Increased H-Bond Stability Relates to Altered \u03b5-Cleavage Efficiency and A\u03b2 Levels in the I45T Familial Alzheimer\u2019s Disease Mutant of APP

Alexander Götz,Claudia Muhle-Goll,Philipp Högel,Iro Chaitoglou,Burkhard Luy,Mara Silber,Christina Scharnagl,Dieter Langosch

doi:10.1038/s41598-019-41766-1

Abstract

Cleavage of the amyloid precursor protein’s (APP) transmembrane domain (TMD) by γ-secretase is a crucial step in the aetiology of Alzheimer’s Disease (AD). Mutations in the APP TMD alter cleavage and lead to familial forms of AD (FAD). The majority of FAD mutations shift the preference of initial cleavage from ε49 to ε48, thus raising the AD-related Aβ42/Aβ40 ratio. The I45T mutation is among the few FAD mutations that do not alter ε-site preference, while it dramatically reduces the efficiency of ε-cleavage. Here, we investigate the impact of the I45T mutation on the backbone dynamics of the substrate TMD. Amide exchange experiments and molecular dynamics simulations in solvent and a lipid bilayer reveal an increased stability of amide hydrogen bonds at the ζ- and γ-cleavage sites. Stiffening of the H-bond network is caused by an additional H-bond between the T45 side chain and the TMD backbone, which alters dynamics within the cleavage domain. In particular, the increased H-bond stability inhibits an upward movement of the ε-sites in the I45T mutant. Thus, an altered presentation of ε-sites to the active site of γ-secretase as a consequence of restricted local flexibility provides a rationale for reduced ε-cleavage efficiency of the I45T mutant.

Highlights

The intramembrane aspartyl protease γ-secretase cleaves the transmembrane domains (TMD) of ~90 bitopic type I transmembrane proteins[1,2]
Since hydrogen bond networks determine the mechanical and thermodynamic properties of proteins, their alteration in forms of AD (FAD) mutants of the amyloid precursor protein (APP) TMD has been proposed to perturb the fine-tuned interplay of the conformational dynamics of the enzyme and substrate[29], which is a key factor for substrate recognition and subsequent relaxation steps[37,38]
In order to provide a rationale for the altered γ-secretase cleavage of the C99 I45T FAD mutant, we probed the structure and flexibility of its TMD in comparison to the WT TMD

Summary

Introduction

The intramembrane aspartyl protease γ-secretase cleaves the transmembrane domains (TMD) of ~90 bitopic type I transmembrane proteins[1,2]. Subsequent cleavage of C99 by γ-secretase results in the release of the APP intracellular domain (AICD) and β-amyloid (Aβ) peptides of various lengths[5] Such step-wise cleavage starts at one of two ε-sites located at the cytosolic border of the C99 TMD between either residues L49 and V50 (ε49) or T48 and L49 (ε48). Exhaustive sampling of the conformational space allowed us to correlate the impact of the I45T mutation on the H-bond network with altered bending and twisting motions around a flexible hinge located one turn upstream of the ε-sites These motions may control the vertical position of the ε-cleavage sites within presenilin and provide a mechanistic interpretation for changed ε-cleavage efficiency but unaffected ε-site preference, in the I45T mutant

Methods

Results

Conclusion