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1.  Toward the structure of presenilin/γ-secretase and homologs☆ 
Biochimica et biophysica acta  2013;1828(12):2886-2897.
Presenilin is the catalytic component of the γ-secretase complex, a membrane-embedded aspartyl protease that plays a central role in biology and in the pathogenesis of Alzheimer's disease. Upon assembly with its three protein cofactors (nicastrin, Aph-1 and Pen-2), presenilin undergoes autoproteolysis into two subunits, each of which contributes one of the catalytic aspartates to the active site. A family of presenilin homologs, including signal peptide peptidase, possess proteolytic activity without the need for other protein factors, and these simpler intramembane aspartyl proteases have given insight into the action of presenilin within the γ-secretase complex. Cellular and molecular studies support a nine-transmembrane topology for presenilins and their homologs, and small-molecule inhibitors and cysteine scanning with crosslinking have suggested certain presenilin residues and regions that contribute to substrate recognition and handling. Identification of partial complexes has also offered clues to protein–protein interactions within the γ-secretase complex. Biophysical methods have allowed 3D views of the γ-secretase complex and presenilins. Most recently, the crystal structure of amicrobial presenilin homolog has confirmed a nine-transmembrane topology and intramembranous location and proximity of the two conserved and essential aspartates. The crystal structure also provides a platform for the formulation of specific hypotheses regarding substrate interaction and catalysis as well as the pathogenic mechanism of Alzheimer-causing presenilin mutations.
PMCID: PMC3801419  PMID: 24099007
Protease; Membrane; Alzheimer's disease; Signal peptide peptidase
2.  Development and Mechanism of γ-Secretase Modulators for Alzheimer Disease 
Biochemistry  2013;52(19):10.1021/bi400377p.
γ-Secretase is an aspartyl intramembranal protease composed of presenilin, Nicastrin, Aph1 and Pen2 with 19 transmembrane domains. γ-Secretase cleaves the amyloid precursor proteins (APP) to release Aβ peptides that likely play a causative role in the pathogenesis of Alzheimer disease (AD). In addition, γ-secretase cleaves Notch and other type I membrane proteins. γ-Secretase inhibitors (GSIs) have been developed and used for clinical studies. However, clinical trials have shown adverse effects of GSIs that are potentially linked with non-discriminatory inhibition of Notch signaling, overall APP processing and other substrate cleavages. Therefore, these findings call for the development of disease modifying agents that target γ-secretase activity to lower Aβ42 production without blocking the overall processing of γ-secretase substrates. γ-Secretase modulators (GSMs) originally derived from non-steroidal anti-inflammatory drugs (NSAIDs) display such characteristics and are the focus of this review. However, first generation GSMs have limited potential due to low potency and undesired neuropharmacokinetic properties. This generation of GSMs has been suggested to interact with the APP substrate, γ-secretase or both. To improve the potency and brain availability, second generation GSMs including NSAID-derived carboxylic acid and non-NSAID-derived heterocyclic chemotypes as well as natural product-derived GSMs have been developed. Animal studies of this generation of GSMs have shown encouraging preclinical profiles. Moreover, using potent GSM photoaffinity probes, multiple studies unambiguously have showed that both carboxylic acid and heterocyclic GSMs specifically target presenilin, the catalytic subunit of γ-secretase. In addition, two types of GSMs have distinct binding sites within the γ-secretase complex and exhibit different Aβ profiles. GSMs induce a conformational change of γ-secretase to achieve modulation. Various models are proposed and discussed. Despite the progress of GSM research, many outstanding issues remain to be investigated to achieve the ultimate goal of developing GSMs as effective AD therapies.
PMCID: PMC3796170  PMID: 23614767
3.  Structure, Mechanism and Inhibition of γ-Secretase and Presenilin-Like Proteases 
Biological chemistry  2010;391(8):839-847.
Presenilin is the catalytic component of γ-secretase, a complex aspartyl protease and a founding member of intramembrane-cleaving proteases. γ-Secretase is involved in the pathogenesis of Alzheimer’s disease and a top target for therapeutic intervention. However, the protease complex processes a variety of transmembrane substrates, including the Notch receptor, raising concerns about toxicity. Nevertheless, γ-secretase inhibitors and modulators have been identified that allow Notch processing and signalling to continue, and promising compounds are entering clinical trials. Molecular and biochemical studies offer a model for how this protease hydrolyzes transmembrane domains in the confines of the lipid bilayer. Progress has also been made toward structure elucidation of presenilin and the γ-secretase complex by electron microscopy as well as by studying cysteine-mutant presenilins. The signal peptide peptidase (SPP) family of proteases are distantly related to presenilins. However, the SPPs work as single polypeptides without the need for cofactors and otherwise appear to be simple model systems for presenilin in the γ-secretase complex. SPP biology, structure, and inhibition will also be discussed.
PMCID: PMC2997569  PMID: 20482315
amyloid; Notch receptor; peptidomimetics; signal peptide peptidase; substrate analogues; substrate recognition
4.  Substrate docking to γ-secretase allows access of γ-secretase modulators to an allosteric site 
Nature Communications  2010;1:130-.
γ-Secretase generates the peptides of Alzheimer's disease, Aβ40 and Aβ42, by cleaving the amyloid precursor protein within its transmembrane domain. γ-Secretase also cleaves numerous other substrates, raising concerns about γ-secretase inhibitor off-target effects. Another important class of drugs, γ-secretase modulators, alter the cleavage site of γ-secretase on amyloid precursor protein, changing the Aβ42/Aβ40 ratio, and are thus a promising therapeutic approach for Alzheimer's disease. However, the target for γ-secretase modulators is uncertain, with some data suggesting that they function on γ-secretase, whereas others support their binding to the amyloid precursor. In this paper we address this controversy by using a fluorescence resonance energy transfer-based assay to examine whether γ-secretase modulators alter Presenilin-1/γ-secretase conformation in intact cells in the absence of its natural substrates such as amyloid precursor protein and Notch. We report that the γ-secretase allosteric site is located within the γ-secretase complex, but substrate docking is needed for γ-secretase modulators to access this site.
