Inhibitor Fingerprinting of Rhomboid Proteases by Activity-Based

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ACS Chemical Biology

Inhibitor fingerprinting of rhomboid proteases by activity-based protein profiling reveals inhibitor selectivity and rhomboid autoprocessing

Eliane V. Wolf,† Annett Zeissler,† Steven H. L. Verhelst*,†,‡,§

†

Chair for Chemistry of Biopolymers, Technische Universität München, Weihenstephaner Berg

3, 85354, Freising, Germany ‡

Leibniz Institute for Analytical Sciences ISAS, e.V., Otto-Hahn-Str. 6b, 44227 Dortmund,

Germany §

Laboratory of Chemical Biology, Department of Cellular and Molecular Medicine, University of

Leuven, Herestr. 49, 3000 Leuven, Belgium

ACS Paragon Plus Environment


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Abstract Rhomboid proteases were discovered almost 15 years ago and are structurally the best characterized intramembrane proteases. Apart from the general serine protease inhibitor 3,4dichloro-isocoumarin (DCI) and a few crystal structures of the Escherichia coli rhomboid GlpG with other inhibitors, there is surprisingly little information about inhibitors of rhomboids from other species, probably because of a lack of general methods to measure inhibition against different rhomboid species. We here present activity-based protein profiling (ABPP) as a general method to screen rhomboids for their activity and inhibition. Using ABPP, we compare the inhibitory capacity of 50 small molecules against 13 different rhomboids. We find one new pan rhomboid inhibitor and several inhibitors that display selectivity. We also demonstrate that inhibition profile and sequence similarity of rhomboids are not related, which suggests that related rhomboids may be selectively inhibited. Finally, by making use of the here discovered inhibitors, we were able to show that two bacterial rhomboids autoprocess themselves in their N-terminal part.


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Intramembrane proteases (IMPs) are structurally different from soluble proteases. Their active sites are formed by residues in different transmembrane segment (TMs) and are buried within the lipid bilayer of a membrane. Nevertheless, they share mechanistic characteristics with soluble proteases: the main classes of IMPs consist of metallo IMPs (site-2 proteases), aspartyl IMPs (signal peptide peptidases and -secretase), glutamyl IMPs and serine IMPs (also known as rhomboid proteases). IMPs cleave their substrates, which are also membrane proteins, in a TM or in a juxtamembrane region. The cleavage often results in the release of the N- or Cterminal substrate fragment (or both) from the membrane. IMPs display a wide range of biological functions and have been implicated to play a role in the biochemistry of several human diseases.1 Rhomboid proteases are amongst the most widespread IMPs and occur in all three domains of life: bacteria, archaea and eukaryotes. Structurally, rhomboid proteases are the best characterized IMPs. The basic type of rhomboid protease consists of 6 TMs and has its Nterminus in the cytosol (Figure 1a). Some rhomboids have an extra seventh TM located at the N-terminal or C-terminal end of the 6 TM core.

2

Several structures of the E. coli GlpG

(EcGlpG)3-5 and the related Haemophilus influenzae GlpG (HiGlpG)6 have contributed considerably to our structural understanding of intramembrane proteolysis. Although rhomboids have been implicated in some medically relevant processes, it remains unclear what their exact role is and to what extent they are relevant for the manifestation of disease or for possible treatment. In 2001, the general serine protease inhibitor 3,4-dichloro-isocoumarin (DCI) was the very first molecule reported as a rhomboid inhibitor in experiments on Drosophila melanogaster Rhomboid-1.7 In later studies, DCI has been shown to inhibit rhomboids from a small number of species besides drosophila.8-13 For EcGlpG, several other inhibitors have been reported (Figure 1b), including 4-chloro-isocoumarin derivatives (ICs),14, 15 fluorophosphonates,16, 17 -lactams,18,



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19

-lactones,20 and chloromethyl ketones.21 Some of these inhibitors have been used for further

structural characterization of rhomboids. Specifically, crystal structures of EcGlpG in covalent complex with isocoumarins,14, 15 fluorophosphonates,16 and chloromethyl ketones21 have created insights into the possible binding of a substrate to the rhomboid active site. In addition, a couple of studies have used labeled forms of inhibitors as activity-based probes (ABPs) to specifically label the active EcGlpG species, illustrating the versatile use of inhibitors as research tools for rhomboids.

