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

Abstract

Intramembrane cleavage of the b-amyloid precursor protein C99 substrate by g-secretase is implicated in Alzheimer's disease pathogenesis. Biophysical data have suggested that the N-terminal part of the C99 transmembrane domain (TMD) is separated from the C-terminal cleavage domain by a di-glycine hinge. Because the flexibility of this hinge might be critical for g-secretase cleavage, we mutated one of the glycine residues, G38, to a helix-stabilizing leucine and to a helix-distorting proline. Both mutants impaired g-secretase cleavage and also altered its cleavage specificity. Circular dichroism, NMR, and backbone amide hydrogen/deuterium exchange measurements as well as molecular dynamics simulations showed that the mutations distinctly altered the intrinsic structural and dynamical properties of the substrate TMD. Although helix destabilization and/or unfolding was not observed at the initial ε-cleavage sites of C99, subtle changes in hinge flexibility were identified that substantially affected helix bending and twisting motions in the entire TMD. These resulted in altered orientation of the distal cleavage domain relative to the N-terminal TMD part. Our data suggest that both enhancing and reducing local helix flexibility of the di-glycine hinge may decrease the occurrence of enzyme-substrate complex conformations required for normal catalysis and that hinge mobility can thus be conducive for productive substrate-enzyme interactions.

Non-canonical Shedding of TNFα by SPPL2a Is Determined by the Conformational Flexibility of Its Transmembrane Helix

“…Since the destabilizing function of glycine results from a packing defect due to its missing side chain ( Högel et al, 2018 ), we reasoned that the small side chain of both neighboring alanine residues might also contribute to local helix flexibility. This compares to the sequence context of the G37G38 hinge of the C99 TM helix, where the GG motif is flanked by insufficiently packing VAL residues V36, V39, V40 ( Götz et al., 2019b ; Hitzenberger et al., 2020 ). Consequently, we replaced the A 42 G 43 A 44 motif by leucine residues (TNFα AGA/LLL).…”

“…Earlier studies suggested that intramembrane proteolysis by GxGD-aspartyl proteases is affected by the conformational flexibility of a substrate's TM helix ( Fluhrer et al., 2012 ; Götz et al., 2019b ; Langosch et al., 2015 ; Langosch and Steiner, 2017 ). To elucidate whether similar determinants also influence SPPL2a-mediated non-canonical shedding of TNFα, all serines (S34, S37), glycines (G43), cysteine (C49), and histidines (H52) in the TNFα TM domain were substituted by alanine, leucine, or proline.…”

“…Similar to the G37/G38 hinge of the APP TM helix ( Götz et al., 2019b ), G43 may increase helix flexibility at the center of the TNFα TM domain. Interestingly, an even stronger helix destabilization at this position by the G43P mutation caused the most pronounced increase in non-canonical shedding.…”

“…Other than most soluble proteases, intramembrane proteases in the context of RIP typically do not recognize their substrates by consensus sequences but rather seem to sense structural properties, including the dynamics of the substrate's TM helix ( Hitzenberger et al., 2020 ; Langosch et al., 2015 ; Langosch and Steiner, 2017 ). For instance, cleavage of the C-terminal β-amyloid precursor protein (APP) fragment (C99) by presenilin is influenced by the local helical flexibility at the G37G38 hinge located in the N-terminal half of its TM domain ( Barrett et al., 2012 ; Götz et al., 2019b ; Scharnagl et al., 2014 ). It has been proposed that a flexible TM helix facilitates translocation of a substrate from an initial binding site of the enzyme toward its catalytic center ( Langosch et al., 2015 ; Langosch and Steiner, 2017 ).…”

The C99 domain of the amyloid precursor protein is a disordered membrane phase-preferring protein

“…First, both experimental and computational studies have shown that the TMD of C99 is flexible (45,(102)(103)(104)(105)(106)(107). At the same time, C99 has been shown to be adaptable to changes in bilayer thickness, wherein the Cterminus of its TMD has a fixed transmembraneend site (defined by three consecutive lysine residues) but the N-terminus of the TMD helix seems to be energetically tolerant of being either membrane-buried (in thicker bilayers) or water exposed (in thinner bilayers) (108).…”