γ-Secretase modulators have promise in the treatment of Alzheimer's disease, but their molecular target is uncertain. Here, fluorescence resonance energy transfer is used to determine that the γ-secretase allosteric site is within the γ-secretase complex and that substrate docking is required for modulators to access the site.
PMCID: PMC3060602  PMID: 21119643
5.  Binding of longer Aβ to transmembrane domain 1 of presenilin 1 impacts on Aβ42 generation 
Amyloid-β peptide ending at 42nd residue (Aβ42) is believed as a pathogenic peptide for Alzheimer disease. Although γ-secretase is a responsible protease to generate Aβ through a processive cleavage, the proteolytic mechanism of γ-secretase at molecular level is poorly understood.
We found that the transmembrane domain (TMD) 1 of presenilin (PS) 1, a catalytic subunit for the γ-secretase, as a key modulatory domain for Aβ42 production. Aβ42-lowering and -raising γ-secretase modulators (GSMs) directly targeted TMD1 of PS1 and affected its structure. A point mutation in TMD1 caused an aberrant secretion of longer Aβ species including Aβ45 that are the precursor of Aβ42. We further found that the helical surface of TMD1 is involved in the binding of Aβ45/48 and that the binding was altered by GSMs as well as TMD1 mutation.
Binding between PS1 TMD1 and longer Aβ is critical for Aβ42 production.
PMCID: PMC3896738  PMID: 24410857
Presenilin; Secretases; Alzheimer disease; Intramembrane proteolysis; γ-Secretase modulator
6.  Structural biology of presenilin 1 complexes 
The presenilin genes were first identified as the site of missense mutations causing early onset autosomal dominant familial Alzheimer's disease. Subsequent work has shown that the presenilin proteins are the catalytic subunits of a hetero-tetrameric complex containing APH1, nicastrin and PEN-2. This complex (variously termed presenilin complex or gamma-secretase complex) performs an unusual type of proteolysis in which the transmembrane domains of Type I proteins are cleaved within the hydrophobic compartment of the membrane. This review describes some of the molecular and structural biology of this unusual enzyme complex. The presenilin complex is a bilobed structure. The head domain contains the ectodomain of nicastrin. The base domain contains a central cavity with a lateral cleft that likely provides the route for access of the substrate to the catalytic cavity within the centre of the base domain. There are reciprocal allosteric interactions between various sites in the complex that affect its function. For instance, binding of Compound E, a peptidomimetic inhibitor to the PS1 N-terminus, induces significant conformational changes that reduces substrate binding at the initial substrate docking site, and thus inhibits substrate cleavage. However, there is a reciprocal allosteric interaction between these sites such that prior binding of the substrate to the initial docking site paradoxically increases the binding of the Compound E peptidomimetic inhibitor. Such reciprocal interactions are likely to form the basis of a gating mechanism that underlies access of substrate to the catalytic site. An increasingly detailed understanding of the structural biology of the presenilin complex is an essential step towards rational design of substrate- and/or cleavage site-specific modulators of presenilin complex function.
PMCID: PMC4326451  PMID: 25523933
Presenilin; Nicastrin; APH1; PEN-2; Gamma-secretase; Epsilon secretase; Notch; APP; Abeta; Structural biology; Gamma-secretase inhibitor compounds; Gamma-secretase modulator compounds; Lateral gate
7.  Specific domains in anterior pharynx-defective 1 determine its intramembrane interactions with nicastrin and presenilin 
Neurobiology of aging  2010;33(2):277-285.
γ-Secretase, a multi-subunit transmembrane protease comprised of presenilin, nicastrin, presenilin enhancer 2, and anterior pharynx-defective 1, participates in the regulated intramembrane proteolysis of Type I membrane proteins including the amyloid precursor protein (APP). Although Aph-1 is thought to play a structural role in the assembly of γ-secretase complex and several transmembrane domains (TMDs) of Aph-1 have been shown to be critical for its function, the importance of the other domains of Aph-1 remains elusive. We screened a series of Aph-1 mutants and focused on 9 mutations distributed in 6 different TMDs of human APH-1aS, assessing their ability to complement mouse embryonic fibroblasts lacking Aph-1. We showed that mutations in TMD4 (G126) and TMD5 (H171) of Aph-1a prevented the formation of the Nct/Aph-1 subcomplex. Importantly, although mutations in TMD3 (Q83/E84/R85) and TMD6 (H197) of APH-1aS did not affect Nct/Aph-1 subcomplex formation, both mutations prevented further association/endoproteolysis of PS1. We propose a model that identifies critical TMDs of Aph-1 for associations with Nct and PS for the stepwise assembly of γ-secretase components.
PMCID: PMC2904414  PMID: 20382452
γ-Secretase; Aph-1; Nct; PS; mutagenesis; transmembrane domain
8.  Identification of an Archaeal Presenilin-Like Intramembrane Protease 
PLoS ONE  2010;5(9):e13072.
