Structural and Functional Determinants of γ-Secretase,an Intramembrane Protease Implicated in Alzheimer’s Disease

Alzheimer’s disease is the most common form of neurodegenerative diseases in humans, characterized by the progressive accumulation and aggregation of amyloid-β peptides (Aβ) in brain regions subserving memory and cognition. These 39-43 amino acids long peptides are generated by the sequential proteolytic cleavages of the amyloid-β precursor protein (APP) by β- and γ-secretases, with the latter being the founding member of a new class of intramembrane-cleaving proteases (I-CliPs) characterized by their intramembranous catalytic residues hydrolyzing the peptide bonds within the transmembrane regions of their respective substrates. These proteases include the S2P family of metalloproteases, the Rhomboid family of serine proteases, and two aspartyl proteases: the signal peptide peptidase (SPP) and γ-secretase. In sharp contrast to Rhomboid and SPP that function as a single component, γ-secretase is a multi-component protease with complex assembly, maturation and activation processes. Recently, two low-resolution three-dimensional structures of γ-secretase and three high-resolution structures of the GlpG rhomboid protease have been obtained almost simultaneously by different laboratories. Although these proteases are unrelated by sequence or evolution, they seem to share common functional and structural mechanisms explaining how they catalyze intramembrane proteolysis. Indeed, a water-containing chamber in the catalytic cores of both γ-secretase and GlpG rhomboid provides the hydrophilic environment required for proteolysis and a lateral gating mechanism controls substrate access to the active site. The studies that have identified and characterized the structural determinants critical for the assembly and activity of the γ-secretase complex are reviewed here.     

Sequence features of substrates required for cleavage by GlpG, an Escherichia coli rhomboid protease

Rhomboids are a family of serine proteases belonging to intramembrane cleaving proteases, which are supposed to catalyse proteolysis of a substrate protein within the membrane. It remains unclear whether substrates of the rhomboid proteases have a common sequence feature that allows specific cleavage by rhomboids. We showed previously that GlpG, the Escherichia coli rhomboid, can cleave a type I model membrane protein Bla-LY2-MBP having the second transmembrane region of lactose permease (LY2) at the extramembrane region in vivo and in vitro, and that determinants for proteolysis reside within the LY2 sequence. Here we characterized sequence features in LY2 that allow efficient cleavage by GlpG and identified two elements, a hydrophilic region encompassing the cleavage site and helix-destabilizing residues in the downstream hydrophobic region. Importance of the positioning of helix-destabilizers relative to the cleavage site was suggested. These two elements appear to co-operatively promote proteolysis of substrates by GlpG. Finally, random mutagenesis of the cleavage site residues in combination with in vivo screening revealed that GlpG prefers residues with a small side chain and a negative charge at the P1 and P1' sites respectively.     

Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate–peptide complex structures

The mechanisms of intramembrane proteases are incompletely understood due to the lack of structural data on substrate complexes. To gain insight into substrate binding by rhomboid proteases, we have synthesised a series of novel peptidyl-chloromethylketone (CMK) inhibitors and analysed their interactions with Escherichia coli rhomboid GlpG enzymologically and structurally. We show that peptidyl-CMKs derived from the natural rhomboid substrate TatA from bacterium Providencia stuartii bind GlpG in a substrate-like manner, and their co-crystal structures with GlpG reveal the S1 to S4 subsites of the protease. The S1 subsite is prominent and merges into the ‘water retention site’, suggesting intimate interplay between substrate binding, specificity and catalysis. Unexpectedly, the S4 subsite is plastically formed by residues of the L1 loop, an important but hitherto enigmatic feature of the rhomboid fold. We propose that the homologous region of members of the wider rhomboid-like protein superfamily may have similar substrate or client-protein binding function. Finally, using molecular dynamics, we generate a model of the Michaelis complex of the substrate bound in the active site of GlpG.     

