Specific Binding of Cholesterol to C99 Domain of Amyloid Precursor Protein Depends Critically on Charge State of Protein

Abstract

Recent NMR chemical shift measurements of the 99 residue C-terminal fragment of Amyloid Precursor Protein (APP-C99) in the presence of cholesterol provide evidence of binary complex formation between C99 and cholesterol in membrane mimetic environments. It has also been observed that the production of Aβ protein is enhanced under conditions of high cholesterol concentration. In this study, we investigated the impact of the charge state of C99 on the structure and stability of the C99-cholesterol complex. We observed that the binding of C99 to cholesterol depends critically on the charge state of Glu 693 (E22) and Asp 694 (D23). Evaluation of the pKa values of the Asp and Glu sidechains suggests that these residues may be predominantly neutral in existing experimental observations of a stable C99-cholesterol complex at lower pH (characteristic of the endosomal environment), while binding is destabilized near neutral pH (characteristic of the cytoplasm). These observations suggest that specific binding of cholesterol to C99 is a sensitive function of the pH encountered in vivo, with key E22 and D23 residues serving as a “pH switch” controlling C99-cholesterol binding.

Structure of APP-C991–99 and implications for role of extra-membrane domains in function and oligomerization

“…[10] C99 features a glycine zipper motif, G 29 xxxG 33 xxxG 37 , in the TMD, which is frequently observed in dimer-prone single-pass TM proteins. [11,12] It is further evidenced to be a component of putative cholesterol binding site on C99,[13,14] a finding that is important because cholesterol has been hypothesized to recruit C99 to γ-secretase. [14–16] Mutation of G 29 and G 33 in this motif reduces Aβ 42 production,[17] and is expected to reduce C99 dimerization.…”

“…[34] Residues 15 to 21 (LVFFAED of the N-Helix domain), sometimes referred to as the juxta-membrane (JM) region, is plays a role in inhibiting γ-secretase binding[36] and binding with cholesterol. [8,13,15] Furthermore, membrane insertion of residues in the JM region appears to sensitively depend on pH. [13] The N-Helix also features mutants A21G,[37] E22Q,[38] E22K,[39] E22G,[40] E22Δ,[41] and D23N,[42] all found to occur in AD patients.…”

“…[8,13,15] Furthermore, membrane insertion of residues in the JM region appears to sensitively depend on pH. [13] The N-Helix also features mutants A21G,[37] E22Q,[38] E22K,[39] E22G,[40] E22Δ,[41] and D23N,[42] all found to occur in AD patients. Within the NTD, the mutation K16N is known to make APP untenable for binding by α-secretase[43,44] and the E11K mutation was found to enhance Aβ production.…”

Structure of APP-C991-99and Implications for Role of Extra-Membrane Domains in Function and Oligomerization

“…[34] Residues 15 to 21 (LVFFAED of the N-Helix domain), sometimes referred to as the juxtamembrane (JM) region, is plays a role in inhibiting γ-secretase binding [36] and binding with cholesterol. [8,13,15] Furthermore, membrane insertion of residues in the JM region appears to sensitively depend on pH. [13] The N-Helix also features mutants A21G, [37] E22Q, [38] E22K, [39] E22G, [40] E22Δ, [41] and D23N, [42] all found to occur in AD patients.…”

Regimes of Complex Lipid Bilayer Phases Induced by Cholesterol Concentration in MD Simulation

“…However, the transition of domain overlap, Λ, to below Λ random indicates the onset of anti-registration at 42 mol% Chol (Figure 3(B)), and there is a clear signature of this transition in ) and | f | (Figure 4 There may be biological implications of the s oc phase for determination of protein structure and function, as proteins can preferentially partition to particular lipid domains. For examples, amyloid precursor processing (APP) is known to change structure due to binding cholesterol (116)(117)(118)(119) or changes in membrane thickness, (120)(121)(122) which depend on lipid domain composition and structure. APP is processed by α-or βsecretase which residue in different lipid domains.…”