The GXGD-type diaspartyl intramembrane protease, presenilin, constitutes the catalytic core of the γ-secretase multi-protein complex responsible for activating critical signaling cascades during development and for the production of β-amyloid peptides (Aβ) implicated in Alzheimer's disease. The only other known GXGD-type diaspartyl intramembrane proteases are the eukaryotic signal peptide peptidases (SPPs). The presence of presenilin-like enzymes outside eukaryots has not been demonstrated. Here we report the existence of presenilin-like GXGD-type diaspartyl intramembrane proteases in archaea.
Methodology and Principal Findings
We have employed in vitro activity assays to show that MCMJR1, a polytopic membrane protein from the archaeon Methanoculleus marisnigri JR1, is an intramembrane protease bearing the signature YD and GXGD catalytic motifs of presenilin-like enzymes. Mass spectrometry analysis showed MCMJR1 could cleave model intramembrane protease substrates at several sites within their transmembrane region. Remarkably, MCMJR1 could also cleave substrates derived from the β-amyloid precursor protein (APP) without the need of protein co-factors, as required by presenilin. Two distinct cleavage sites within the transmembrane domain of APP could be identified, one of which coincided with Aβ40, the predominant site processed by γ-secretase. Finally, an established presenilin and SPP transition-state analog inhibitor could inhibit MCMJR1.
Conclusions and Significance
Our findings suggest that a primitive GXGD-type diaspartyl intramembrane protease from archaea can recapitulate key biochemical properties of eukaryotic presenilins and SPPs. MCMJR1 promises to be a more tractable, simpler system for in depth structural and mechanistic studies of GXGD-type diaspartyl intramembrane proteases.
PMCID: PMC2947513  PMID: 20927381
9.  An allosteric Sec61 inhibitor traps nascent transmembrane helices at the lateral gate 
eLife  2014;3:e01483.
Membrane protein biogenesis requires the coordinated movement of hydrophobic transmembrane domains (TMD) from the cytosolic vestibule of the Sec61 channel into the lipid bilayer. Molecular insight into TMD integration has been hampered by the difficulty of characterizing intermediates during this intrinsically dynamic process. In this study, we show that cotransin, a substrate-selective Sec61 inhibitor, traps nascent TMDs in the cytosolic vestibule, permitting detailed interrogation of an early pre-integration intermediate. Site-specific crosslinking revealed the pre-integrated TMD docked to Sec61 near the cytosolic tip of the lateral gate. Escape from cotransin-arrest depends not only on cotransin concentration, but also on the biophysical properties of the TMD. Genetic selection of cotransin-resistant cancer cells uncovered multiple mutations clustered near the lumenal plug of Sec61α, thus revealing cotransin’s likely site of action. Our results suggest that TMD/lateral gate interactions facilitate TMD transfer into the membrane, a process that is allosterically modulated by cotransin binding to the plug.
eLife digest
Cells are surrounded by a plasma membrane that acts like a barrier around the cell—keeping the cell’s boundaries distinct from surrounding cells and helping to regulate the contents of the cell. This plasma membrane is made up mostly of two layers of fatty molecules, and is also studded with proteins. Some of these membrane proteins act as channels that allow nutrients and other chemicals to enter and leave the cell, while others allow the cell to communicate with other cells and the outside environment.
Like all proteins, membrane proteins are chains of amino acids that are linked together by a molecular machine called a ribosome. The ribosomes that make membrane proteins are located on the outside of a membrane-enclosed compartment within the cell called the endoplasmic reticulum. To eventually become embedded within a membrane, a new protein must—at the same time as it is being built—enter a channel within the membrane of the endoplasmic reticulum. The newly synthesized protein chain enters this channel, called Sec61, via an entrance near the ribosome and then threads its way toward the inside of the endoplasmic reticulum. However, there is also a ‘side-gate’ in Sec61 that allows specific segments the new protein to escape the channel and become embedded within the membrane. From here, the membrane protein can be trafficked to other destinations within the cell, including the plasma membrane. However, how the newly forming protein chain passes through the side-gate of Sec61 is not well understood.
Now MacKinnon, Paavilainen et al. have used a small molecule called cotransin—which is known to interfere with the passage of proteins through Sec61—to observe the interactions between the Sec61 channel and the new protein. Cotransin appears to trap the new protein chain within the Sec61 channel by essentially ‘locking’ the side-gate.
MacKinnon, Paavilainen et al. observed that the trapped protein interacts with the inside of the channel at the end closest to the ribosome—which is the likely location of the side-gate. In contrast, cotransin likely binds at the other end of the channel, to a piece of Sec61 that serves to plug the exit into the endoplasmic reticulum; and this plug is directly connected to the side-gate. By preventing the plug from moving out of the way, cotransin can somehow stop the new protein from passing through the side-gate. However, MacKinnon, Paavilainen et al. did find that some membrane proteins with certain physical and chemical properties could get through the gate, despite the presence of cotransin. The next challenge is to resolve exactly how interactions between cotransin and the Sec61 plug can block the escape of new proteins into the membrane.
PMCID: PMC3913039  PMID: 24497544
cotransin; transmembrane domain; endoplasmic reticulum; human
10.  Proteomic Profiling of γ-Secretase Substrates and Mapping of Substrate Requirements 
PLoS Biology  2008;6(10):e257.