Structure and Mechanism of Rhomboid Protease

Rhomboid protease was first discovered in Drosophila. Mutation of the fly gene interfered with growth factor signaling and produced a characteristic phenotype of a pointed head skeleton. The name rhomboid has since been widely used to describe a large family of related membrane proteins that have diverse biological functions but share a common catalytic core domain composed of six membrane-spanning segments. Most rhomboid proteases cleave membrane protein substrates near the N terminus of their transmembrane domains. How these proteases function within the confines of the membrane is not completely understood. Recent progress in crystallographic analysis of the Escherichia coli rhomboid protease GlpG in complex with inhibitors has provided new insights into the catalytic mechanism of the protease and its conformational change. Improved biochemical assays have also identified a substrate sequence motif that is specifically recognized by many rhomboid proteases.     

Influence of hydrophobic mismatch on the catalytic activity of Escherichia coli GlpG rhomboid protease

Rhomboids comprise a broad family of intramembrane serine proteases that are found in a wide range of organisms and participate in a diverse array of biological processes. High-resolution structures of the catalytic transmembrane domain of the Escherichia coli GlpG rhomboid have provided numerous insights that help explain how hydrolytic cleavage can be achieved below the membrane surface. Key to this are observations that GlpG hydrophobic domain dimensions may not be sufficient to completely span the native lipid bilayer. This formed the basis for a model where hydrophobic mismatch Induces thinning of the local membrane environment to promote access to transmembrane substrates. However, hydrophobic mismatch also has the potential to alter the functional properties of the rhomboid, a possibility we explore in the current work. For this purpose, we purified the catalytic transmembrane domain of GlpG into phosphocholine or maltoside detergent micelles of varying alkyl chain lengths, and assessed proteolytic function with a model water-soluble substrate. Catalytic turnover numbers were found to depend on detergent alkyl chain length, with saturated chains containing 10–12 carbon atoms supporting maximal activity. Similar results were obtained in phospholipid bicelles, with no proteolytic activity being detected in longer-chain lipids. Although differences in thermal stability and GlpG oligomerization could not explain these activity differences, circular dichroism spectra suggest that mismatch gives rise to a small change in structure. Overall, these results demonstrate that hydrophobic mismatch can exert an inhibitory effect on rhomboid activity, with the potential for changes in local membrane environment to regulate activity in vivo.     

Discovery and validation of 2-styryl substituted benzoxazin-4-ones as a novel scaffold for rhomboid protease inhibitors

Rhomboids are intramembrane serine proteases with diverse physiological functions in organisms ranging from archaea to humans. Crystal structure analysis has provided a detailed understanding of the catalytic mechanism, and rhomboids have been implicated in various disease contexts. Unfortunately, the design of specific rhomboid inhibitors has lagged behind, and previously described small molecule inhibitors displayed insufficient potency and/or selectivity. Using a computer-aided approach, we focused on the discovery of novel scaffolds with reduced liabilities and the possibility for broad structural variations. Docking studies with the E. coli rhomboid GlpG indicated that 2-styryl substituted benzoxazinones might comprise novel rhomboid inhibitors. Protease in vitro assays confirmed activity of 2-styryl substituted benzoxazinones against GlpG but not against the soluble serine protease α-chymotrypsin. Furthermore, mass spectrometry analysis demonstrated covalent modification of the catalytic residue Ser201, corroborating the predicted mechanism of inhibition and the formation of an acyl enzyme intermediate. In conclusion, 2-styryl substituted benzoxazinones are a novel rhomboid inhibitor scaffold with ample opportunity for optimization.     

How does the exosite of rhomboid protease affect substrate processing and inhibition?

Rhomboid proteases constitute a family of intramembrane serine proteases ubiquitous in all forms of life. They differ in many aspects from their soluble counterparts. We applied molecular dynamics (MD) computational approach to address several challenging issues regarding their catalytic mechanism: How does the exosite of GlpG rhomboid protease control the kinetics efficiency of substrate hydrolysis? What is the mechanism of inhibition by the non‐competitive peptidyl aldehyde inhibitors bound to the GlpG rhomboid active site (AS)? What is the underlying mechanism that explains the hypothesis that GlpG rhomboid protease is not adopted for the hydrolysis of short peptides that do not contain a transmembrane domain (TMD)? Two fundamental features of rhomboid catalysis, the enzyme recognition and discrimination of substrates by TMD interactions in the exosite, and the concerted mechanism of non‐covalent pre‐catalytic complex to covalent tetrahedral complex (TC) conversion, provide answers to these mechanistic questions.     

