By solving the crystal structure of nicastrin—one of the quartet of proteins that form γ-secretase—scientists reveal more detail than ever before about this protease complex. Yigong Shi and colleagues at Tsinghua University in Beijing zoomed in to atomic resolution to see the intricate details of nicastrin. The results offer new evidence that the subunit captures substrates that are then chopped up by the catalytic component, presenilin. With these new structural details in hand, the authors propose a mechanism for how the protease works. The results appeared September 16 in the Proceedings of the National Academy of Sciences.
Recent evidence strengthens the argument that nicastrin binds substrates for γ-secretase (see May 2012 news story and Shah et al., 2005), but that idea still stirs debate. A clearer view of nicastrin’s structure could help settle the matter. Shi’s group previously used cryo-electron microscopy to visualize human γ-secretase at 4.5 Å (see Lu et al. 2014). However, components of this enzyme complex have proven difficult to crystallize and study at atomic-level detail. Instead, the authors turned to the slime mold Dictyostelium purpureum, a single-celled eukaryote with a γ-secretase that contains four subunits homologous to the human version. This amoeba version of the enzyme can process human APP to Aβ40 or Aβ42. Using X-ray crystallography, the researchers now resolve structural details down to 1.95 Å.
First authors Tian Xie and Chuangye Yan report that nicastrin from D. purpureum has one small and one large lobe, each consisting of numerous α-helices and β-strands. At the interface, hydrophobic phenyl rings from the large lobe protrude into a “greasy” pocket formed by lipophilic side chains from the small one, like a bolt in a receiver. This hinge likely allows the two subunits to pivot relative to one another, wrote the authors. The group also discovered that a loop extending from the small lobe covers the putative substrate-binding site on the large one, much like a lid (see image above). Prior low-resolution techniques overlooked this feature.
To help elucidate the function of nicastrin, the authors searched for similar structures in the Protein Data Bank. The closest was a bacterial aminopeptidase with homologous large and small lobes. The large one contains two zinc ions essential for the catalytic activity of this enzyme. The homologous site on nicastrin lacks amino acids that can bind zinc. This likely explains why nicastrin has no protease activity of its own, the authors wrote. However, they suggest that the protein could still bind substrates, and potentially deliver them to presenilin.
In contrast to nicastrin's lobes, those in the aminopeptidase keep the “lid” away from the active site, leaving it permanently open. The authors predict that the lid in nicastrin might have a regulatory role. “Our structural analysis of nicastrin provides a tantalizing clue about its function,” they write. They speculate that in an inactive state, the lid covers the substrate-binding pocket. When the two lobes pivot around the hydrophobic link, the lid opens, allowing nicastrin to bind substrates (see image below).
Open Sesame. At left, the “lid” (red) covers the substrate-binding site (green) on nicastrin. When the large lobe rotates around the hydrophobic pivot (blue), the lid opens, allowing substrate (yellow) to bind. [Courtesy of Xie et al., PNAS.]
“This new study fuels speculation that nicastrin may indeed serve as a receptor of γ-secretase substrates,” wrote David Bolduc and Michael Wolfe, Brigham and Women’s Hospital, Boston, in an accompanying commentary published online September 29. More work will be required to prove that hypothesis, they wrote. “Gaining a detailed mechanistic understanding of how γ-secretase processes APP—and its other substrates, such as Notch receptors—is critically important for biology and medicine.” Scientists should obtain high-resolution structures of the other components of γ-secretase as well, especially the catalytic subunit presenilin, they added, which is the focus of modulators that are being tested as therapeutics (see Jul 2014 news story). More details will illuminate ways to target this enzyme for Alzheimer’s disease, they wrote.
“This nicastrin structural model clearly supports previous reports that γ-secretase accesses its substrates through nicastrin,” wrote Xulun Zhang, University of Chicago, to Alzforum in an email (see full comment below). He was particularly interested in the lid, calling it unexpected and wondering if it serves only as an on/off switch, or if it could play additional roles in recruiting substrates.—Gwyneth Dickey Zakaib
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