The presenilin/γ-secretase complex, an unusual intramembrane aspartyl protease, plays an essential role in cellular signaling and membrane protein turnover. Its ability to liberate numerous intracellular signaling proteins from the membrane and also mediate the secretion of amyloid-β protein (Aβ) has made modulation of γ-secretase activity a therapeutic goal for cancer and Alzheimer disease. Although the proteolysis of the prototypical substrates Notch and β-amyloid precursor protein (APP) has been intensely studied, the full spectrum of substrates and the determinants that make a transmembrane protein a substrate remain unclear. Using an unbiased approach to substrate identification, we surveyed the proteome of a human cell line for targets of γ-secretase and found a relatively small population of new substrates, all of which are type I transmembrane proteins but have diverse biological roles. By comparing these substrates to type I proteins not regulated by γ-secretase, we determined that besides a short ectodomain, γ-secretase requires permissive transmembrane and cytoplasmic domains to bind and cleave its substrates. In addition, we provide evidence for at least two mechanisms that can target a substrate for γ cleavage: one in which a substrate with a short ectodomain is directly cleaved independent of sheddase association, and a second where a substrate requires ectodomain shedding to instruct subsequent γ-secretase processing. These findings expand our understanding of the mechanisms of substrate selection as well as the diverse cellular processes to which γ-secretase contributes.
Author Summary
All cells face the challenge of removing transmembrane proteins from the lipid bilayer for the purpose of signaling or degradation. One molecular solution to this problem is the multiprotein enzyme complex γ-secretase, which is able to hydrolyze several known transmembrane proteins within the hydrophobic lipid environment. Due to its central role in the pathogenesis of Alzheimer disease, modulation of γ-secretase activity has become a therapeutic goal. However, the number and diversity of proteins that can be cleaved by this protease remain unknown, and the attributes that target these proteins to γ-secretase are unclear. In this study, we used an unbiased approach to substrate identification and surveyed the proteome for targets of γ-secretase. Of the thousands of proteins detectable, only a relative few were substrates of γ-secretase, all of which were type I transmembrane proteins. In addition to validating several of these novel substrates, we compared them to other proteins that we identified as nonsubstrates and determined that there are specific domains that can activate or inhibit γ-secretase processing. These findings should advance our understanding of the many cellular processes regulated by γ-secretase and may offer insights into how γ-secretase can be exploited for therapeutic purposes.
Using an unbiased quantitative proteomics approach, novel substrate targets for the protease γ-secretase are identified and analyzed to determine which domains enable their cleavage.
PMCID: PMC2570425  PMID: 18942891
11.  The Amyloid Precursor Protein has a Flexible Transmembrane Domain and Binds Cholesterol 
Science (New York, N.Y.)  2012;336(6085):1168-1171.
C99 is the transmembrane carboxyl-terminal domain of the amyloid precursor protein that is cleaved by γ-secretase to release the amyloid-β polypeptides, which are associated with Alzheimer’s disease. Nuclear magnetic resonance and electron paramagnetic resonance spectroscopy show that the extracellular amino terminus of C99 includes a surface-embedded “N-helix” followed by a short “N-loop” connecting to the transmembrane domain (TMD). The TMD is a flexibly curved α helix, making it well suited for processive cleavage by γ-secretase. Titration of C99 reveals a binding site for cholesterol, providing mechanistic insight into how cholesterol promotes amyloidogenesis. Membrane-buried GXXXG motifs (G, Gly; X, any amino acid), which have an established role in oligomerization, were also shown to play a key role in cholesterol binding. The structure and cholesterol binding properties of C99 may aid in the design of Alzheimer’s therapeutics.
PMCID: PMC3528355  PMID: 22654059
12.  γ-Secretase Substrate Selectivity Can Be Modulated Directly via Interaction with a Nucleotide-binding Site*s 
The Journal of biological chemistry  2005;280(51):41987-41996.
γ-Secretase is an unusual protease with an intramembrane catalytic site that cleaves many type I membrane proteins, including the amyloid β-protein (Aβ) precursor (APP) and the Notch receptor. Genetic and biochemical studies have identified four membrane proteins as components of γ-secretase: heterodimeric presenilin composed of its N- and C-terminal fragments, nicastrin, Aph-1, and Pen-2. Here we demonstrated that certain compounds, including protein kinase inhibitors and their derivatives, act directly on purified γ-secretase to selectively block cleavage of APP- but not Notch-based substrates. Moreover, ATP activated the generation of the APP intracellular domain and Aβ, but not the generation of the Notch intracellular domain by the purified protease complex, and was a direct competitor of the APP-selective inhibitors, as were other nucleotides. In accord, purified γ-secretase bound specifically to an ATP-linked resin. Finally, a photoactivable ATP analog specifically labeled presenilin 1-C-terminal fragments in purified γ-secretase preparations; the labeling was blocked by ATP itself and APP-selective γ-secretase inhibitors. We concluded that a nucleotide-binding site exists within γ-secretase, and certain compounds that bind to this site can specifically modulate the generation of Aβ while sparing Notch. Drugs targeting the γ-secretase nucleotide-binding site represent an attractive strategy for safely treating Alzheimer disease.
PMCID: PMC1523323  PMID: 16236717
13.  Cellular mechanisms of γ-secretase substrate selection, processing and toxicity 
Progress in Neurobiology  2012;98(2):166-175.
Presenilins (PSs) are catalytic components of the γ-secretase proteolytic complexes that produce Aβ and cell signaling peptides. γ-Secretase substrates are mostly membrane-bound peptides derived following proteolytic cleavage of the extracellular domain of typeI transmembrane proteins. Recent work reveals that γ-secretase substrate processing is regulated by proteins termed γ-Secretase Substrate Recruiting Factors (γSSRFs) that bridge substrates to γ-secretase complexes. These factors constitute novel targets for pharmacological control of specific γ-secretase products such as Aβ and signaling peptides. PS familial Alzheimer’s disease (FAD) mutants cause a loss of γ-secretase cleavage function at epsilon sites of substrates thus inhibiting production of cell signaling peptides while promoting accumulation of uncleaved toxic substrates. Importantly, γ-secretase inhibitors may cause toxicity in vivo by similar mechanisms. Here we review novel mechanisms that control γ-secretase substrate selection and cleavage and examine their relevance to AD.
