Permissive Conformations of a Transmembrane Helix Allow Intramembrane Proteolysis by γ-Secretase.

The intramembrane protease γ-secretase activates important signaling molecules, such as Notch receptors. It is still unclear, however, how different elements within the primary structure of substrate transmembrane domains (TMDs) contribute to their cleavability. Using a newly developed yeast-based cleavage assay, we identified three crucial regions within the TMDs of the paralogs Notch1 and Notch3 by mutational and gain-of-function approaches. The AAAA or AGAV motifs within the N-terminal half of the TMDs were found to confer strong conformational flexibility to these TMD helices, as determined by mutagenesis coupled to deuterium/hydrogen exchange. Crucial amino acids within the C-terminal half may support substrate docking into the catalytic cleft of presenilin, the enzymatic subunit of γ-secretase. Further, residues close to the C-termini of the TMDs may stabilize a tripartite ß-sheet in the substrate/enzyme complex. NMR structures reveal different extents of helix bending as well as an ability to adopt widely differing conformational substates, depending on the sequence of the N-terminal half. The difference in cleavability between Notch1 and Notch3 TMDs is jointly determined by the conformational repertoires of the TMD helices and the sequences of the C-terminal half, as suggested by mutagenesis and building molecular models. In sum, cleavability of a γ-secretase substrate is enabled by different functions of cooperating TMD regions, which deepens our mechanistic understanding of substrate/non-substrate discrimination in intramembrane proteolysis.

Cooperation of N- and C-terminal substrate transmembrane domain segments in intramembrane proteolysis by γ-secretase.

Intramembrane proteases play a pivotal role in biology and medicine, but how these proteases decode cleavability of a substrate transmembrane (TM) domain remains unclear. Here, we study the role of conformational flexibility of a TM domain, as determined by deuterium/hydrogen exchange, on substrate cleavability by γ-secretase in vitro and in cellulo. By comparing hybrid TMDs based on the natural amyloid precursor protein TM domain and an artificial poly-Leu non-substrate, we find that substrate cleavage requires conformational flexibility within the N-terminal half of the TMD helix (TM-N). Robust cleavability also requires the C-terminal TM sequence (TM-C) containing substrate cleavage sites. Since flexibility of TM-C does not correlate with cleavage efficiency, the role of the TM-C may be defined mainly by its ability to form a cleavage-competent state near the active site, together with parts of presenilin, the enzymatic component of γ-secretase. In sum, cleavability of a γ-secretase substrate appears to depend on cooperating TM domain segments, which deepens our mechanistic understanding of intramembrane proteolysis.

Helical stability of the GnTV transmembrane domain impacts on SPPL3 dependent cleavage.

Signal-Peptide Peptidase Like-3 (SPPL3) is an intramembrane cleaving aspartyl protease that causes secretion of extracellular domains from type-II transmembrane proteins. Numerous Golgi-localized glycosidases and glucosyltransferases have been identified as physiological SPPL3 substrates. By SPPL3 dependent processing, glycan-transferring enzymes are deactivated inside the cell, as their active site-containing domain is cleaved and secreted. Thus, SPPL3 impacts on glycan patterns of many cellular and secreted proteins and can regulate protein glycosylation. However, the characteristics that make a substrate a favourable candidate for SPPL3-dependent cleavage remain unknown. To gain insights into substrate requirements, we investigated the function of a GxxxG motif located in the transmembrane domain of N-acetylglucosaminyltransferase V (GnTV), a well-known SPPL3 substrate. SPPL3-dependent secretion of the substrate's ectodomain was affected by mutations disrupting the GxxxG motif. Using deuterium/hydrogen exchange and NMR spectroscopy, we studied the effect of these mutations on the helix flexibility of the GnTV transmembrane domain and observed that increased flexibility facilitates SPPL3-dependent shedding and vice versa. This study provides first insights into the characteristics of SPPL3 substrates, combining molecular biology, biochemistry, and biophysical techniques and its results will provide the basis for better understanding the characteristics of SPPL3 substrates with implications for the substrates of other intramembrane proteases.

Non-canonical Shedding of TNFα by SPPL2a Is Determined by the Conformational Flexibility of Its Transmembrane Helix.