Insights into substrate gating in H. influenzae rhomboid

Rhomboids are a remarkable class of serine proteases that are embedded in lipid membranes. These membrane-bound enzymes play key roles in cellular signaling events, and disruptions in these events can result in numerous disease pathologies, including hereditary blindness, type 2 diabetes, Parkinson's disease, and epithelial cancers. Recent crystal structures of rhomboids from Escherichia coli have focused on how membrane-bound substrates gain access to a buried active site. In E. coli, it has been shown that movements of loop 5, with smaller movements in helix 5 and loop 4, act as substrate gate, facilitating inhibitor access to rhomboid catalytic residues. Herein we present a new structure of the Haemophilus influenzae rhomboid hiGlpG, which reveals disorder in loop 5, helix 5, and loop 4, indicating that, together, they represent mobile elements of the substrate gate. Substrate cleavage assays by hiGlpG with amino acid substitutions in these mobile regions demonstrate that the flexibilities of both loop 5 and helix 5 are important for access of the substrates to the catalytic residues. Mutagenesis indicates that less mobility by loop 4 is required for substrate cleavage. A reexamination of the reaction mechanism of rhomboid substrates, whereby cleavage of the scissile bond occurs on the si-face of the peptide bond, is discussed.     

Large Lateral Movement of Transmembrane Helix S5 Is Not Required for Substrate Access to the Active Site of Rhomboid Intramembrane Protease

Rhomboids represent an evolutionarily ancient protease family. Unlike most other proteases, they are polytopic membrane proteins and specialize in cleaving transmembrane protein substrates. The polar active site of rhomboid protease is embedded in the membrane and normally closed. For the bacterial rhomboid GlpG, it has been proposed that one of the transmembrane helices (S5) of the protease can rotate to open a lateral gate, enabling substrate to enter the protease from inside the membrane. Here, we studied the conformational change in GlpG by solving the cocrystal structure of the protease with a mechanism-based inhibitor. We also examined the lateral gating model by cross-linking S5 to a neighboring helix (S2). The crystal structure shows that inhibitor binding displaces a capping loop (L5) from the active site but causes only minor shifts in the transmembrane helices. Cross-linking S5 and S2, which not only restricts the lateral movement of S5 but also prevents substrate from passing between the two helices, does not hinder the ability of the protease to cleave a membrane protein substrate in detergent solution and in reconstituted membrane vesicles. Taken together, these data suggest that a large lateral movement of the S5 helix is not required for substrate access to the active site of rhomboid protease.     

The structural basis for catalysis and substrate specificity of a rhomboid protease

Rhomboids are intramembrane proteases that use a catalytic dyad of serine and histidine for proteolysis. They are conserved in both prokaryotes and eukaryotes and regulate cellular processes as diverse as intercellular signalling, parasitic invasion of host cells, and mitochondrial morphology. Their widespread biological significance and consequent medical potential provides a strong incentive to understand the mechanism of these unusual enzymes for identification of specific inhibitors. In this study, we describe the structure of Escherichia coli rhomboid GlpG covalently bound to a mechanism‐based isocoumarin inhibitor. We identify the position of the oxyanion hole, and the S₁‐ and S₂′‐binding subsites of GlpG, which are the key determinants of substrate specificity. The inhibitor‐bound structure suggests that subtle structural change is sufficient for catalysis, as opposed to large changes proposed from previous structures of unliganded GlpG. Using bound inhibitor as a template, we present a model for substrate binding at the active site and biochemically test its validity. This study provides a foundation for a structural explanation of rhomboid specificity and mechanism, and for inhibitor design.     