PMCID: PMC3404154  PMID: 22622135
Alzheimer’s disease; Presenilin; Familial AD mutations; γ-Secretase Substrate Recruiting Factors (γSSRFs); metalloproteinases; ADAMs; toxic substrates; γ-Secretase-produced signaling peptides
14.  Allosteric Modulation of PS1/γ-Secretase Conformation Correlates with Amyloid β42/40 Ratio 
PLoS ONE  2009;4(11):e7893.
Presenilin 1(PS1) is the catalytic subunit of γ-secretase, the enzyme responsible for the Aβ C-terminal cleavage site, which results in the production of Aβ peptides of various lengths. Production of longer forms of the Aβ peptide occur in patients with autosomal dominant Alzheimer disease (AD) due to mutations in presenilin. Many modulators of γ-secretase function have been described. We hypothesize that these modulators act by a common mechanism by allosterically modifying the structure of presenilin.
Methodology/Principal Findings
To test this hypothesis we generated a genetically encoded GFP-PS1-RFP (G-PS1-R) FRET probe that allows monitoring of the conformation of the PS1 molecule in its native environment in live cells. We show that G-PS1-R can be incorporated into the γ-secretase complex, reconstituting its activity in PS1/2 deficient cells. Using Förster resonance energy transfer (FRET)-based approaches we show that various pharmacological and genetic manipulations that target either γ-secretase components (PS1, Pen2, Aph1) or γ-secretase substrate (amyloid precursor protein, APP) and are known to change Aβ42 production are associated with a consistent conformational change in PS1.
These results strongly support the hypothesis that allosteric changes in PS1 conformation underlie changes in the Aβ42/40 ratio. Direct measurement of physiological and pathological changes in the conformation of PS1/γ-secretase may provide insight into molecular mechanism of Aβ42 generation, which could be exploited therapeutically.
PMCID: PMC2773935  PMID: 19924286
15.  Helix-Destabilizing, β-Branched, and Polar Residues in the Baboon Reovirus p15 Transmembrane Domain Influence the Modularity of FAST Proteins ▿  
Journal of Virology  2011;85(10):4707-4719.
The fusogenic reoviruses induce syncytium formation using the fusion-associated small transmembrane (FAST) proteins. A recent study indicated the p14 FAST protein transmembrane domain (TMD) can be functionally replaced by the TMDs of the other FAST proteins but not by heterologous TMDs, suggesting that the FAST protein TMDs are modular fusion units. We now show that the p15 FAST protein is also a modular fusogen, as indicated by the functional replacement of the p15 ectodomain with the corresponding domain from the p14 FAST protein. Paradoxically, the p15 TMD is not interchangeable with the TMDs of the other FAST proteins, implying that unique attributes of the p15 TMD are required when this fusion module is functioning in the context of the p15 ecto- and/or endodomain. A series of point substitutions, truncations, and reextensions were created in the p15 TMD to define features that are specific to the functioning of the p15 TMD. Removal of only one or two residues from the N terminus or four residues from the C terminus of the p15 TMD eliminated membrane fusion activity, and there was a direct correlation between the fusion-promoting function of the p15 TMD and the presence of N-terminal, hydrophobic β-branched residues. Substitution of the glycine residues and triserine motif present in the p15 TMD also impaired or eliminated the fusion-promoting activity of the p15 TMD. The ability of the p15 TMD to function in an ecto- and endodomain-specific context is therefore influenced by stringent sequence requirements that reflect the importance of TMD polar residues and helix-destabilizing residues.
PMCID: PMC3126213  PMID: 21367887
16.  Highly Conserved Cysteines Are Involved in the Oligomerization of Occludin—Redox Dependency of the Second Extracellular Loop 
Antioxidants & Redox Signaling  2014;20(6):855-867.
The tight junction (TJ) marker occludin is a 4-transmembrane domain (TMD) protein with unclear physiological and pathological functions, interacting with other TJ proteins. It oligomerizes and is redox sensitive. However, oligomerization sites and mechanisms are unknown. Aims: To identify hypoxia-sensitive binding sites, we investigated the consequences of amino-acid substitutions of highly conserved cysteines in human occludin, under normal and hypoxic incubations. Results: (i) The extracellular loop 2 (ECL2) showed homophilic trans- and cis-association between opposing cells and along the cell membrane, respectively, caused by a loop properly folded via an intraloop disulfide bridge between the shielded C216 and C237. Hypoxia and reductants prevented the associations. (ii) C82 in TMD1 directly cis-associated without disulfide formation. (iii) C76 in TMD1 and C148 in TMD2 limited the trans-interaction; C76 also limited occludin-related paracellular tightness and changed the strand morphology of claudin-1. (iv) The diminished binding strength found after substituting C82, C216, or C237 was accompanied by increased occludin mobility in the cell membrane. Innovation: The data enable the first experimentally proven structural model of occludin and its homophilic interaction sites, in which the ECL2, via intraloop disulfide formation, has a central role in occludin's hypoxia-sensitive oligomerization and to regulate the structure of TJs. Conclusion: Our findings support the new concept that occludin acts as a hypoxiasensor and contributes toward regulating the TJ assembly redox dependently. This is of pathogenic relevance for tissue barrier injury with reducing conditions. The ECL2 disulfide might be a model for four TMD proteins in TJs with two conserved cysteines in an ECL. Antioxid. Redox Signal. 20, 855–867.
