Several single-span membrane proteins are cleaved within their transmembrane domains (TMDs) by intramembrane-cleaving proteases, although the structure of the active site executing intramembrane cleavage remains unknown. Here we use the substituted cysteine accessibility method to examine the structure of presenilin-1, a catalytic subunit of gamma-secretase, involved in amyloid beta protein generation in Alzheimer's disease and Notch signaling. We show that TMD6 and TMD7 of presenilin-1 contribute to the formation of a hydrophilic pore within the membrane. Residues at the luminal portion of TMD6 are predicted to form a subsite for substrate or inhibitor binding on the alpha-helix facing a hydrophilic milieu, whereas those around the GxGD catalytic motif within TMD7 are highly water accessible, suggesting formation of a hydrophilic structure within the pore. Collectively, our data suggest that the active site of gamma-secretase resides in a catalytic pore filled with water within the lipid bilayer and is tapered around the catalytic aspartates.
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"However, other substrates, including Notch, N-cadherin and ErbB4, link csecretase activities to development, cancer and immunity (De Strooper and Annaert, 2010; Selkoe and Wolfe, 2007). Presenilin 1 (PS1), nicastrin (NCT), presenilin enhancer 2 (PEN-2) and anterior pharynx defective 1A (APH-1A) assemble in a tetrameric complex (Fig. 1A) (De Strooper, 2003) and PS1 autoproteolysis results in an active pentameric c-secretase (Thinakaran et al., 1996), in which the catalytic center is structured at the interface between the N-terminal and the Cterminal fragments of PS1 (NTF and CTF respectively) (Esler et al., 2000; Li et al., 2013; Li et al., 2000; Wolfe et al., 1999) and connected to the intracellular aqueous environment (Sato et al., 2006; Tolia et al., 2006). NCT, a type 1 integral membrane glycoprotein, plays a crucial role in complex maturation and stabilization (Gutiérrez et al., 2008) and might be involved in substrate binding (Shah et al., 2005; Zhang et al., 2012), although this is debated (Chávez-Gutiérrez et al., 2008). "
[Show abstract][Hide abstract]ABSTRACT: The structure and function of the γ-secretase proteases are of vast interest because of their critical roles in cellular and disease processes. We established a novel purification protocol for γ-secretase complex that involves a conformation and complex-specific nanobody, yielding highly pure and active enzyme. Using single particle electron microscopy, we analyzed the γ-secretase structure and its conformational variability. Under steady state conditions the complex adopts three major conformations, which are different in overall compactness and relative position of the nicastrin ectodomain. Occupancy of the active or substrate binding sites by inhibitors differentially stabilize sub-populations of particles with compact conformations, whereas a Familial Alzheimer Disease-linked mutation results in enrichment of extended-conformation complexes with increased flexibility. Our study presents the γ-secretase complex as a dynamic population of inter-converting conformations, involving rearrangements at the nanometer scale and high level of structural interdependence between subunits. The fact that protease inhibition or clinical mutations, which affect Aβ generation, enrich for particular subpopulations of conformers indicates the functional relevance of the observed dynamic changes, which are likely instrumental for highly allosteric behavior of the enzyme.
Full-text · Article · Dec 2014 · Journal of Cell Science
"Eight kDa N-terminal fragment of PS1 NTF generated by thrombin cleavage of PS1-Th1 after Fen-B crosslinking was specifically precipitated in a similar fashion to that by phenylpiperidine-type photoprobes, suggesting that Fen-B also targets to the most N-terminal region of PS1, including TMD1 (Figure
2B and C). We showed that the cytosolic side of TMD1 participates in the catalytic hydrophilic pore
[7,11]. To analyze the relationship between the fenofibrate binding site and the catalytic site within TMD1, we employed the cross-competition analysis in Fen-B labeling using different classes of compounds. "
[Show abstract][Hide abstract]ABSTRACT: Amyloid-beta peptide ending at 42nd residue (Abeta42) is believed as a pathogenic peptide for Alzheimer disease. Although gamma-secretase is a responsible protease to generate Abeta through a processive cleavage, the proteolytic mechanism of gamma-secretase at molecular level is poorly understood.
We found that the transmembrane domain (TMD) 1 of presenilin (PS) 1, a catalytic subunit for the gamma-secretase, as a key modulatory domain for Abeta42 production. Abeta42-lowering and -raising gamma-secretase modulators (GSMs) directly targeted TMD1 of PS1 and affected its structure. A point mutation in TMD1 caused an aberrant secretion of longer Abeta species including Abeta45 that are the precursor of Abeta42. We further found that the helical surface of TMD1 is involved in the binding of Abeta45/48 and that the binding was altered by GSMs as well as TMD1 mutation.
Binding between PS1 TMD1 and longer Abeta is critical for Abeta42 production.
Full-text · Article · Jan 2014 · Molecular Neurodegeneration
"Other structural features of the γ-secretase complex, specifically of the presenilin component, have been revealed by cysteine mutagenesis with crosslinking of chemical probes [145,146]. The generation of a cysteine-less version of presenilin that retains the ability to assemble with other complex members, to undergo endoproteolysis to NTF and CTF, and to process APP allowed incorporation of single cysteine resides at various sites near the key aspartates. "
[Show abstract][Hide abstract]ABSTRACT: Presenilin is the catalytic component of the γ-secretase complex, a membrane-embedded aspartyl protease that plays a central role in biology and in the pathogenesis of Alzheimer's disease. Upon assembly with its three protein cofactors (nicastrin, Aph-1 and Pen-2), presenilin undergoes autoproteolysis into two subunits, each of which contributes one of the catalytic aspartates to the active site. A family of presenilin homologs, including signal peptide peptidase, possess proteolytic activity without the need for other protein factors, and these simpler intramembane aspartyl proteases have given insight into the action of presenilin within the γ-secretase complex. Cellular and molecular studies support a nine-transmembrane topology for presenilins and their homologs, and small-molecule inhibitors and cysteine scanning with crosslinking have suggested certain presenilin residues and regions that contribute to substrate recognition and handling. Identification of partial complexes has also offered clues to protein-protein interactions within the γ-secretase complex. Biophysical methods have allowed 3D views of the γ-secretase complex and presenilins. Most recently, the crystal structure of a microbial presenilin homolog has confirmed a nine-transmembrane topology and intramembranous location and proximity of the two conserved and essential aspartates. The crystal structure also provides a platform for the formulation of specific hypotheses regarding substrate interaction and catalysis as well as the pathogenic mechanism of Alzheimer-causing presenilin mutations. This article is part of a Special Issue entitled: Intramembrane Proteases.
Preview · Article · Dec 2013 · Biochimica et Biophysica Acta