The first atomic-level structure of γ-secretase reveals how individual amino acids interact within the four-unit complex that churns out Aβ. Described August 17 in Nature, the 3.4 Angstrom resolution cryo-electron microscopy (cryo-EM) structure illuminated the configuration of the 20 transmembrane regions that weave in and out of the cell, and also offered hints about how the complex contorts to snip substrates. The researchers, led by Yigong Shi at Tsinghua University in Beijing and Sjors Scheres at Cambridge University in England, used the structure to map out many of the known presenilin mutations linked to familial Alzheimer’s disease, pinpointing “mutation hotspots” that face the enzyme’s core. They measured Aβ production from 10 of the mutants, and found that while some trigger an uptick in γ-secretase activity and others turn it down, they all tipped the balance toward production of Aβ42 over Aβ40.
Sam Sisodia of the University of Chicago called the structure a tour de force. “A 3.4Å resolution structure via cryo-EM is virtually unheard of; it doesn’t get much better than this,” he said. Sisodia added that while the paper allows researchers to dissect the structure of the complex like never before, further studies that examine how the complex changes shape when bound to substrates or inhibitors will be crucial to understand how it catalyzes proteolysis in the membrane.
This sub-four Angstrom feat tops the 4.32Å structure unveiled by Shi’s group this spring (see May 2015 news). The researchers also obtained the previous structure using cryo-EM, which freezes proteins in place before zooming in on them with a beam of electrons. That structure allowed the researchers to assign each of the 20 transmembrane regions in the complex to one of the secretase’s four subunits: presenilin 1 (the catalytic core of the enzyme), nicastrin, APH-1, or PEN-2.
Covered Horseshoe. The four subunits of γ-secretase make a lopsided horseshoe with a nicastrin lid (green). A previous study assigned each of the 20 transmembrane domains to a specific subunit, and the new atomic-level structure confirmed those assignments. [Courtesy of Bai et al., Nature 2015.]
With the better resolution the researchers confirmed that γ-secretase forms a lopsided horseshoe-shaped structure with thick and thin ends, lying on its side in the membrane and adorned with an extracellular lid. Except for its single transmembrane domain jutting into APH1, nicastrin was primarily extracellular and formed a cap over the horseshoe. APH1 made up the bulk of the horseshoe’s thick end, and one of presenilin’s nine transmembrane (TM) regions fit snugly into a pocket made up of two APH1 TM domains. Presenilin rounded out the horseshoe and linked up on the thin end with PEN2, containing three TMs. A key finding from Shi’s previous paper was that the two TM domains (TM6/7) that contain PS1’s catalytic aspartate residues reside on the convex, not the concave, side of the horseshoe. This suggested that substrates enter the complex not through the middle of the horseshoe, which would be intuitively satisfying, but rather through a lateral route on the outside of the structure. The new structure confirmed this configuration, and offered up new insights about the enzymatic acrobatics that could be necessary to accommodate a substrate.
To move beyond TM assignments and view γ-secretase at the atomic level, co-first authors Xiao-chen Bai at Cambridge and Guanghui Yang at Tsinghua ramped up the magnification of EM and bombarded millions of γ-secretase particles. The massive data set of more than 400,000 particles that revealed a clear structure yielded the 3.5Å map of the complex. The researchers then employed a new algorithm they developed to single out the most homogenous 160,000 particles for analysis. This allowed them to squeeze out that extra 0.1Å resolution from the data. In their prized 3.4Å structure, most of the amino acids in the transmembrane regions of the complex could be identified, except for much of TM2 and TM6 of presenilin, which were poorly resolved. The fuzzy resolution of these two TMs, which the researchers had also noted in their previous study, suggested these regions were highly flexible and could assume a variety of configurations. This flexibility could also help explain how the secretase manages to accommodate such a variety of substrates with wildly different sequences.
Activity with Help from a PAL?
The atomic structure revealed the surprising location of presenilin’s catalytic residues. Not only were they on the convex side of the horseshoe, as had been observed before, they were also more than 10Å apart: Asp257 was located in the middle of TM6 slightly toward the extracellular side, while Asp385 sat near the cytoplasmic side of TM7. This is much too far for the hydrogen bonding needed for catalysis to occur, which indicates that the complex must undergo a conformational change to bring the two residues together and snip a substrate. The authors speculated that this could occur via a proline-alanine-leucine motif that resides on TM9. The structure revealed that this motif, which was previously reported to facilitate substrate binding and activate the enzyme, lies between the two catalytic aspartates (see Wang et al., 2006; Sato et al., 2008).