PMCID: PMC3924802  PMID: 23923978
17.  Changed membrane integration and catalytic site conformation are two mechanisms behind the increased Aβ42/Aβ40 ratio by presenilin 1 familial Alzheimer-linked mutations 
FEBS Open Bio  2014;4:393-406.
•Familial Alzheimer disease (FAD) mutations affect presenilin membrane integration.•The transmembrane domains around the catalytic site are vulnerable to changes.•All FAD mutations cause changes in the active site of the γ-secretase complex.•The FAD mutants lead to a complex processing pattern of the amyloid precursor protein.
The enzyme complex γ-secretase generates amyloid β-peptide (Aβ), a 37–43-residue peptide associated with Alzheimer disease (AD). Mutations in presenilin 1 (PS1), the catalytical subunit of γ-secretase, result in familial AD (FAD). A unifying theme among FAD mutations is an alteration in the ratio Aβ species produced (the Aβ42/Aβ40 ratio), but the molecular mechanisms responsible remain elusive. In this report we have studied the impact of several different PS1 FAD mutations on the integration of selected PS1 transmembrane domains and on PS1 active site conformation, and whether any effects translate to a particular amyloid precursor protein (APP) processing phenotype. Most mutations studied caused an increase in the Aβ42/Aβ40 ratio, but via different mechanisms. The mutations that caused a particular large increase in the Aβ42/Aβ40 ratio did also display an impaired APP intracellular domain (AICD) formation and a lower total Aβ production. Interestingly, seven mutations close to the catalytic site caused a severely impaired integration of proximal transmembrane/hydrophobic sequences into the membrane. This structural defect did not correlate to a particular APP processing phenotype. Six selected FAD mutations, all of which exhibited different APP processing profiles and impact on PS1 transmembrane domain integration, were found to display an altered active site conformation. Combined, our data suggest that FAD mutations affect the PS1 structure and active site differently, resulting in several complex APP processing phenotypes, where the most aggressive mutations in terms of increased Aβ42/Aβ40 ratio are associated with a decrease in total γ-secretase activity.
PMCID: PMC4050182  PMID: 24918054
APP, amyloid precursor protein; Aβ, amyloid-β peptide; AICD, amyloid precursor protein intracellular domain; AD, Alzheimer disease; FAD, familial AD; TMD, transmembrane domains; PS, presenilin; NTF, N-terminal fragment; CTF, C-terminal fragment; ER, endoplasmic reticulum; Lep, leader peptidase; BD8, blastocyst-derived embryonic stem cells; GVP, Gal4VP16; GCB, γ-secretase inhibitor coupled to biotin; WT, wild type; FLIM/FRET, Fluorescence Lifetime Imaging/ Fluorescence Resonance Energy Transfer; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid; Bis-Tris, 2-(bis(2-hydroxyethyl)amino)-2-(hydroxymethyl)propane-1,3-diol; MSD, Meso Scale Discovery; RM, rough microsomes; CRM, column-washed dog pancreas rough microsomes; Endo H, endoglycosidase H; MGD, minimal glycosylation distance; Alzheimer disease; γ-Secretase; Membrane integration; Amyloid β-peptide; Protein structure
18.  Beyond Anchoring: the Expanding Role of the Hendra Virus Fusion Protein Transmembrane Domain in Protein Folding, Stability, and Function 
Journal of Virology  2012;86(6):3003-3013.
While work with viral fusion proteins has demonstrated that the transmembrane domain (TMD) can affect protein folding, stability, and membrane fusion promotion, the mechanism(s) remains poorly understood. TMDs could play a role in fusion promotion through direct TMD-TMD interactions, and we have recently shown that isolated TMDs from three paramyxovirus fusion (F) proteins interact as trimers using sedimentation equilibrium (SE) analysis (E. C. Smith, et al., submitted for publication). Immediately N-terminal to the TMD is heptad repeat B (HRB), which plays critical roles in fusion. Interestingly, addition of HRB decreased the stability of the trimeric TMD-TMD interactions. This result, combined with previous findings that HRB forms a trimeric coiled coil in the prefusion form of the whole protein though HRB peptides fail to stably associate in isolation, suggests that the trimeric TMD-TMD interactions work in concert with elements in the F ectodomain head to stabilize a weak HRB interaction. Thus, changes in TMD-TMD interactions could be important in regulating F triggering and refolding. Alanine insertions between the TMD and HRB demonstrated that spacing between these two regions is important for protein stability while not affecting TMD-TMD interactions. Additional mutagenesis of the C-terminal end of the TMD suggests that β-branched residues within the TMD play a role in membrane fusion, potentially through modulation of TMD-TMD interactions. Our results support a model whereby the C-terminal end of the Hendra virus F TMD is an important regulator of TMD-TMD interactions and show that these interactions help hold HRB in place prior to the triggering of membrane fusion.
PMCID: PMC3302297  PMID: 22238302
19.  Probing the structural basis for differential KCNQ1 modulation by KCNE1 and KCNE2 
The Journal of General Physiology  2012;140(6):653-669.
