30 April 2010. Creating inhibitors for γ-secretase has felt a bit like trying to slay an unseen beast. That’s because this fearsome protein complex—which slashes a precursor into the Aβ peptides that clog the brains of Alzheimer disease patients—has resisted years of intense effort to unravel its structure. At long last, one piece of the massive γ-secretase has succumbed. Researchers led by Volker Dötsch of Goethe University, Frankfurt, Germany, have zeroed in on the catalytic subunit, presenilin 1 (PS1), and report the structure of its C-terminal fragment in this week’s PNAS Early Edition. "It's a pretty big deal,” Michael Wolfe, Brigham and Women’s Hospital, Boston, told ARF. “This is the first detailed structure of any γ-secretase component.” Applying a complementary approach, a Japanese research team led by Taisuke Tomita and Takeshi Iwatsubo, University of Tokyo, has used biochemical experiments to tease out the function of individual transmembrane domains in PS1. Those findings were published online 23 April in the Journal of Biological Chemistry. Together, the two studies nudge the field closer to the possibility of designing γ-secretase inhibitors in a rational manner.
Previous electron microscopy studies gave tantalizing glimpses of various nooks and crannies (Lazarov et al., 2006; Ogura et al., 2006), and more recent EM work revealed the overall shape of γ-secretase to 12-angstrom resolution (Osenkowski et al., 2009)—still a far cry from the fine detail offered by crystallography. Nevertheless, these EM studies, combined with biochemical approaches, have converged on a fairly consistent picture of PS1’s N-terminal fragment. By and large, this fragment appears to have classical transmembrane topology, with six alpha-helices spanning the length of the membrane. The C-terminal fragment (CTF) of PS1 is comparatively smaller but more complex, inspiring continued debate over how many times it crosses the membrane and what those transmembrane regions look like.
To help resolve the controversy, first author Solmaz Sobhanifar and colleagues performed nuclear magnetic resonance (NMR) microscopy on PS1’s C-terminal fragment (CTF) in detergent-solubilized micelles. They describe in their PNAS paper a structural model suggesting that CTF passes through the membrane three times (see figure below). One transmembrane region (shown in green) is a half helix with the catalytic aspartate 385 poking into PS1’s water-filled cavity, consistent with the idea that γ-secretase cleaves amyloid precursor protein (APP) within an intramembrane hydrophilic pore. The second transmembrane domain (shown in yellow) resembles a classical alpha-helix. The third region does not seem to traverse the membrane so much as dip into it. This element actually consists of two short helical regions (shown in red and orange)—one parallel to the membrane surface, the other perpendicular—with a loop between them. Unexpectedly, the model revealed an additional alpha-helix (shown in blue) outside the membrane in the N-terminal region of CTF. Curiously, this new structural element contains several amino acids that are mutated in familial AD, suggesting it could play a functional role.
Spectroscopy gives first structural model of presenilin 1’s C-terminal fragment (CTF). Three transmembrane regions are shown—a half-helix (green) containing the active site aspartate, a classical alpha-helix (yellow), and a pair of perpendicular shorter helices (red and orange). In this model, CTF has an additional alpha-helix (blue) located away from the membrane. Image credit: Volker Dötsch
Though an important step toward understanding how γ-secretase acts at an atomic level, the structural data should not be taken “as gospel,” Wolfe cautioned. “Like other structural studies, this provides a model. We can now use the model to generate more specific hypotheses and test them.”
The authors themselves acknowledge that their NMR investigation of highly concentrated, SDS-solubilized CTF raises concern about the biological relevance of these structures. However, given biochemical data hinting that PS1’s cleavage activity happens inside a watery pore (Sato et al., 2006; Tolia et al., 2006), they reasoned that SDS micelles might in fact provide a more natural environment compared to solid membranes that lack other components of the hydrophilic cavity. “They expressed the protein and had to re-dissolve it, and you worry that you wouldn't get homogeneous conformers,” Wolfe told ARF. “But apparently they did, and what they saw by NMR seems largely consistent with other reports using biochemical and molecular approaches, which makes one even more confident in their results.” Furthermore, the authors note that other NMR studies of detergent-solubilized membrane proteins have yielded biologically relevant information.
Given the challenges of probing PS1 through crystallographic or NMR approaches, the Japanese researchers used clever biochemical methods to do structure-function analysis of the protein. As reported in their JBC paper, one strategy entails exchanging individual helices in PS1 with helices of unrelated transmembrane proteins, and then measuring function of the modified PS1s. The researchers have used this transmembrane domain (TMD)-swap approach to show that all six TMDs within the NTF are needed for γ-secretase activity (Watanabe et al., 2005). In the current study—which includes some data initially reported at a 2008 Keystone meeting (see ARF conference story)—first author Naoto Watanabe and colleagues apply the same technique across the entire PS1, including two transmembrane helices in the CTF.
They used the swap method to figure out which transmembrane domains contribute to substrate binding. Prior research reveals an unusual feature of PS1—namely, that initial substrate binding occurs at a site distinct from the enzyme’s active site. Substrates are believed to dock onto PS1 somewhere outside the lipid bilayer before gaining access to the active site in the middle of the membrane, where the cutting takes place.
To determine if a specific transmembrane region is required for PS1 to bind substrate, the scientists used an assay where binding was measured by how well PS1 is labeled with a photoreactive APP helical probe. In this set-up, the probe did not label the TMD2 and TMD6 swap mutants, suggesting that these two PS1 regions are involved in forming the initial substrate-binding site. Furthermore, chemical crosslinking experiments with the photoreactive APP probes revealed that TMD2 and TMD6 are found near TMD9, a region prior research has implicated in substrate binding. Wolfe noted in an email that while TMD2 and TMD6 do appear critical to formation of the initial binding site, whether each region is in fact a part of the docking site is unclear. The uncertainty stems from somewhat weak evidence for TMD2’s proximity to TMD9, he wrote. (See full comment below.)
Taken together, the two papers suggest that a dual-pronged approach may eventually provide the structural information required for rational design of inhibitors to the monstrous and multi-faceted γ-secretase. “Only through a combination of biochemical assays, as in the Watanabe et al. paper, and structural studies, as in ours, might it be possible to design inhibitors that selectively suppress the cutting of the precursor protein without affecting its other targets,” Dötsch wrote (see full comment below).—Esther Landhuis.
Sobhanifar S, Schneider B, Löhr F, Gottstein D, Ikeya T, Mlynarczyk K, Pulawski W, Ghoshdastider U, Kolinski M, Filipek S, Guntert P, Bernhard F, Dötsch V. Structural investigation of the C-terminal catalytic fragment of presenilin 1. PNAS Early Edition. April 2010. Abstract
Watanabe N, Takagi S, Tominaga A, Tomita T, Iwatsubo T. Functional analysis of the transmembrane domains of presenilin 1: Participation of transmembrane domains 2 and 6 in the formation of initial substrate-binding site of gamma-secretase. J. Biol. Chem. 23 April 2010. Abstract