Structural interactions between inhibitor and substrate docking sites give insight into mechanisms of human PS1 complexes - PubMed (original) (raw)

. 2014 Jan 7;22(1):125-35.

doi: 10.1016/j.str.2013.09.018. Epub 2013 Nov 7.

Stephen Hsueh-Jeng Lu 1, Ching-Ju Tsai 1, Christopher Bohm 2, Seema Qamar 1, Roger B Dodd 1, William Meadows 1, Amy Jeon 2, Adam McLeod 2, Fusheng Chen 2, Muriel Arimon 3, Oksana Berezovska 3, Bradley T Hyman 3, Taisuke Tomita 4, Takeshi Iwatsubo 4, Christopher M Johnson 5, Lindsay A Farrer 6, Gerold Schmitt-Ulms 2, Paul E Fraser 2, Peter H St George-Hyslop 7

Affiliations

Structural interactions between inhibitor and substrate docking sites give insight into mechanisms of human PS1 complexes

Yi Li et al. Structure. 2014.

Abstract

Presenilin-mediated endoproteolysis of transmembrane proteins plays a key role in physiological signaling and in the pathogenesis of Alzheimer disease and some cancers. Numerous inhibitors have been found via library screens, but their structural mechanisms remain unknown. We used several biophysical techniques to investigate the structure of human presenilin complexes and the effects of peptidomimetic γ-secretase inhibitors. The complexes are bilobed. The head contains nicastrin ectodomain. The membrane-embedded base has a central channel and a lateral cleft, which may represent the initial substrate docking site. Inhibitor binding induces widespread structural changes, including rotation of the head and closure of the lateral cleft. These changes block substrate access to the catalytic pocket and inhibit the enzyme. Intriguingly, peptide substrate docking has reciprocal effects on the inhibitor binding site. Similar reciprocal shifts may underlie the mechanisms of other inhibitors and of the "lateral gate" through which substrates access to the catalytic site.

Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

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Figures

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Graphical abstract

Figure 1

Figure 1

Compound E Binding Induces Conformational Changes in PS1 Complexes that Prevent Detergent-Mediated Dissociation of PS1 Complexes into Hemi-Complexes (A) Cartoon depicting the hemi-complexes. Substrates have been previously shown to bind to both PS1-NTF and PS1-CTF. (B) In 0.1% DDM, nicastrin coimmunoprecipitates all complex components: PS1-NTF, PS1-CTF, aph1, and pen2. With increasing detergent concentration, nicastrin coimmunoprecipitates only aph1 and PS1-CTF. (C) Incubation of complexes with compound E stabilizes complexes across a range of detergent concentrations. (D) The compound E-mediated stabilization of the PS1 complexes is dose dependent.

Figure 2

Figure 2

FLIM-FRET Analysis of Purified, Mature, Catalytically Active, GFP- and RFP-Tagged PS1 Complexes Confirms that Compound E Binding Causes Conformational Changes (A) FRET efficiencies of GFP/RFP-tagged PS1 complexes are improved after incubation in 10 μM compound E (DMSO control: 15.46 ± 0.69% mean ± SEM; 10 μM compound E: 18.02 ± 0.95%, n = three independent experiments). ∗p ≤ 0.05. (B) Representative fluorescence decay curves for donor GFP fluorescence under each experimental condition. G-PS1 and G-PS1-R decay curves and fluorescence lifetimes were derived from fitting the experimentally observed photon counts by single exponential decay and double exponential decay models, respectively. The relationship of the FRET donor-receiver pair on the two hemi-complexes is displayed in Figure S1A. A western blot demonstrating complete endoproteolysis and maturation of the GFP/RFP-tagged PS1 complexes is displayed in Figure S1B.

Figure 3

Figure 3

Noncleavable Peptidic D-Helical Substrate Mimic and Compound E Have Reciprocal Allosteric Effects on Each Other’s Binding to PS1 Complexes and Compound E Inhibits Binding of the D-Amino-Acid Helical Substrate to the Initial Substrate Docking Site of PS1 Complexes (A) A representative blot showing progressive inhibition of D-helical photoprobe binding to PS1-NTF in the presence of increasing concentrations of compound E. (B) Quantitative results of four independent experiments expressed as percentage of DMSO control. Error bars are SEM.

Figure 4

Figure 4

Noncleavable Peptidic D-Helical Substrate Mimic and Compound E Have Reciprocal Allosteric Effects on Each Other’s Binding to PS1 Complexes Preincubation of the PS1 complexes with the D-helical substrate mimic enhanced binding of compound E to the PS1 complex.

Figure 5

Figure 5

Raw Particles and 3D Model Validation of Human PS1 Complex (A) CCD image of native PS1 complexes. Representative particle shapes are highlighted by white boxes; boxes 1–3, bilobed shapes; boxes 4–8, round or oval shapes; boxes 6–8 show suggestive central cavities. Scale bar, 20 nm. (B) Classums of native PS1 particles compared with 2D projections of the final model for native PS1 complexes. The classums and corresponding 2D projections are highly similar in size, shape, and internal density distribution. Bilobed, oval, and round shapes are seen that are similar to the raw particle images. Central cavities in the base domain are apparent in most classum/2D-projection pairs. Scale bar, 100 Å. Supplemental information is available, including a detailed flowchart of the complex purification algorithm (Figure S2), silver-stained SDS-PAGE gel showing the presence of all complex components and blue native PAGE showing their monodispersity and catalytic activity that can be inhibited by compound E (Figure S3), mass analysis of the complex using size exclusion chromatography with SEC-MALS (Figure S4), detailed flowchart of the model-building algorithm (Figure S5), and additional classum images (Figure S6).

Figure 6

Figure 6

Resolution of the Final Maps as Evaluated by gsFSC Method The 0.143 threshold resolution of both the native and compound E-bound PS1 map was 17.4 Å resolution.

Figure 7

Figure 7

Top-Down Vertical, Lateral, and Cross-Sectional Views of 3D Reconstructions of PS1 Complexes Reveal that Both Native PS1 Complexes and Compound E-Bound Complexes Have an Irregular Bilobed Shape (A) The native PS1 complex contains a head domain and a base domain. The base domain has a lateral cleft and central cavity/channel, which appears to open onto the upper/extracellular surface and also onto the lower surface via a smaller pore. The shaded lipid bilayer represents the boundaries of a putative membrane. (B) 3D reconstructions of compound E-bound PS1 complexes reveal a similar structure, with the rotation and tilting of the head. Density shifts on the external surface of the base result in closure of the lateral cleft and of the lower pore of central channel. (C) Corresponding vertical and lateral views of the difference map, which was calculated using the UCSF Chimera package. Blue mesh is the native PS1 complex, and the pink mesh is compound E-bound PS1. Positive density is represented in green. Negative density is displayed in red. A detailed flowchart of the model-building algorithm is available in Figure S5. A rotating animated video of the native complex built in chimera (Movie S1) and an animated video comparing the native and compound E complexes (Movie S2) are available in online supplemental data files.

Figure 8

Figure 8

Antibodies to the Ectodomain of Nicastrin Label the Head Domain, Indicating that the Head Domain Is Lumenal/Extracellular Representative class average images of native and anti-nicastrin antibody-labeled PS1 complexes reveal that anti-nicastrin antibody-labeled complexes have an increased density of the head domain. The box width is 261.12 Å.

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