KCNE1 associates with KCNQ1 to increase its current amplitude and slow the activation gating process, creating the slow delayed rectifier channel that functions as a “repolarization reserve” in human heart. The transmembrane domain (TMD) of KCNE1 plays a key role in modulating KCNQ1 pore conductance and gating kinetics, and the extracellular juxtamembrane (EJM) region plays a modulatory role by interacting with the extracellular surface of KCNQ1. KCNE2 is also expressed in human heart and can associate with KCNQ1 to suppress its current amplitude and slow the deactivation gating process. KCNE1 and KCNE2 share the transmembrane topology and a high degree of sequence homology in TMD and surrounding regions. The structural basis for their distinctly different effects on KCNQ1 is not clear. To address this question, we apply cysteine (Cys) scanning mutagenesis to TMDs and EJMs of KCNE1 and KCNE2. We analyze the patterns of functional perturbation to identify high impact positions, and probe disulfide formation between engineered Cys side chains on KCNE subunits and native Cys on KCNQ1. We also use methanethiosulfonate reagents to probe the relationship between EJMs of KCNE subunits and KCNQ1. Our data suggest that the TMDs of both KCNE subunits are at about the same location but interact differently with KCNQ1. In particular, the much closer contact of KCNE2 TMD with KCNQ1, relative to that of KCNE1, is expected to impact the allosteric modulation of KCNQ1 pore conductance and may explain their differential effects on the KCNQ1 current amplitude. KCNE1 and KCNE2 also differ in the relationship between their EJMs and KCNQ1. Although the EJM of KCNE1 makes intimate contacts with KCNQ1, there appears to be a crevice between KCNQ1 and KCNE2. This putative crevice may perturb the electrical field around the voltage-sensing domain of KCNQ1, contributing to the differential effects of KCNE2 versus KCNE1 on KCNQ1 gating kinetics.
PMCID: PMC3514736  PMID: 23183700
20.  Structural Interactions between Inhibitor and Substrate Docking Sites Give Insight into Mechanisms of Human PS1 Complexes 
Structure(London, England:1993)  2014;22(1):125-135.
Presenilin-mediated endoproteolysis of transmembrane proteins plays a key role in physiological signaling and in the pathogenesis of Alzheimer disease and some cancers. Numerous inhibitors have been found via library screens, but their structural mechanisms remain unknown. We used several biophysical techniques to investigate the structure of human presenilin complexes and the effects of peptidomimetic γ-secretase inhibitors. The complexes are bilobed. The head contains nicastrin ectodomain. The membrane-embedded base has a central channel and a lateral cleft, which may represent the initial substrate docking site. Inhibitor binding induces widespread structural changes, including rotation of the head and closure of the lateral cleft. These changes block substrate access to the catalytic pocket and inhibit the enzyme. Intriguingly, peptide substrate docking has reciprocal effects on the inhibitor binding site. Similar reciprocal shifts may underlie the mechanisms of other inhibitors and of the “lateral gate” through which substrates access to the catalytic site.
Graphical Abstract
•The head contains nicastrin ectodomain and overhangs a solute-accessible cavity in base•The base has a central channel and a lateral cleft (putative substrate docking site)•Inhibitors close the cleft and channel and rotate the head, blocking substrate access
Presenilin complexes mediate proteolysis of transmembrane proteins during physiological signaling and disease. Li et al. describe the architecture of human presenilin complex PS1 and inhibitor-induced structural changes. They propose that similar shifts likely underlie substrate access to the catalytic site.
PMCID: PMC3887256  PMID: 24210759
21.  Effect of Metal Chelators on γ-Secretase Indicates That Calcium and Magnesium Ions Facilitate Cleavage of Alzheimer Amyloid Precursor Substrate 
Gamma-secretase is involved in the production of Aβ amyloid peptides. It cleaves the transmembrane domain of the amyloid precursor protein (APP) at alternative sites to produce Aβ and the APP intracellular domain (AICD). Metal ions play an important role in Aβ aggregation and metabolism, thus metal chelators and ligands represent potential therapeutic agents for AD treatment. A direct effect of metal chelators on γ-secretase has not yet been investigated. The authors used an in vitro  γ-secretase assay consisting of cleavage of APP C100-3XFLAG by endogenous γ-secretase from rodent brains and human neuroblastoma SH-SY5Y, and detected AICD production by western blotting. Adding metalloprotease inhibitors to the reaction showed that clioquinol, phosphoramidon, and zinc metalloprotease inhibitors had no significant effect on γ-secretase activity. In contrast, phenanthroline, EDTA, and EGTA markedly decreased γ-secretase activity that could be restored by adding back calcium and magnesium ions. Mg2+ stabilized a 1,000 kDa presenilin 1 complex through blue native gel electrophoresis and size-exclusion chromatography. Data suggest that Ca2+ and Mg2+ stabilize γ-secretase and enhance its activity.
PMCID: PMC3021864  PMID: 21253550
22.  Snorkeling Basic Amino Acid Side Chains Regulate Transmembrane Integrin Signalling 
Nature  2011;481(7380):209-213.
Side chains of Lys/Arg near transmembrane domain (TMD)1–3 membrane-water interfaces can “snorkel” placing their positive charge near negatively-charged phospholipid head groups4–6; however, snorkeling's functional effects are obscure. Integrin β TMDs exhibit such conserved basic amino acids; here we used nuclear magnetic resonance (NMR) spectroscopy7, 8 to show that integrin β3(Lys716) helps determine β3 TMD topography. The αIIbβ3 TMD structure suggests that precise β3 TMD crossing angles enable the assembly of outer and inner membrane “clasps” (OMC and IMC) that hold the αβ TMD together to limit transmembrane signalling9 . Mutation of β3(Lys716) caused dissociation of αIIbβ3 TMDs and integrin activation. To confirm that altered topography of β3(Lys716) mutants activated αIIbβ3, we utilized directed evolution of β3(K716A) to identify substitutions restoring default state. Introduction Pro(711) at the midpoint of β3 TMD (A711P) increased αIIbβ3 TMD association and inactivated integrin αIIbβ3(A711P,K716A). β3(Pro711) introduced a TMD kink of 30 ± 1° precisely at the OMC/IMC border, thereby decoupling the tilt between these segments. Thus, widely-occurring snorkeling residues in TMDs can help maintain TMD topography and membrane-embedding thereby regulating transmembrane signalling.
