The AICD/Fe65-PTB2 complex. (A) Overall structure of the complex in a view on the binding cleft with the AICD shown in blue and Fe65-PTB2 shown in yellow to red. The phosphorylation-amenable residues of the AICD are either on the side (T⁶⁶⁸ and Y⁶⁸⁷) or central (Y⁶⁸²) to the interface. (B) Numbered primary sequences and labelled secondary structures of the peptides in the same colour code used in (A). Residues of the cloned constructs (AICD-C32 and Fe65-PTB2) are shown in bold and underlined if included in the model. The crucial complex-regulating residue T⁶⁶⁸ is highlighted in red, the N⁶⁸⁴PTY consensus PTB-binding motif in orange and Fe65 residues involved in the interface in blue. (C) Electrostatic surface potential of Fe65-PTB2 in the same view as in (A), highlighting the general hydrophobicity of the interaction and showing the charge clamp for the helix dipole of AICD helix αN (indicated by arrows). (D) Surface of Fe65 showing the conservation (green) of helix α3 and of the binding sites for both AICD helices. The central part of the interface corresponding to strand β5 and the Y⁶⁸⁷ binding pocket is less conserved. AICD, amyloid precursor protein intracellular domain; PTB2, C-terminal phosphotyrosine-binding domain.

The AICD/Fe65-PTB2 interface. (A) The interaction of AICD helix αC including the N⁶⁸⁴PTY sequence with Fe65-PTB2. (B) The central interface including the antiparallel β sheet in trans formed by the AICD Y⁶⁸²E sequence and Fe65-PTB2 strand β5. The hydrophobic binding cavity for Y⁶⁸² created by helix α3 and the hinge glycine G⁶⁸¹ (with encircled peptide bond) linking helix αN and the β-strand of the AICD are highlighted. The final 2mFo-DFc map at a 1.5 σ level is given for G⁶⁸¹ and Y⁶⁸². (C) The complex-regulating interaction of the T⁶⁶⁸PEE capping box and helix αN of the AICD with Fe65-PTB2. T⁶⁶⁸ points inside the helix, P⁶⁶⁹ in trans configuration, and the glutamates are well separated and stabilize the whole arrangement. H⁶⁷³ is shown in its alternative interactions, and L⁶⁷⁴ and M⁶⁷⁷ are accommodated in hydrophobic cavities separated by V⁶¹⁴. Residues E⁶⁴⁸ and R⁶¹⁶ clamping the helix dipole as shown in Figure 1C are given. AICD, amyloid precursor protein intracellular domain; PTB2, C-terminal phosphotyrosine-binding domain.

‘Molecular switch' model of APP/Fe65 complex regulation. (A) Scheme for complex regulation by the phosphorylation of T⁶⁶⁸. The two-helix capping sequences are labelled by their phosphorylation-amenable residues (T for T⁶⁶⁸PEE and Y for N⁶⁸⁴PTY). The hinge glycine G⁶⁸¹ is indicated by an encircled letter G. The destruction of the helix cap T by T⁶⁶⁸ phosphorylation (pT) is shown by the transition from a square to a circle. The destabilization of helix αN is shown by the transition of the box to a simple line. (B) Simplified sketch of the structural rearrangements in the T⁶⁶⁸PEE helix capping box. The middle panel corresponds to the crystal structure in the unphosphorylated state (edges correspond to the stable cap). The structure of the T668E mutant (left, same for T668A) shows a destabilization of the helix cap (as indicated by round corners and dashed lines). Phosphorylation of T⁶⁶⁸ (right panels) leads to a partitioning in trans and cis conformers of P⁶⁶⁹ (Ramelot & Nicholson, 2001) and results in a decrease in Fe65-PTB2 binding affinity. In the trans conformation, Fe65-PTB2 binding is reduced owing to electrostatic repulsion (indicated by dashed lines for Fe65). In the cis conformation the helix cap is destroyed (circle and simple line), resulting in a low binding affinity (dotted lines for Fe65). APP, amyloid precursor protein.