Increased H-Bond Stability Relates to Altered ε-Cleavage Efficiency and Aβ Levels in the I45T Familial Alzheimer’s Disease Mutant of APP

Abstract

1Cleavage of the amyloid precursor protein's (APP) transmembrane domain (TMD) by γ-2 secretase is a crucial step in the etiology of Alzheimer's Disease (AD). Mutations in the APP 3 TMD alter cleavage and lead to familial forms of AD (FAD). The majority of FAD mutations 4 shifts the preference of initial cleavage from ε49 to ε48, thus raising the AD-related Aβ42/Aβ40 5 ratio. The I45T mutation is among the few FAD mutations that do not alter ε-site preference, 6 while it dramatically reduces the efficiency of ε-cleavage. Here we investigate the impact of 7 the I45T mutation on the backbone dynamics of the substrate TMD. Amide exchange 8 experiments and molecular dynamics simulations in solvent and a lipid bilayer reveal an 9 increased stability of amide hydrogen bonds at the ζ-and γ-cleavage sites. Stiffening of the H-10 bond network is caused by an additional H-bond between the T45 side chain and the TMD 11 backbone, which alters dynamics within the cleavage domain. In particular, the increased H-12 bond stability inhibits an upward movement of the ε-sites in the I45T mutant. Thus, an altered 13 presentation of ε-sites to the active site of γ-secretase as a consequence of restricted local 14 flexibility provides a rationale for reduced ε-efficiency of the I45T FAD mutant.15 16 Introduction 1The intramembrane aspartyl protease γ-secretase cleaves the transmembrane domains (TMD) 2 of ~90 bitopic type I transmembrane proteins. 1,2 Due to its involvement in Alzheimer's disease 3 (AD), the amyloid-precursor-protein (APP) is the most extensively studied γ-secretase 4 substrate. 3,4 Prior to cleavage by γ-secretase, APP's ectodomain is shed by β-secretase, thus 5 generating the C99 fragment. Subsequent cleavage of C99 by γ-secretase results in release of 6 the APP intracellular domain (AICD) and β-amyloid (Aβ) peptides of various length. 5 Such 7 step-wise cleavage starts at one of two ε-sites located at the cytosolic border of the C99 TMD 8 between either residue L49 and V50 (ε49) or T48 and L49 (ε48). Cleavage gradually proceeds 9 towards the N-terminus, releasing fragments of three or four residues and leaving Aβ peptides 10 of different length. 6-11 Aβ40, the most abundant Aβ peptide produced from wild-type (WT) 11 C99, is generated by cleavage along the ε49-ζ46-γ43-γ40 pathway. A smaller amount of C99 is 12 processed via cleavage at ε48 and ζ45, leading to Aβ42 and Aβ38 peptides. Of these species, 13Aβ42 is the most aggregation-prone and forms neurotoxic oligomers and plaques. 12,13 The 14 accumulation of plagues consisting of such cell-toxic Aβ peptides in the brain is a central 15 hallmark of AD. 14 In familial forms of AD (FAD) an increased Aβ42/Aβ40 ratio correlates with 16 early onset and fast progression of AD and results from point mutations in C99 or presenilin, 17 the catalytic subunit of γ-secretase. 1,[15][16][17][18][19][20][21] In most mutants, alterations in Aβ42/Aβ40 ratios are 18 linked to shifting the preferential initial cleavage site from ε49 to ε48. However, switching of 19 pathways after ε-cl...

Modulating Hinge Flexibility in the APP Transmembrane Domain Alters γ-Secretase Cleavage

“…The C99 26-55 WT, C99 26-55 G38L, and C99 26-55 G38P model peptide (for sequences, see Table 1) were investigated in a fully hydrated POPC bilayer and in a mixture of 80% TFE with 20% TIP3 water (v/v). Because no experimental structures were available for the G38 mutants, we used a stochastic sampling protocol to generate a set of 78 initial start conformations (for details, see (75)).…”

“…All-atom simulations in 80% TFE and 20% TIP3 (v/v) were set up as described previously (75). Each start conformation was simulated for 200 ns using settings as described in (46).…”

“…To obtain insight into local amide H-bond strength, we next measured residue-specific amide DHX rate constants (k exp,DHX ) using ETD of our TMD peptides in the gas phase after various periods of exchange (MS-ETD-DHX) (47,72). To enhance fragmentation efficiency, we substituted the N-terminal SNK sequence of the C99 26-55 TMD by KKK (75). The rate constants shown in Fig.…”

Mechanism of Tripeptide Trimming by γ-Secretase

“…However, artificial structural constraints were included that could affect the enzyme-substrate interactions. Molecular dynamics (MD) simulations have proven useful in understanding the structural dynamics of γ-secretase, notably the enzyme-substrate interactions (13)(14)(15)(16)(17). Recently, we computationally restored the wildtype (WT) enzyme-substrate co-structure and applied all-atom simulations using the Gaussian accelerated molecular dynamics (GaMD) method to build the first dynamic model of γ-secretase activation (18).…”

γ‐Secretase cleavage of the Alzheimer risk factor TREM 2 is determined by its intrinsic structural dynamics

“…Indeed, mutations that affect transmembrane domain flexibility in the cleavage site region of APP can markedly alter γ‐secretase cleavage (Fernandez et al , ). Likewise, flexibility via a naturally occurring hinge in the TMH as in the case of APP can also be critical for γ‐secretase cleavage (Götz et al , ). Recent structural studies of γ‐secretase in complex with APP or Notch1 substrates suggest that unfolding of the cleavage region is caused by formation of a hybrid β‐strand between substrate and enzyme (Yang et al , ; Zhou et al , ).…”