Ectodomain (EC) shedding defines the proteolytic removal of a membrane protein EC and acts as an important molecular switch in signaling and other cellular processes. Using tumor necrosis factor (TNF)α as a model substrate, we identify a non-canonical shedding activity of SPPL2a, an intramembrane cleaving aspartyl protease of the GxGD type. Proline insertions in the TNFα transmembrane (TM) helix strongly increased SPPL2a non-canonical shedding, while leucine mutations decreased this cleavage. Using biophysical and structural analysis, as well as molecular dynamic simulations, we identified a flexible region in the center of the TNFα wildtype TM domain, which plays an important role in the processing of TNFα by SPPL2a. This study combines molecular biology, biochemistry, and biophysics to provide insights into the dynamic architecture of a substrate's TM helix and its impact on non-canonical shedding. Thus, these data will provide the basis to identify further physiological substrates of non-canonical shedding in the future.

Conformationally Flexible Sites within the Transmembrane Helices of Amyloid Precursor Protein and Notch1 Receptor.

Intramembrane proteases typically cleave multiple substrates within their transmembrane domains (TMDs). Because substrate TMDs lack a consensus sequence around their scissile sites, it remains unclear how the enzyme discriminates substrates from nonsubstrates at the level of their TMDs. Here, we compare the previously well investigated TMDs of γ-secretase substrates C99 and Notch1 in terms of helix flexibility. Our results reveal that the low-stability site neigboring a functionally relevant diglycine hinge of C99 has an equivalent in the Notch1 TMD. This suggests that the tetra-alanine motif of Notch1 also functions as a hinge which may facilitate its cleavage.

The Metastable XBP1u Transmembrane Domain Defines Determinants for Intramembrane Proteolysis by Signal Peptide Peptidase.

Unspliced XBP1 mRNA encodes XBP1u, the transcriptionally inert variant of the unfolded protein response (UPR) transcription factor XBP1s. XBP1u targets its mRNA-ribosome-nascent-chain-complex to the endoplasmic reticulum (ER) to facilitate UPR activation and prevents overactivation. Yet, its membrane association is controversial. Here, we use cell-free translocation and cellular assays to define a moderately hydrophobic stretch in XBP1u that is sufficient to mediate insertion into the ER membrane. Mutagenesis of this transmembrane (TM) region reveals residues that facilitate XBP1u turnover by an ER-associated degradation route that is dependent on signal peptide peptidase (SPP). Furthermore, the impact of these mutations on TM helix dynamics was assessed by residue-specific amide exchange kinetics, evaluated by a semi-automated algorithm. Based on our results, we suggest that SPP-catalyzed intramembrane proteolysis of TM helices is not only determined by their conformational flexibility, but also by side-chain interactions near the scissile peptide bond with the enzyme's active site.

Glycine Perturbs Local and Global Conformational Flexibility of a Transmembrane Helix.

Flexible transmembrane helices frequently support the conformational transitions between different functional states of membrane proteins. While proline is well known to distort and destabilize transmembrane helices, the role of glycine is still debated. Here, we systematically investigated the effect of glycine on transmembrane helix flexibility by placing it at different sites within the otherwise uniform leucine/valine repeat sequence of the LV16 model helix. We show that amide deuterium/hydrogen exchange kinetics are increased near glycine. Molecular dynamics simulations reproduce the measured exchange kinetics and reveal, at atomic resolution, a severe packing defect at glycine that enhances local hydration. Furthermore, glycine alters H-bond occupancies and triggers a redistribution of α-helical and 310-helical H-bonds. These effects facilitate local helix bending at the glycine site and change the collective dynamics of the helix.

Signal peptide peptidase functions in ERAD to cleave the unfolded protein response regulator XBP1u.

