Structural Interactions between Inhibitor and Substrate Docking Sites Give Insight into Mechanisms of Human PS1 Complexes

Li, Yang; Lu, Stephen Hsueh-Jeng; Tsai, Ching‐Ju; Böhm, Christopher; Qamar, Seema; Dodd, Roger B.; Meadows, William; Jeon, Amy Hye Won; McLeod, A.R.; Chen, Fusheng; Arimon, Muriel; Berezovska, Oksana; Hyman, Bradley T.; Tomita, Taisuke; Iwatsubo, Takeshi; Johnson, Christopher M.; Farrer, Lindsay A.; Schmitt‐Ulms, Gerold; Fraser, Paul E.; George-Hyslop, Peter St

doi:10.1016/j.str.2013.09.018

Cited by 57 publications

(98 citation statements)

References 49 publications

(61 reference statements)

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“…This model predicts that, in the activated conformation of nicastrin, the lid opens and is relocated away from the putative substrate-binding site, thus allowing substrate recruitment. Supporting this conjecture, nicastrin in γ-secretase was reported to undergo a marked conformational switch in response to inhibitor/substrate binding (24), resulting in the closure of the upper subdomain (likely the large lobe) onto the lower subdomain (the small lobe) and compaction of the overall conformation.…”

Section: Resultsmentioning

confidence: 94%

“…γ-Secretase is thought to consist of two subcomplexes (24,29), one containing Pen-2 and PS1-NTF (the N-terminal six TMs), bearing eight TMs, and the other comprising PS1-CTF (the C-terminal three TMs), Aph-1, and nicastrin, possessing 11 TMs. The putative assignment of the lone TM from nicastrin suggests that the subcomplexes Pen-2/PS1-NTF and PS1-CTF/Aph-1/ nicastrin may occupy the thin and thick ends, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…The limited structural information comes from low-resolution electron microscopic (EM) analysis of γ-secretase (20)(21)(22)(23)(24), an NMR structure of the C-terminal three TMs of PS1 (25), and a crystal structure of a PS homolog from archaea (12). Consequently, mechanistic understanding of γ-secretase has been slow to emerge.…”

mentioning

confidence: 99%

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Crystal structure of the γ-secretase component nicastrin

Xie

Yan

Zhou

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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γ-Secretase is an intramembrane protease responsible for the generation of amyloid-β (Aβ) peptides. Aberrant accumulation of Aβ leads to the formation of amyloid plaques in the brain of patients with Alzheimer's disease. Nicastrin is the putative substrate-recruiting component of the γ-secretase complex. No atomicresolution structure had been identified on γ-secretase or any of its four components, hindering mechanistic understanding of γ-secretase function. Here we report the crystal structure of nicastrin from Dictyostelium purpureum at 1.95-Å resolution. The extracellular domain of nicastrin contains a large lobe and a small lobe. The large lobe of nicastrin, thought to be responsible for substrate recognition, associates with the small lobe through a hydrophobic pivot at the center. The putative substrate-binding pocket is shielded from the small lobe by a lid, which blocks substrate entry. These structural features suggest a working model of nicastrin function. Analysis of nicastrin structure provides insights into the assembly and architecture of the γ-secretase complex.

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Section: Resultsmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

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Crystal structure of the γ-secretase component nicastrin

Xie

Yan

Zhou

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…This result suggests a γ-secretase component other than the catalytic presenilin is responsible for this selective cleavage function. Several EM structures of γ-secretase dating back as far as a decade have demonstrated that the ectodomain of nicastrin sits on top of the extracellular side of the γ-secretase TMDs (24,(49)(50)(51). It would therefore stand to reason that nicastrin itself might act to sterically block substrates with large ectodomains from entering the γ-secretase complex for catalysis.…”

Section: Resultsmentioning

confidence: 99%

Nicastrin functions to sterically hinder γ-secretase–substrate interactions driven by substrate transmembrane domain

Bolduc

Montagna

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

126

152

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γ-Secretase is an intramembrane-cleaving protease that processes many type-I integral membrane proteins within the lipid bilayer, an event preceded by shedding of most of the substrate's ectodomain by α-or β-secretases. The mechanism by which γ-secretase selectively recognizes and recruits ectodomain-shed substrates for catalysis remains unclear. In contrast to previous reports that substrate is actively recruited for catalysis when its remaining short ectodomain interacts with the nicastrin component of γ-secretase, we find that substrate ectodomain is entirely dispensable for cleavage. Instead, γ-secretase-substrate binding is driven by an apparent tight-binding interaction derived from substrate transmembrane domain, a mechanism in stark contrast to rhomboid-another family of intramembrane-cleaving proteases. Disruption of the nicastrin fold allows for more efficient cleavage of substrates retaining longer ectodomains, indicating that nicastrin actively excludes larger substrates through steric hindrance, thus serving as a molecular gatekeeper for substrate binding and catalysis.