15, 17, 20

However, knowledge about the inhibition of rhomboids from species other

than E. coli is almost non-existent. Only two previous studies have compared inhibition of different rhomboids – both using EcGlpG and Providencia stuartii AarA (PsAarA). In these studies, -lactams and 4-chloro-isocoumarins (ICs) were shown to selectively inhibit one of these rhomboids.15, 18 Although this suggests that rhomboids are able to discriminate between different small molecule ligands, little is known about selective inhibition of rhomboid proteases. In this work we investigate, for the above stated reasons, the inhibition profile of a variety of rhomboids. We demonstrate that activity-based protein profiling (ABPP) represents a general and easy to perform assay for investigation of the activity and inhibition of rhomboids from different species. By screening of 50 small molecules against 13 different rhomboid proteases we obtain ‘fingerprints’ revealing that some compounds display selectively inhibition rhomboid proteases from certain species. These compounds mainly comprised -lactones. In addition to the known DCI, we find another ‘pan’ rhomboid protease inhibitor based on the isocoumarin scaffold. Some of the novel rhomboid inhibitors discovered in this work contain a small alkyne tag and can be used as ABPs in future studies. In the course of the fingerprint experiments, we observed that some rhomboids appeared as multiple bands in polyacrylamide gels. Using inhibitors discovered in this study, we were able to show that these rhomboids autoprocess themselves in their N-terminal part.


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RESULTS AND DISCUSSION ABPP of rhomboid proteases. For our experiments, we selected 13 rhomboid proteases: the rhomboids

from

Aquifex

aeolicus

(AaROM),

Archaeoglobus

fulgidus

(AfROM),

Methanocaldococcus jannaschii (MjROM), Pyrococcus horikoshii (PhROM), Thermotoga maritima (TmROM), Vibrio cholerae (VcROM), AarA from Providencia stuartii (PsAarA), GlpG from Escherichia coli (EcGlpG) and Haemophilus influenzae (HiGlpG), YqgP from Bacillus subtilis (BsYqgP), mouse RHBDL1 and RHBDL3 (MmRHBDL1 and MmRHBDL3), and Rhomboid-1 from the fruit fly Drosophila melanogaster (DmRho1). This set includes rhomboid proteases from all three phylogenetic domains (bacteria, archaea and eukaryota) and displays two distinct topologies: the basic type rhomboid with a 6 TM structure and the rhomboid type with an additional seventh TM C-terminal to the 6 TM core (Figure 1a).2 We recombinantly expressed all 13 rhomboids in E. coli and purified them in dodecyl maltoside (DDM) micelles according to literature procedures. 9, 22 Since no universal rhomboid protease substrate has been reported, we turned our attention to activity-based protein profiling (ABPP) for the detection of activity. ABPP is a chemical proteomics technique in which active enzymes, but not their inactive counterparts, are labeled by small molecule activity-based probes (ABPs).

23, 24

In short, ABPP gives a direct read-out of

the chemical reactivity of the active site machinery by means of a mechanism-based reaction between the ABP and the enzyme’s active site nucleophile. In the past, ABPP has proven especially useful for the study of proteases,25,

26

and has been applied in imaging, profiling

experiments and inhibitor screening (Figure 2a). It conveniently circumvents the need for a peptide or protein substrate and therefore allows the analysis of ill-characterized enzymes. Inhibitor screening is conducted by competitive ABPP, in which samples are first treated with small molecules and then probed for residual activity. Recently, we and others have reported on ABPs for EcGlpG,15,

17, 20

and PsAarA.15 One of

these ABPs is the commercially available fluorophosphonate FP-Rh (Figure 2b).27 FP-Rh labels



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wild type EcGlpG, but not the inhibited or active site mutated form (Figure 2c). Gratifyingly, all other rhomboids were also labeled with FP-Rh (Supplementary Figure 1), which is probably a result of their comparable catalytic mechanism. Hence, FP-Rh is a suitable probe for the measurement of inhibition of all 13 expressed rhomboids. A rhomboid protease inhibitor fingerprint. Previously, we have observed differences in the inhibitory profile of EcGlpG and PsAarA using a set of structurally diverse isocoumarins.15 We here selected a panel of 50 small molecules (Supplementary Table 1) in order to obtain an ‘inhibitor fingerprint’ for the rhomboid proteases. All small molecules in the panel contain an electrophile that can potentially react with the active site serine residue. They have been selected from a larger set of molecules that were screened before against GlpG.20 Additionally, we have recently shown that the inhibitory profile of these small molecules against GlpG in micelles and in a lipid bilayer is identical.28 To measure inhibition, the rhomboid proteases were treated in duplicate with 50 small molecules (100 M) or DMSO as a vehicle control (see Supplementary Table 1 for their chemical structures). Subsequently, the ABP FP-Rh was added to detect the residual rhomboid activity. Inhibition was quantified by gel band densitometry and their numerical values were color-coded with black and red tiles (Figure 3a; for representative gels of all rhomboids, see Supplementary Figure 2). In order to investigate possible similarities between the different rhomboids and also to better visualize the selective inhibitors, the numerical values were analyzed using software developed for clustering and visualization of microarray data.29, 30.The resulting heat map (Figure 3b) shows several black and red areas that allow to distinguish different types of compounds: (1) The two isocoumarins 2 (DCI) and 21 (4-chloro-7-nitro-3-(5phenyl-pentyl)-isocoumarin) act as ‘pan’ inhibitors that inhibit virtually all 13 rhomboids; (2) Small molecules that do not or only weakly inhibit rhomboids, indicated as ‘weak or inactive compounds’; (3) Inhibitors that display a certain selectivity for a subselection of rhomboids. For the rhomboids, three groups can be observed in the heat map: (1) rhomboids inhibited almost


