binding site for cholesterol, providing mechanistic insight into how cholesterol promotes amyloidogenesis. Membrane-buried GXXXG motifs (G, Gly; X, any amino acid), which have an established role in oligomerization, were also shown to play a key role in cholesterol binding. The structure and cholesterol binding properties of C99 may aid in the design of Alzheimer's therapeutics.

Figures

Fig. 1
Fig. 1
Structure of C99 in lyso-myristoylphosphatidylcholine (LMPG) micelles. (A) Representative structure from a preliminary structural ensemble that illustrates the disorder of the N terminus and C loop and the interfacial location of the C helix. The 35 Å sphere inscribed on the structures of this figure represents a detergent micelle. (B) The 30 lowest-energy structures in which the disordered N terminus (residues 672 to 685) and cytosolic domain (726 to 770) are omitted. (C) Representative structure from (B) with atoms for glycine residues 700, 704, 708, and 709 highlighted in van der Waals mode.
Fig. 2
Fig. 2
EPR studies of C99 in 1:4 1-palmitoyl-2-oleoyl-phosphatidylglycerol:1-palmitoyl-2-oleoyl-phosphatidylcholine lipid vesicles. (A) Bilayer depth parameters measured for spin-labeled sites on C99 as determined from power saturation EPR measurements. Positive values indicate burial in the membrane, whereas negative values indicate exposure to water. Though the samples used for NMR structural determination contained a C-terminal purification tag (see caption to fig. S1), the samples used for measurements involving the C-terminal sites (752 to 770) did not contain this tag, such that these measurements verify that the membrane association of the C terminus is not the result of the presence of a non-native tag sequence. (B) X-band DEER time evolutions measured at 80 K for C99 that was spin-labeled at the ends of its TMD (at sites 700 and 723). Results are shown for C99 with its wild-type (WT) sequence, except for the two spin-labeled sites, and for C99 that was additionally subjected to Gly-to-Leu mutations at G708 and G709. (C) Distance distributions between the spin labels measured from the DEER data from (B). The SD associated with each average distance relates to the uncertainty of the average, not to the population distribution around the average.
Fig. 3
Fig. 3
Cholesterol binding to C99 in bicelles. (A) Cholesterol titration of U-15N-C99 in dihexanoylphosphatidylcholine-dimyristoylphosphatidylcholine (DHPC-DMPC) bicelles, as monitored by 1H,15N–transverse relaxation optimized spectroscopy NMR. Cholesterol was varied from 0 to 20 mol % (relative to total moles of lipid). ppm, parts per million. T, Thr; V, Val; I, Ile; F, Phe; M, Met; D, Asp; A, Ala; Y, Tyr. (B) Changes in amide 1H NMR chemical shifts for E693 (black), G700 (light blue), and G704 (red) in response to cholesterol titration of WT C99. Also shown are the fits of a 1:1 binding model to each data set, with resulting Kd values for complex formation indicated as well. Units of mole percent are appropriate to describe the binding of two molecules that are both associated with model membranes {mole percent = [moles cholesterol/(moles DMPC + moles cholesterol)] × 100}. (C) Changes in 1H NMR chemical shifts for WT C99 in response to the addition of cholesterol to 20 mol % concentration. Results are shown for residues at or near the cholesterol binding site. (D) Titration of the G700A mutant form of C99 with cholesterol. (Data for other C99 mutants are shown in fig. S8). (E) Results of Ala-scanning mutagenesis. Residue color indicates the impact on cholesterol binding of substituting each position in the 690 to 710 range, as assessed by NMR. Red indicates that mutation to Ala for that site eliminates binding, yellow indicates significantly attenuated binding, and green indicates that mutation results in little change in cholesterol binding affinity.

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