Foamy Virus Envelope Protein Is a Substrate for Signal Peptide Peptidase-like 3 (SPPL3)

Signal peptide peptidase (SPP), its homologs, the SPP-like proteases SPPL2a/b/c and SPPL3, as well as presenilin, the catalytic subunit of the γ-secretase complex, are intramembrane-cleaving aspartyl proteases of the GxGD type. In this study, we identified the 18-kDa leader peptide (LP18) of the foamy virus envelope protein (FVenv) as a new substrate for intramembrane proteolysis by human SPPL3 and SPPL2a/b. In contrast to SPPL2a/b and γ-secretase, which require substrates with an ectodomain shorter than 60 amino acids for efficient intramembrane proteolysis, SPPL3 cleaves mutant FVenv lacking the proprotein convertase cleavage site necessary for the prior shedding. Moreover, the cleavage product of FVenv generated by SPPL3 serves as a new substrate for consecutive intramembrane cleavage by SPPL2a/b. Thus, human SPPL3 is the first GxGD-type aspartyl protease shown to be capable of acting like a sheddase, similar to members of the rhomboid family, which belong to the class of intramembrane-cleaving serine proteases.     

In vivo analysis reveals substrate-gating mutants of a rhomboid intramembrane protease display increased activity in living cells

Intramembrane proteases hydrolyze peptide bonds within cell membranes. Recent crystal structures revealed that rhomboid intramembrane proteases contain a hydrated active site that opens to the outside of the cell, but is protected laterally from membrane lipids by protein segments. Using Escherichia coli rhomboid (GlpG) structures as a guide, we previously took a mutational approach to identify the GlpG gating mechanism that allows substrates to enter the active site laterally from the membrane. Mutations that weaken contacts keeping the gate closed increase enzyme activity and implicate transmembrane segment 5 as the substrate gate. Since these analyses were performed in vitro with pure proteins in detergent micelles, we have now examined GlpG in its natural environment, within the membrane of live E. coli cells. In striking congruity with in vitro analysis, gate-opening mutants in transmembrane segment 5 display up to a 10-fold increase in protease activity in living cells. Conversely, mutations in other parts of the protease, including the membrane-inserted L1 loop previously thought to be the gate, decrease enzyme activity. These observations provide evidence for the existence of both closed and open forms of GlpG in cells, and show that inter-conversion between them via substrate gating is rate limiting physiologically.     

Catalytic mechanism of rhomboid protease GlpG probed by 3,4-dichloroisocoumarin and diisopropyl fluorophosphonate

Rhomboid proteases have many important biological functions. Unlike soluble serine proteases such as chymotrypsin, the active site of rhomboid protease, which contains a Ser-His catalytic dyad, is submerged in the membrane and surrounded by membrane-spanning helices. Previous crystallographic analyses of GlpG, a bacterial rhomboid protease, and its complex with isocoumarin have provided insights into the mechanism of the membrane protease. Here, we studied the interaction of GlpG with 3,4-dichloroisocoumarin and diisopropyl fluorophosphonate, both mechanism-based inhibitors for the serine protease, and describe the crystal structure of the covalent adduct between GlpG and diisopropyl fluorophosphonate, which mimics the oxyanion-containing tetrahedral intermediate of the hydrolytic reaction. The crystal structure confirms that the oxyanion is stabilized by the main chain amide of Ser-201 and by the side chains of His-150 and Asn-154. The phosphorylation of the catalytic Ser-201 weakens its interaction with His-254, causing the catalytic histidine to rotate away from the serine. The rotation of His-254 is accompanied by further rearrangement of the side chains of Tyr-205 and Trp-236 within the substrate-binding groove. The formation of the tetrahedral adduct is also accompanied by opening of the L5 cap and movement of transmembrane helix S5 toward S6 in a direction different from that predicted by the lateral gating model. Combining the new structural data with those on the isocoumarin complex sheds further light on the plasticity of the active site of rhomboid membrane protease.     

