Letter pubs.acs.org/JPCL
Specific Binding of Cholesterol to the Amyloid Precursor Protein: Structure of the Complex and Driving Forces Characterized in Molecular Detail Łukasz Nierzwicki and Jacek Czub* Department of Physical Chemistry, Gdansk University of Technology, Narutowicza St. 11/12, 80-233 Gdansk, Poland S Supporting Information *
ABSTRACT: C99 is the C-terminal membrane-bound fragment of the amyloid precursor protein that is cleaved by γ-secretase to release Aβ peptides, the hallmark of Alzheimer’s disease (AD). Specific interactions of C99 with cholesterol have been proposed to underlie the recognized role of cholesterol in promoting amyloidogenesis. By using molecular dynamics simulations, we studied cholesterol binding to C99 in a lipid bilayer. We determined the freeenergy profile of binding and analyzed the structure of C99/cholesterol complexes in two lowenergy binding modes. We also examined the complexation driving forces and found, unexpectedly, that the interactions between the GxxxG dimerization motif and the cholesterol ring system are not sufficient for binding and that further stabilization mediated by the C99 Nterminal domain is essential. Taken together, our results strongly support the view that C99 specifically binds cholesterol in the cell membrane; the detailed information on the structure and energetics of the complex may assist in the design of new anti-AD drugs.
found to cocluster β- and γ-secretases with their substrates, APP and C99, within lipid rafts.25,26 Recently, it has been reported that APP and C99 specifically bind cholesterol in neuronal membranes and that this interaction appears to favor the amyloidogenic pathway.27,28 Using NMR-controlled titration experiments, Barrett et al. found that in dihexanoylphosphatidylcholine/dimyristoylphosphatidylcholine (DHPC/DMPC) bicelles C99 and cholesterol form 1:1 binary complexes with a dissociation constant of ∼5 mol % (of cholesterol).29 Furthermore, they used Ala-scanning mutagenesis along with the NMR-derived structure of C99 in lysomyristoylphosphatidylglycerol micelles to examine the structural properties of C99−cholesterol complexes.29 In particular, they proposed that the cholesterol binding site of C99 is formed by residues from the extracellular N-terminal helix and the interhelical N-loop and, most importantly, by the tandem GxxxG motif of the N-terminal portion of the transmembrane domain.29 On the basis of their findings and previous data, Barrett et al. further proposed that binding of cholesterol may promote partitioning of APP/C99 into lipid rafts or may facilitate substrate recognition and processing by β-secretase.29,30 It has also been suggested that cholesterol binding competes with C99 homodimerization,30 which, in turn, may affect the flexibility of the transmembrane domain and consequently modulate the accessibility of the cleavage site to γ-secretase,31 as predicted by recent molecular dynamics simulations.32,33
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lzheimer’s disease (AD) is the most common neurodegenerative disorder, characterized clinically as a progressive dementia and affecting >10% of the population over the age of 65 years.1,2 It is widely thought that AD is caused by the accumulation and deposition of the β-amyloid peptides (Aβ) in extracellular plaques, eventually leading to synaptic failure and neuronal loss.2−5 Soluble Aβ oligomers have also been found to contribute to the cognitive dysfunction in AD, which may be due to the specific binding of the oligomers to the receptors essential for synaptic plasticity.6−8 Aβ is the product of sequential proteolytic cleavage of the amyloid precursor protein (APP).2,3 This amyloidogenic pathway is initiated by β-secretase cleavage of APP generating the membrane-bound C99 fragment (known also as β-CTF), which is subsequently cleaved by γ-secretase to release Aβ peptides.9 In an alternative nonamyloidogenic pathway, αsecretase cleavage of APP within the Aβ sequence prevents the formation of neurotoxic Aβ by γ-secretase.10,11 It is well known that elevated levels of cholesterol in neuronal membranes lead to increased production of Aβ,12,13 yet the molecular mechanism of this enhancement remains unclear.13,14 It has been suggested that the observed proamyloidogenic effect is linked to the role of cholesterol in the formation of liquid-ordered membrane microdomains, termed lipid rafts.12,14 Indeed, within cholesterol-rich lipid rafts APP seems to be processed mostly by the β-secretase-mediated amyloidogenic pathway, whereas in nonraft membrane fractions the processing of APP is predominantly nonamyloidogenic.13,15−17 This appears to be due to the association of β- and γsecretase with lipid rafts,18−21 which also serve as an appropriate microenvironment for the proteolytic activity of the enzymes.15,22−24 Furthermore, exposure to cholesterol was © XXXX American Chemical Society
Received: January 28, 2015 Accepted: February 11, 2015
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DOI: 10.1021/acs.jpclett.5b00197 J. Phys. Chem. Lett. 2015, 6, 784−790
Letter
The Journal of Physical Chemistry Letters Indeed, the GxxxG motif that is presumably involved in the interaction with cholesterol is also known to promote association of α-helices in lipid membranes and therefore plays an essential role in dimerization/oligomerization and folding of numerous transmembrane proteins.34,35 By forming a flat surface on one side of the helix, two (or more) glycine residues of the dimerization motif allow tight packing and drive helix−helix association. In particular, it has been demonstrated by NMR study36 and confirmed by molecular dynamics37,38 that the tandem GxxxG motif is involved in homodimerization of C99. Thus, the previous hypotheses suggest a mechanism by which cholesterol accelerates amyloidogenesis and also provides a potential framework for the rational design of novel anti-AD drugs.29 The structure and energetics of the complexes formed by APP/C99 with cholesterol have to be accurately described to develop and refine this framework. Here we use all-atom molecular dynamics (MD) to study the specific binding of cholesterol to the C-terminal domain of APP (C99) in a 1:1 binary complex, as identified by the previous NMR experiments.29 We determine the free-energy profile governing the association of C99 with cholesterol in a lipid bilayer and characterize the structure of the resulting complex. We further analyze the binding free energy in terms of individual enthalpic contributions to provide a detailed description of the association driving forces and the binding mechanism. To test our predictions, we also show that the E693A mutation abolishes binding of cholesterol in accordance with the experimental data.29 The simulation model contained a C99 fragment (Val683− Tyr728) embedded in a liquid-ordered (lo) dimyristoylphosphatidylcholine (DMPC) bilayer with 40 mol % cholesterol. MD simulations were carried out using NAMD39 and CHARMM36 force field.40 For a full description of the simulation protocol, see the Supporting Information. To examine the process of cholesterol binding to C99, we used umbrella sampling41 to calculate the free-energy profile for the reaction coordinate defined as the separation distance between the centers of mass of the N-terminal portion of the C99 transmembrane helix (Gly700−Gly708 segment) and of a selected cholesterol molecule, projected on the bilayer plane (Supporting Information Figure S1). The resulting free-energy profile for the binding of cholesterol to C99 (Figure 1A) shows a clear minimum indicating that the bound complex configurations (xy distance 0.4) are H bonds with the backbone amino and carbonyl groups of Phe691 and Val689, respectively. Phe691 and Val689 are anchored to the membrane by their hydrophobic side chains (Figure 1B) and participate in the formation of the loosely bound complex (at 8.5 and 11 Å, respectively), and, to a lesser extent, of the tightly bound one (
