Open the curtains to a clearer picture of who plays footsy with whom in the γ-secretase complex. On April 27 in the Proceedings of the National Academy of Sciences, researchers led by Yigong Shi at Tsinghua University in Beijing described the highest resolution structure of the presenilin-containing apparatus to date. The structure one-upped the group’s previous work, which had yielded a slightly lower-resolution image that could not distinguish between the transmembrane (TM) regions of the four-subunit complex. The new findings illuminate interactions forged between members of the horseshoe-shaped protease, and point to flexible regions of the conglomerate that may bend and tilt to accommodate substrates such as APP.
Four protein subunits interact within and outside of the plasma membrane to form γ-secretase. Presenilin 1 (PS1) is the catalytic core. Nicastrin is proposed to help capture enzyme substrates, while PEN2 and APH1 are thought to help form and stabilize the complex. A majority of familial AD (FAD) mutations occur in PS1, and most researchers believe they cause AD by reducing the processing efficiency of the enzyme, which leads to a predominance of amyloidogenic Aβ42. Loss of PS1 function may also cause neuronal damage by limiting the processing of other substrates, such as Notch1.
Researchers have tried to resolve the complex’s structure for the past 15 years, but the intricate entanglement of the four proteins within the membrane has made it a tough nut to crack. Previous studies employed single-particle electron microscopy to generate a low-resolution image (see Lazarov et al., 2006). Together with other biochemical methods, this technique suggested that the complex could exist in different conformations depending upon substrate engagement, bound inhibitors, or FAD mutations (see Elad et al., 2014, and Li et al., 2014). A subsequent study used cryo-EM, which freezes the protein in its native conformation, to resolve the structure to 12A resolution (see Osenkowski et al., 2009). Last year, Shi’s lab used cryo-EM to go even deeper, reporting a structure at 4.5Ǻ (see Lu et al., 2014). However, while this resolved the extracellular regions of the protein well, the TM regions of the complex—where the real action takes place—were at lower resolution and the researchers could not determine which domain belonged to which subunit.
For the current study, first author Linfeng Sun and colleagues set their sights on that intramembrane landscape. To identify the TM domains belonging to PS1, the researchers expressed T4 lysozyme protein fused to the PS1 N-terminus, which juts into the cytoplasm. This would allow them to identify the first TM domain of PS1; then, given sufficient resolution, they planned to trace the rest of the protein as it snaked through the membrane. They used the mild detergent digitonin to purify the complex from HEK293 cells, and found that it processed the C99 fragment of APP in vitro, suggesting it was in an active conformation, even with T4 attached.
The researchers performed cryo-EM and made three-dimensional electron density maps to resolve the structure to 4.32Ǻ . Unlike in their previous study, the resolution was more uniform throughout the extracellular and TM domains. The basic structure agreed with what the researchers had seen before when they had used a different detergent: a horseshoe-shaped complex lying flat within the membrane. The horseshoe peeped out of the extracellular side of the membrane to bind nicastrin, an extracellular subunit. The researchers used the T4 lysozyme to identify the nine TM regions of PS1. They had already reported the crystal structure of nicastrin, which contains a single TM domain (see Oct 2014 news). Shi told Alzforum that the three transmembrane domains belonging to PEN2 were obvious due to their position within the complex and their small number. That left APH1 to account for the remaining seven TM domains.
Using tagging techniques and prior structural knowledge, researchers assigned each of the 20 TM domains in the 4.32 Ǻ γ-secretase structure to one of the four subunits in the complex. [Image courtesy of Sun et al., PNAS 2015.]
With all TM domains accounted for, the researchers could then paint a clearer picture of protein interactions. Nicastrin, an extracellular subunit save its single TM region, interacted with the extracellular portion of PEN2 on the thin end of the horseshoe, and with Aph1 on the thick end. Nicastrin’s lone TM region stacked closely against three of seven TM domains from APH1. The researchers found that while most of APH1’s TM domains were stacked roughly perpendicular to the membrane, two rogue TM domains were set askew, creating a V-shaped pocket that cradled the C-terminus of PS1.