Membrane Proteases in the Bacterial Protein Secretion and Quality Control Pathway

Summary: Proteolytic cleavage of proteins that are permanently or transiently associated with the cytoplasmic membrane is crucially important for a wide range of essential processes in bacteria. This applies in particular to the secretion of proteins and to membrane protein quality control. Major progress has been made in elucidating the structure-function relationships of many of the responsible membrane proteases, including signal peptidases, signal peptide hydrolases, FtsH, the rhomboid protease GlpG, and the site 1 protease DegS. These enzymes employ very different mechanisms to cleave substrates at the cytoplasmic and extracytoplasmic membrane surfaces or within the plane of the membrane. This review highlights the different ways that bacterial membrane proteases degrade their substrates, with special emphasis on catalytic mechanisms and substrate delivery to the respective active sites.     

Untangling structure–function relationships in the rhomboid family of intramembrane proteases

Rhomboid proteases are a family of integral membrane proteins that have been implicated in critical regulatory roles in a wide array of cellular processes and signaling events. The determination of crystal structures of the prokaryotic rhomboid GlpG from Escherichia coli and Haemophilus influenzae has ushered in an era of unprecedented understanding into molecular aspects of intramembrane proteolysis by this fascinating class of protein. A combination of structural studies by X-ray crystallography, and biophysical and spectroscopic analyses, combined with traditional enzymatic and functional analysis has revealed fundamental aspects of rhomboid structure, substrate recognition and the catalytic mechanism. This review summarizes these remarkable advances by examining evidence for the proposed catalytic mechanism derived from inhibitor co-crystal structures, conflicting models of rhomboid-substrate interaction, and recent work on the structure and function of rhomboid cytosolic domains. In addition to exploring progress on aspects of rhomboid structure, areas for future research and unaddressed questions are emphasized and highlighted. This article is part of a Special Issue entitled: Intramembrane Proteases.     

Proteolytic action of GlpG, a rhomboid protease in the Escherichia coli cytoplasmic membrane

We characterized Escherichia coli GlpG as a membrane-embedded protease and a possible player in the regulated intramembrane proteolysis in this organism. From the sequence features, it belongs to the widely conserved rhomboid family of membrane proteases. We verified the expected topology of GlpG, and it traverses the membrane six times. A model protein having an N-terminal and periplasmically localized beta-lactamase (Bla) domain, a LacY-derived transmembrane region, and a cytosolic maltose binding protein (MBP) mature domain was found to be GlpG-dependently cleaved in vivo. This proteolytic reaction was reproduced in vitro using purified GlpG and purified model substrate protein, and the cleavage was shown to occur between Ser and Asp in a region of high local hydrophilicity, which might be located in a juxtamembrane rather than an intramembrane position. The conserved Ser and His residues of GlpG were essential for the proteolytic activities. Our results using several variant forms of the model protein suggest that GlpG recognizes features of the transmembrane regions of substrates. These results point to a detailed molecular mechanism and cellular analysis of this interesting class of membrane-embedded proteases.     

Role of rhomboid proteases in bacteria

The first member of the rhomboid family of intramembrane serine proteases in bacteria was discovered almost 20 years ago. It is now known that rhomboid proteins are widely distributed in bacteria, with some bacteria containing multiple rhomboids. At the present time, only a single rhomboid-dependent function in bacteria has been identified, which is the cleavage of TatA in Providencia stuartii. Mutational analysis has shown that loss of the GlpG rhomboid in Escherichia coli alters cefotaxime resistance, loss of the YqgP (GluP) rhomboid in Bacillus subtilis alters cell division and glucose uptake, and loss of the MSMEG_5036 and MSMEG_4904 genes in Mycobacterium smegmatis results in altered colony morphology, biofilm formation and antibiotic susceptibilities. However, the cellular substrates for these proteins have not been identified. In addition, analysis of the rhombosortases, together with their possible Gly-Gly CTERM substrates, may shed new light on the role of these proteases in bacteria. This article is part of a Special Issue entitled: Intramembrane Proteases.     