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“…It seemed possible that the interaction of the substrate with the enzyme might change its oligomeric state and/or its interactions with lipids in a way that facilitated solubilization from membranes. In recent studies of other types of IPs, small substrate mimics or inhibitors induced conformational changes ranging from modest shifts of TMSs and loops seen in rhomboid GlpG crystal structures (69 -71) to widespread domain movements observed in presenilin-containing ␥-secretase complexes using cyroelectron microscopy methods (72). Our results show that the SpoIVFB-TEV-FLAG 2 E44Q⅐Pro-K (1-126)-His 6 complex is efficiently solubilized from membranes with nonionic detergents, as well as with lipid-like zwitterionic detergents ( Fig.…”

Section: Solubilization Of An Immp⅐substratementioning

confidence: 99%

Complex Formed between Intramembrane Metalloprotease SpoIVFB and Its Substrate, Pro-σK

Zhang

Halder

Kerr

et al. 2016

Journal of Biological Chemistry

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Intramembrane metalloproteases (IMMPs) are conserved from bacteria to humans and control many important signaling pathways, but little is known about how IMMPs interact with their substrates. SpoIVFB is an IMMP that cleaves Pro-K during Bacillus subtilis endospore formation. When catalytically inactive SpoIVFB was coexpressed with C-terminally truncated Pro-K (1-126) (which can be cleaved by active SpoIVFB) in Escherichia coli, the substrate dramatically improved solubilization of the enzyme from membranes with mild detergents. Both the Pro(1-20) and K (21-126) parts contributed to improving SpoIVFB solubilization from membranes, but only the K part was needed to form a stable complex with SpoIVFB in a pulldown assay. The last 10 residues of SpoIVFB were required for improved solubilization from membranes by Pro-K (1-126) and for normal interaction with the substrate. The inactive SpoIVFB⅐Pro-K (1-126)-His 6 complex was stable during affinity purification and gel filtration chromatography. Disulfide cross-linking of the purified complex indicated that it resembled the complex formed in vivo. Ion mobility-mass spectrometry analysis resulted in an observed mass consistent with a 4:2 SpoIVFB⅐Pro-K (1-126)-His 6 complex. Stepwise photobleaching of SpoIVFB fused to a fluorescent protein supported the notion that the enzyme is tetrameric during B. subtilis sporulation. The results provide the first evidence that an IMMP acts as a tetramer, give new insights into how SpoIVFB interacts with its substrate, and lay the foundation for further biochemical analysis of the enzyme⅐substrate complex and future structural studies.Many critical cellular processes are regulated by intramembrane proteolysis (1). Intramembrane proteases (IPs) 3 cleave their substrates within a transmembrane segment (TMS) or near the membrane surface. There are three classes of IPs: rhomboids, aspartyl IPs, and IMMPs (often called site-2 proteases or S2Ps). Rhomboids are serine IPs that promote animal cellular signaling, coordinate bacterial quorum sensing, regulate mitochondrial homeostasis, and control protozoan infection (2-5). Presenilin, an aspartyl IP, is the catalytic component of ␥-secretase, which is involved in the processing of the amyloid precursor protein, Notch, and many other subtrates (6, 7). Dysfunction of ␥-secretase contributes to the pathogenesis of Alzheimer disease (8) and many other diseases (7). Aspartyl IPs also include preflagellin and prepilin peptidases involved in bacterial pathogenesis (9), and signal peptide peptidases, which facilitate the clearance of signal peptides from membranes, participate in viral infection, and generate small peptides as signal molecules for immune systems (10, 11). IMMPs also play critical roles in a wide variety of biological functions. In eukaryotes, cholesterol metabolism, the unfolded protein response, and the acute-phase response are regulated by IMMPs (1, 12, 13). In bacteria, IMMPs control sporulation, envelope stress responses, mating signal production, polar morphogenesis, virulence, ...

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Structural Interactions between Inhibitor and Substrate Docking Sites Give Insight into Mechanisms of Human PS1 Complexes

Cited by 57 publications

References 49 publications

Crystal structure of the γ-secretase component nicastrin

Crystal structure of the γ-secretase component nicastrin

Nicastrin functions to sterically hinder γ-secretase–substrate interactions driven by substrate transmembrane domain

Complex Formed between Intramembrane Metalloprotease SpoIVFB and Its Substrate, Pro-σK

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