“Clearly, this is the model of an inactive γ-secretase complex that must go through extensive conformational changes to bring the catalytic aspartate residues to hydrogen bonding distance,” commented Lucia Chavez-Gutierrez of KU Leuven in Belgium. “This snapshot supports the view that γ-secretase exists as an ensemble of multiple conformations and their equilibrium is functionally relevant.”
The atomic structure of nicastrin revealed no surprises, and supported current hypotheses about this subunit’s role in substrate binding. Nicastrin consists of two lobes—a large one harboring a proposed substrate binding pocket containing Glu22 and Tyr337, and a small one containing a lid that stretches out to cover this pocket (see red sequence in image below). The researchers previously proposed that this lid rotates around a pivot point centered at Phe287. Now they report that a greasy hydrophobic pocket surrounds this phenylalanine, which may keep the hinge well-oiled. They proposed that entering substrates somehow trigger the lid to pivot and lift, which then opens up the binding pocket to position the substrate for processing.
Open Sesame. Researchers propose that nicastrin (small lobe, green; large lobe, blue) binds substrates via Tyr337 and Glu333. A lid covering this binding pocket (red) may flip open to allow substrates to bind (right). [Courtesy of Bai et al., Nature 2015.]
The high-resolution structure unveiled many interactions between residues in the complex. Van der Waals contacts between hydrophobic residues made up the bulk of bonding within the membrane. In addition, two phospholipids—one wedged in between TM1/TM8 of PS1 and TM4 of APH1, and the other between APH1 and nicastrin’s lone TM—stabilize interactions between the subunits, suggest Bai and colleagues. Taisuke Tomita of the University of Tokyo wondered about the lipids’ potential role in γ-secretase function. “It is well known that composition of lipids also affects enzymatic activity,” he wrote. “It would be very interesting to test whether these lipids are essential for γ-secretase structure and activity in a similar manner to that observed in several channels and receptors.”
The researchers used the atomic structure to map out the locations of PS1 mutations linked to familial AD. Some 212 mutations affect 135 residues, of which 53 have more than one mutation. Of these most variable residues, 35 were located in TM domains and resolved clearly in the structure. The researchers found a majority of them fell within two mutational hotspots: TM2-5 and TM6-9. While the mutations were clearly scattered across different regions, most of them faced the inner core of the complex. Interestingly, this alignment of FAD mutations on specific facets of the helices had been proposed more than a decade ago (see image below and Hardy and Crook, 2001). While TM6 and 7 contain the catalytic residues of PS1 and thus could have obvious links to function, mutations in the other TMs could affect substrate recognition or positioning, the researchers speculated.
To get an idea of how different mutations affected γ-secretase function, the researchers generated 10 PS1 mutants, purified them, and mixed them with the C99 fragment of APP to monitor processing in vitro. They found that four of the mutations led to strong reductions in Aβ40 and Aβ42 production, two producing no detectable amounts of Aβ40. Three other mutations had little impact on γ-secretase activity, while three others increased cleavage activity. The researchers concluded that these variations in proteolysis refuted the idea that an overall impairment in γ-secretase function leads to AD in these mutation carriers. All of the mutations that produced detectable levels of Aβ40 and Aβ42 elevated Aβ42/40 ratios to varying degrees, but the researchers said that this did not prove those heightened ratios were the cause of AD either.
The fact that PS1 mutations have differing effects on γ-secretase activity is not a surprise, said John Hardy and Rita Guerreiro of University College London in a joint comment. Similar conclusions have been drawn from other biochemical studies (see Chavez-Gutierrez et al., 2012).
Sisodia wondered why Shi and colleagues selected these 10 particular mutations to measure γ-secretase activity, as none were among the most well-characterized FAD mutations. Shi said the researchers were measuring activity of the remaining mutations using their in vitro assay. They also plan to examine the structure of the complex when bound to substrates as well as γ-secretase modulators, to see how the enzyme changes in response to binding. This information could also help guide rational design of γ-secretase modulators, which aim to block the production of Aβ42 while sparing processing of other important substrates.—Jessica Shugart
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