The intramembrane active site of GlpG, an E. coli rhomboid protease, is accessible to water and hydrolyses an extramembrane peptide bond of substrates

Escherichia coli GlpG is an orthologue of the rhomboid proteases that catalyse intramembrane proteolysis of specific membrane proteins. We previously showed that it can cleave a type I model membrane protein, Bla-LY2-MBP, having the second transmembrane region of lactose permease (LY2) in vivo and in vitro at the predicted periplasm-membrane boundary region of LY2. Here we investigated the environment of the active site regions of GlpG in the membrane-integrated state by examining the modifiability of Cys residues introduced into the regions around the catalytic residues with membrane-permeable and -impermeable alkylating reagents. The results indicate that the enzyme active site is fully open to the external aqueous phase. GlpG also cleaved a similar fusion protein, Bla-GknTM-MBP, having the transmembrane region of Gurken (GknTM), a physiological substrate of Drosophila rhomboids. Engineered Cys residues in the cleavage site regions of the LY2 and GknTM sequences were efficiently modified with a membrane-impermeable alkylating reagent, showing that these regions are exposed to the periplasm. These results suggest that GlpG cleaves an extramembrane region of substrates, unlike the currently prevailing view that this class of membrane proteases acts against a membrane-embedded polypeptide segment after its lateral entrance into the enzyme's active site.     

Structural and Functional Characterization of Cleavage and Inactivation of Human Serine Protease Inhibitors by the Bacterial SPATE Protease EspPα from Enterohemorrhagic E. coli

EspPα and EspI are serine protease autotransporters found in enterohemorrhagic Escherichia coli. They both belong to the SPATE autotransporter family and are believed to contribute to pathogenicity via proteolytic cleavage and inactivation of different key host proteins during infection. Here, we describe the specific cleavage and functional inactivation of serine protease inhibitors (serpins) by EspPα and compare this activity with the related SPATE EspI. Serpins are structurally related proteins that regulate vital protease cascades, such as blood coagulation and inflammatory host response. For the rapid determination of serpin cleavage sites, we applied direct MALDI-TOF-MS or ESI-FTMS analysis of coincubations of serpins and SPATE proteases and confirmed observed cleavage positions using in-gel-digest of SDS-PAGE-separated degradation products. Activities of both serpin and SPATE protease were assessed in a newly developed photometrical assay using chromogenic peptide substrates. EspPα cleaved the serpins α1-protease inhibitor (α1-PI), α1-antichymotrypsin, angiotensinogen, and α2-antiplasmin. Serpin cleavage led to loss of inhibitory function as demonstrated for α1-PI while EspPα activity was not affected. Notably, EspPα showed pronounced specificity and cleaved procoagulatory serpins such as α2-antiplasmin while the anticoagulatory antithrombin III was not affected. Together with recently published research, this underlines the interference of EspPα with hemostasis or inflammatory responses during infection, while the observed interaction of EspI with serpins is likely to be not physiologically relevant. EspPα-mediated serpin cleavage occurred always in flexible loops, indicating that this structural motif might be required for substrate recognition.     

Conformational change in rhomboid protease GlpG induced by inhibitor binding to its S' subsites

Rhomboid protease conducts proteolysis inside the hydrophobic environment of the membrane. The conformational flexibility of the protease is essential for the enzyme mechanism, but the nature of this flexibility is not completely understood. Here we describe the crystal structure of rhomboid protease GlpG in complex with a phosphonofluoridate inhibitor, which is covalently bonded to the catalytic serine and extends into the S' side of the substrate binding cleft. Inhibitor binding causes subtle but extensive changes in the membrane protease. Many transmembrane helices tilt and shift positions, and the gap between S2 and S5 is slightly widened so that the inhibitor can bind between them. The side chain of Phe-245 from a loop (L5) that acts as a cap rotates and uncovers the opening of the substrate binding cleft to the lipid bilayer. A concurrent turn of the polypeptide backbone at Phe-245 moves the rest of the cap and exposes the catalytic serine to the aqueous solution. This study, together with earlier crystallographic investigation of smaller inhibitors, suggests a simple model for explaining substrate binding to rhomboid protease.