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      Sampling the conformational space of the catalytic subunit of human γ-secretase


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          Human γ-secretase is an intra-membrane protease that cleaves many different substrates. Aberrant cleavage of Notch is implicated in cancer, while abnormalities in cutting amyloid precursor protein lead to Alzheimer's disease. Our previous cryo-EM structure of γ-secretase revealed considerable disorder in its catalytic subunit presenilin. Here, we describe an image classification procedure that characterizes molecular plasticity at the secondary structure level, and apply this method to identify three distinct conformations in our previous sample. In one of these conformations, an additional transmembrane helix is visible that cannot be attributed to the known components of γ-secretase. In addition, we present a γ-secretase structure in complex with the dipeptidic inhibitor N-[N-(3,5-difluorophenacetyl)- L-alanyl]- S-phenylglycine t-butyl ester (DAPT). Our results reveal how conformational mobility in the second and sixth transmembrane helices of presenilin is greatly reduced upon binding of DAPT or the additional helix, and form the basis for a new model of how substrate enters the transmembrane domain.

          DOI: http://dx.doi.org/10.7554/eLife.11182.001

          eLife digest

          An enzyme called gamma-secretase cuts other proteins in cells into smaller pieces. Like most enzymes, gamma-secretase is expected to move through several different three-dimensional shapes to perform its role, and identifying these structures could help us to understand how the enzyme works.

          One of the proteins that is targeted by gamma-secretase is called amyloid precursor protein, and cutting this protein results in the formation of so-called amyloid-beta peptides. When gamma-secretase doesn't work properly, these amyloid-beta peptides can accumulate in the brain and large accumulations of these peptides have been observed in the brains of patients with Alzheimer's disease. Earlier in 2015, a group of researchers used a technique called cryo-electron microscopy (cryo-EM) to produce a three-dimensional model of gamma-secretase. This revealed that the active site of the enzyme, that is, the region that is used to cut the other proteins, is particularly flexible.

          Now, Bai et al. – including many of the researchers from the earlier work – studied this flexibility in more detail. For the experiments, gamma-secretase was exposed to an inhibitor molecule that stopped it from cutting other proteins. This meant that the structure of gamma-secretase became more rigid than normal, which made it possible to collect more detailed structural information using cryo-EM. Bai et al. also developed new methods for processing images to separate the images of individual enzyme molecules based on the different shapes they had adopted at the time. These methods make it possible to view a mixture of very similar enzyme structures that differ only in a small region of the protein (in this case the active site).

          In the future, it would be useful to repeat these imaging experiments using a range of different molecules that alter the activity of gamma-secretase. Furthermore, the new image processing methods developed by Bai et al. could be used to study flexibility in the shapes of other proteins.

          DOI: http://dx.doi.org/10.7554/eLife.11182.002

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          Most cited references 33

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          Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.

          We present a statistical model to estimate the accuracy of peptide assignments to tandem mass (MS/MS) spectra made by database search applications such as SEQUEST. Employing the expectation maximization algorithm, the analysis learns to distinguish correct from incorrect database search results, computing probabilities that peptide assignments to spectra are correct based upon database search scores and the number of tryptic termini of peptides. Using SEQUEST search results for spectra generated from a sample of known protein components, we demonstrate that the computed probabilities are accurate and have high power to discriminate between correctly and incorrectly assigned peptides. This analysis makes it possible to filter large volumes of MS/MS database search results with predictable false identification error rates and can serve as a common standard by which the results of different research groups are compared.
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            How cryo-EM is revolutionizing structural biology.

            For many years, structure determination of biological macromolecules by cryo-electron microscopy (cryo-EM) was limited to large complexes or low-resolution models. With recent advances in electron detection and image processing, the resolution by cryo-EM is now beginning to rival X-ray crystallography. A new generation of electron detectors record images with unprecedented quality, while new image-processing tools correct for sample movements and classify images according to different structural states. Combined, these advances yield density maps with sufficient detail to deduce the atomic structure for a range of specimens. Here, we review the recent advances and illustrate the exciting new opportunities that they offer to structural biology research.
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              An atomic structure of human γ-secretase.

              Dysfunction of the intramembrane protease γ-secretase is thought to cause Alzheimer's disease, with most mutations derived from Alzheimer's disease mapping to the catalytic subunit presenilin 1 (PS1). Here we report an atomic structure of human γ-secretase at 3.4 Å resolution, determined by single-particle cryo-electron microscopy. Mutations derived from Alzheimer's disease affect residues at two hotspots in PS1, each located at the centre of a distinct four transmembrane segment (TM) bundle. TM2 and, to a lesser extent, TM6 exhibit considerable flexibility, yielding a plastic active site and adaptable surrounding elements. The active site of PS1 is accessible from the convex side of the TM horseshoe, suggesting considerable conformational changes in nicastrin extracellular domain after substrate recruitment. Component protein APH-1 serves as a scaffold, anchoring the lone transmembrane helix from nicastrin and supporting the flexible conformation of PS1. Ordered phospholipids stabilize the complex inside the membrane. Our structure serves as a molecular basis for mechanistic understanding of γ-secretase function.

                Author and article information

                Role: Reviewing editor
                eLife Sciences Publications, Ltd
                01 December 2015
                : 4
                [1 ]MRC Laboratory of Molecular Biology , Cambridge, United Kingdom
                [2 ]deptMinistry of Education Key Laboratory of Protein Science, Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences , Tsinghua University , Beijing, China
                [3]Max Planck Institute of Biophysics , Germany
                [4]Max Planck Institute of Biophysics , Germany
                Author notes
                © 2015, Bai et al

                This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

                Funded by: FundRef http://dx.doi.org/10.13039/501100000265, Medical Research Council;
                Award ID: MC_UP_A025_1013
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100002855, Ministry of Science and Technology of the People's Republic of China;
                Award ID: 2014ZX09507003006
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: 31130002 and 31321062
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100000780, European Commission;
                Award ID: Marie Curie Fellowship
                Award Recipient :
                The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
                Research Article
                Biophysics and Structural Biology
                Custom metadata
                A masked cryo-EM image classification approach and the structure of an inhibitor-bound complex provide insights into the molecular flexibility of the catalytic subunit of gamma-secretase.

                Life sciences

                electron microscopy, image analysis, gamma-secretase, human


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