PMCID: PMC3257387  PMID: 22178926
23.  His20 Provides the Sole Functionally Significant Side Chain in the Essential TonB Transmembrane Domain▿  
Journal of Bacteriology  2007;189(7):2825-2833.
The cytoplasmic membrane protein TonB couples the protonmotive force of the cytoplasmic membrane to active transport across the outer membrane of Escherichia coli. The uncleaved amino-terminal signal anchor transmembrane domain (TMD; residues 12 to 32) of TonB and the integral cytoplasmic membrane proteins ExbB and ExbD are essential to this process, with important interactions occurring among the several TMDs of all three proteins. Here, we show that, of all the residues in the TonB TMD, only His20 is essential for TonB activity. When alanyl residues replaced all TMD residues except Ser16 and His20, the resultant “all-Ala Ser16 His20” TMD TonB retained 90% of wild-type iron transport activity. Ser16Ala in the context of a wild-type TonB TMD was fully active. In contrast, His20Ala in the wild-type TMD was entirely inactive. In more mechanistically informative assays, the all-Ala Ser16 His20 TMD TonB unexpectedly failed to support formation of disulfide-linked dimers by TonB derivatives bearing Cys substitutions for the aromatic residues in the carboxy terminus. We hypothesize that, because ExbB/D apparently cannot efficiently down-regulate conformational changes at the TonB carboxy terminus through the all-Ala Ser16 His20 TMD, the TonB carboxy terminus might fold so rapidly that disulfide-linked dimers cannot be efficiently trapped. In formaldehyde cross-linking experiments, the all-Ala Ser16 His20 TMD also supported large numbers of apparently nonspecific contacts with unknown proteins. The all-Ala Ser16 His20 TMD TonB retained its dependence on ExbB/D. Together, these results suggest that a role for ExbB/D might be to control rapid and nonspecific folding that the unregulated TonB carboxy terminus otherwise undergoes. Such a model helps to reconcile the crystal/nuclear magnetic resonance structures of the TonB carboxy terminus with conformational changes and mutant phenotypes observed at the TonB carboxy terminus in vivo.
PMCID: PMC1855822  PMID: 17277053
24.  Mutations in Escherichia coli ExbB Transmembrane Domains Identify Scaffolding and Signal Transduction Functions and Exclude Participation in a Proton Pathway 
Journal of Bacteriology  2013;195(12):2898-2911.
The TonB system couples cytoplasmic membrane proton motive force (pmf) to active transport of diverse nutrients across the outer membrane. Current data suggest that cytoplasmic membrane proteins ExbB and ExbD harness pmf energy. Transmembrane domain (TMD) interactions between TonB and ExbD allow the ExbD C terminus to modulate conformational rearrangements of the periplasmic TonB C terminus in vivo. These conformational changes somehow allow energization of high-affinity TonB-gated transporters by direct interaction with TonB. While ExbB is essential for energy transduction, its role is not well understood. ExbB has N-terminus-out, C-terminus-in topology with three TMDs. TMDs 1 and 2 are punctuated by a cytoplasmic loop, with the C-terminal tail also occupying the cytoplasm. We tested the hypothesis that ExbB TMD residues play roles in proton translocation. Reassessment of TMD boundaries based on hydrophobic character and residue conservation among distantly related ExbB proteins brought earlier widely divergent predictions into congruence. All TMD residues with potentially function-specific side chains (Lys, Cys, Ser, Thr, Tyr, Glu, and Asn) and residues with probable structure-specific side chains (Trp, Gly, and Pro) were substituted with Ala and evaluated in multiple assays. While all three TMDs were essential, they had different roles: TMD1 was a region through which ExbB interacted with the TonB TMD. TMD2 and TMD3, the most conserved among the ExbB/TolQ/MotA/PomA family, played roles in signal transduction between cytoplasm and periplasm and the transition from ExbB homodimers to homotetramers. Consideration of combined data excludes ExbB TMD residues from direct participation in a proton pathway.
PMCID: PMC3697263  PMID: 23603742
25.  Reovirus FAST Protein Transmembrane Domains Function in a Modular, Primary Sequence-Independent Manner To Mediate Cell-Cell Membrane Fusion▿  
Journal of Virology  2009;83(7):2941-2950.
The FAST proteins are a unique family of virus-encoded cell-cell membrane fusion proteins. In the absence of a cleavable N-terminal signal peptide, a single-pass transmembrane domain (TMD) functions as a reverse signal-anchor to direct the FAST proteins into the plasma membrane in an Nexo/Ccyt topology. There is little information available on the role of the FAST protein TMD in the cell-cell membrane fusion reaction. We show that in the absence of conservation in the length or primary amino acid sequence, the p14 TMD can be functionally exchanged with the TMDs of the p10 and p15 FAST proteins. This is not the case for chimeric p14 proteins containing the TMDs of two different enveloped viral fusion proteins or a cellular membrane protein; such chimeric proteins were defective for both pore formation and syncytiogenesis. TMD structural features that are conserved within members of the FAST protein family presumably play direct roles in the fusion reaction. Molecular modeling suggests that the funnel-shaped architecture of the FAST protein TMDs may represent such a conserved structural and functional motif. Interestingly, although heterologous TMDs exert diverse influences on the trafficking of the p14 FAST protein, these TMDs are capable of functioning as reverse signal-anchor sequences to direct p14 into lipid rafts in the correct membrane topology. The FAST protein TMDs are therefore not primary determinants of type III protein topology, but they do play a direct, sequence-independent role in the membrane fusion reaction.
PMCID: PMC2655562  PMID: 19129451

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