None of PS1’s nine TM domains sat perpendicular to the membrane plane; all were tilted at various angles and loosely arranged. PS1 splits into N- and C-terminal fragments as a requisite to activation, and the catalytic domain is formed by the interface of these two fragments at TM6 and TM7. The researchers found that these two domains resided on the convex, rather than the concave, side of the horseshoe, suggesting that substrates may enter the complex laterally on the outside, rather than from the center of the horseshoe. Rounding out the horseshoe, PS1’s TM4 rubbed up against TM1 and TM3 of PEN2. This subunit has three, not two, transmembrane domains, the researchers discovered. Two of these, TM1 and TM2, only traversed half the span of the membrane from the intracellular side, whereas TM3 broke through to the extracellular side to interact with nicastrin.
In a comment to Alzforum, David Bolduc, Dennis Selkoe, and Michael Wolfe of Brigham and Women’s Hospital, Boston, wrote that the new structure revealed important new information about the secretase. “With this increased resolution and thus clearer assignment of each of the TMDs come two main surprises: the di-aspartyl active site of presenilin lies on the convex side of the horseshoe, not within the concave cleft as originally presumed; and Pen2 contains not two but three TMDs,” they wrote.
Lucia Chavez-Gutierrez and colleagues at KU Leuven in Belgium had previously reported that PS1 had multiple active conformations. How could Shi’s single structure represent this dynamism? Shi told Alzforum that one clear structure emerging from the cryo-EM study is a strong indicator that one predominant wild-type structure exists. However, he noted that some regions, such as the second and sixth TM domains of PS1, displayed weak electron density and fuzzy EM images. This indicates that these domains may assume more than one configuration, perhaps tilting or twisting to bind substrates.
How might the structure change with an FAD mutation, or in the presence of a γ-secretase inhibitor or modulator? Shi’s lab is investigating these questions. In collaboration with Sjors Scheres at Cambridge University in England. Shi’s lab is also working to resolve the structure of γ-secretase to an atomic level using cryo-EM. Shi hypothesized that such a detailed structure would reveal important functional characteristics about PS1 with relevance to AD. Bolduc, Selkoe, and Wolfe agreed: “[An atomic-level structure] would provide essential clues to the mechanism(s) by which mutations in presenilin found in FAD patients affect amyloid precursor protein processing,” they wrote. “Such information should facilitate the development of modulators of γ-secretase as potential AD therapeutics, an area that has received less attention than it should.”—Jessica Shugart
- Lazarov VK, Fraering PC, Ye W, Wolfe MS, Selkoe DJ, Li H. Electron microscopic structure of purified, active gamma-secretase reveals an aqueous intramembrane chamber and two pores. Proc Natl Acad Sci U S A. 2006 May 2;103(18):6889-94. PubMed.
- Li Y, Lu SH, Tsai CJ, Bohm C, Qamar S, Dodd RB, Meadows W, Jeon A, McLeod A, Chen F, Arimon M, Berezovska O, Hyman BT, Tomita T, Iwatsubo T, Johnson CM, Farrer LA, Schmitt-Ulms G, Fraser PE, St George-Hyslop PH. Structural interactions between inhibitor and substrate docking sites give insight into mechanisms of human PS1 complexes. Structure. 2014 Jan 7;22(1):125-35. Epub 2013 Nov 7 PubMed.
- Osenkowski P, Li H, Ye W, Li D, Aeschbach L, Fraering PC, Wolfe MS, Selkoe DJ. Cryoelectron microscopy structure of purified gamma-secretase at 12 A resolution. J Mol Biol. 2009 Jan 16;385(2):642-52. PubMed.
- Lu P, Bai XC, Ma D, Xie T, Yan C, Sun L, Yang G, Zhao Y, Zhou R, Scheres SH, Shi Y. Three-dimensional structure of human γ-secretase. Nature. 2014 Aug 14;512(7513):166-70. Epub 2014 Jun 29 PubMed.
- Wolfe MS, Selkoe DJ. γ-Secretase: a horseshoe structure brings good luck. Cell. 2014 Jul 17;158(2):247-9. PubMed.