Signal peptide peptidase (SPP) catalyzes intramembrane proteolysis of signal peptides at the endoplasmic reticulum (ER), but has also been suggested to play a role in ER-associated degradation (ERAD). Here, we show that SPP forms a complex with the ERAD factor Derlin1 and the E3 ubiquitin ligase TRC8 to cleave the unfolded protein response (UPR) regulator XBP1u. Cleavage occurs within a so far unrecognized type II transmembrane domain, which renders XBP1u as an SPP substrate through specific sequence features. Additionally, Derlin1 acts in the complex as a substrate receptor by recognizing the luminal tail of XBP1u. Remarkably, this interaction of Derlin1 with XBP1u obviates the need for ectodomain shedding prior to SPP cleavage, commonly required for intramembrane cuts. Furthermore, we show that XBP1u inhibits the UPR transcription factor XBP1s by targeting it toward proteasomal degradation. Thus, we identify an ERAD complex that controls the abundance of XBP1u and thereby tunes signaling through the UPR.

Sequence-dependent backbone dynamics of a viral fusogen transmembrane helix.

The transmembrane domains of membrane fusogenic proteins are known to contribute to lipid bilayer mixing as indicated by mutational studies and functional reconstitution of peptide mimics. Here, we demonstrate that mutations of a GxxxG motif or of Ile residues, that were previously shown to compromise the fusogenicity of the Vesicular Stomatitis virus G-protein transmembrane helix, reduce its backbone dynamics as determined by deuterium/hydrogen-exchange kinetics. Thus, the backbone dynamics of these helices may be linked to their fusogenicity which is consistent with the known over-representation of Gly and Ile in viral fusogen transmembrane helices. The transmembrane domains of membrane fusogenic proteins are known to contribute to lipid bilayer mixing. Our present results demonstrate that mutations of certain residues, that were previously shown to compromise the fusogenicity of the Vesicular Stomatitis virus G-protein transmembrane helix, reduce its backbone dynamics. Thus, the data suggest a relationship between sequence, backbone dynamics, and fusogenicity of transmembrane segments of viral fusogenic proteins.

Sequence-specific conformational flexibility of SNARE transmembrane helices probed by hydrogen/deuterium exchange.

SNARE proteins mediate fusion of intracellular eukaryotic membranes and their alpha-helical transmembrane domains are known to contribute to lipid bilayer mixing. Synthetic transmembrane domain peptides were previously shown to mimic the function of SNARE proteins in that they trigger liposome fusion in a sequence-specific fashion. Here, we performed a detailed investigation of the conformational dynamics of the transmembrane helices of the presynaptic SNAREs synaptobrevin II and syntaxin 1a. To this end, we recorded deuterium/hydrogen-exchange kinetics in isotropic solution as well as in the membrane-embedded state. In solution, the exchange kinetics of each peptide can be described by three different classes of amide deuteriums that exchange with different rate constants. These are likely to originate from exchange at different domains of the helices. Interestingly, the rate constants of each class vary with the TMD sequence. Thus, the exchange rate is position-specific and sequence-specific. Further, the rate constants correlate with the previously determined membrane fusogenicities. In membranes, exchange is retarded and a significant proportion of amide hydrogens are protected from exchange. We conclude that the conformational dynamics of SNARE TMD helices is mechanistically linked to their ability to drive lipid mixing.

De novo design of conformationally flexible transmembrane peptides driving membrane fusion.

Fusion of biological membranes is mediated by distinct integral membrane proteins, e.g., soluble N-ethylmaleimide-sensitive factor attachment protein receptors and viral fusion proteins. Previous work has indicated that the transmembrane segments (TMSs) of such integral membrane proteins play an important role in fusion. Furthermore, peptide mimics of the transmembrane part can drive the fusion of liposomes, and evidence had been obtained that fusogenicity depends on their conformational flexibility. To test this hypothesis, we present a series of unnatural TMSs that were designed de novo based on the structural properties of hydrophobic residues. We find that the fusogenicity of these peptides depends on the ratio of alpha-helix-promoting Leu and beta-sheet-promoting Val residues and is enhanced by helix-destabilizing Pro and Gly residues within their hydrophobic cores. The ability of these peptides to refold from an alpha-helical state to a beta-sheet conformation and backwards was determined under different conditions. Membrane fusogenic peptides with mixed Leu/Val sequences tend to switch more readily between different conformations than a nonfusogenic peptide with an oligo-Leu core. We propose that structural flexibility of these TMSs is a prerequisite of fusogenicity.