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exclusively by the pan inhibitors 2 and 21 and a few selective inhibitors; (2) rhomboids inhibited by -lactones and pan inhibitors, and (3) rhomboids inhibited by pan inhibitors and many isocoumarins. Interestingly, a sequence-based clustering does not match the inhibition profile clustering. For easy comparison these are represented both as hereditary trees in Figure 3c. The differences in these trees indicate that the active site environment probed by the small molecule inhibitors does not correlate to the overall amino acid sequence. New rhomboid inhibitors and ABPs. We selected several inhibitor structures and determined their apparent IC50 by competitive ABPP (Table 1, Table 2, Supplementary Figure 4). Although the pan inhibitors 2 and 21 react with virtually all tested rhomboids, they display a wide activity range of apparent IC50 values. For example, low to sub micromolar apparent IC50 values were found for inhibition of the bacterial rhomboids AaROM, HiGlpG and TmROM by DCI, whereas for MmRHBDL3, the value was approximately two orders of magnitude higher. The more selective inhibitors, including several -lactones, showed inhibition values in the low to mid micromolar range, in line with a previous report on the inhibition of EcGlpG.20 These compounds were only tested against selected rhomboids, since the fingerprint indicated that – at a concentration of 100 M – they poorly reacted with the other rhomboids. For both isocoumarins and -lactones, a covalent inhibition mechanism is expected. The active site serine reacts with the electrophilic carbonyl group and opens the isocoumarin or lactone ring respectively (Figure 4a). Since many of the found inhibitors have an alkyne group amenable for bioorthogonal click chemistry, we set out to prove the covalent inhibition mechanism by incubation of rhomboids with alkynylated inhibitors, followed by conjugation of the covalent complex to an azide-containing fluorophore by copper-catalyzed azide-alkyne cycloaddition. For all but one tested compound, we found a fluorescently labeled rhomboid, indicating that the small molecules had covalently bound (Figure 4b). Hence, compounds 5, 10,



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16, 43, 44, 45, 47, and 48 represent, together with FP-Rh, the first ABPs for AaROM, AfROM, BsYqgP, HiGlpG and VcROM. Of the tested -lactones, some showed a remarkable degree of selectivity. For example, compound 48 inhibits EcGlpG along with the rhomboids from T. maritima (TmROM) and A. aeolicus (AaROM), while compound 45, which is structurally related to compound 48, does not inhibit EcGlpG, but the rhomboid from A. fulgidus (AfROM). These two compounds only differ in the substituent adjacent to the carbonyl group (position 3 of the oxetane/-lactone scaffold, see Figure 5a). Therefore, this substituent must be the source of the compound selectivity. In the past, the structurally related class of -lactams was reported as inhibitor scaffold for rhomboids. Interestingly, a substituent at the 3-position is absent from the -lactam inhibitors previously tested on EcGlpG, such as L29 (Figure 5b).18, 19 In contrast to the -lactones, it was found that the substituent on the nitrogen of the -lactam was responsible for the selectivity of inhibition.18To gain insight into the possible binding mode of the -lactones, we performed molecular docking experiments of -lactone 48 covalently bound in the crystal structure of EcGlpG (Figure 5c). Several differences can be observed compared with the -lactam inhibitor L29 (Figure 5d). For example, the carbonyl group, which is directly bound to the active site serine and resembles the acyl intermediate formed during substrate cleavage, points towards the oxyanion hole, formed by several residues including H150 (in green). This is in contrast to the carbonyl group of lactam inhibitor L29, which is directed away from the oxyanion hole and points towards H254 (part of the serine-histidine catalytic dyad). In L29, the phenyloxycarbonyl substituent on the nitrogen of the lactam ring that is responsible for the inhibitor selectivity, interacts with a pocket that is believed to be the S2’ cavity.19 Obviously, -lactone 48, which does not have a nitrogen in its ring structure, lacks this substituent. Instead, the alkynyl substituent at the 4-position in the four-membered ring scaffold fills the S2’ cavity and overlays in large part with the phenyloxycarbonyl group of the lactam. The substituent on 3-position of


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the -lactone, responsible for the selectivity of the compound, points towards a small hydrophobic patch on TMD5. It thereby adopts a different orientation as the other substituent on the -lactam L29 (the phenyl group at the 4-position), which protrudes through the gap between TMD2 and TMD5 (Figure 5e). Overall, it suggests that the -lactones, although lacking a substituent at the 1-position of the ring system, are flexible enough to adopt a conformation that allows them to interact in a selective manner with the hydrophobic surfaces near the active site. A limitation of the current isocoumarins and -lactones is that they are also reactive against other serine hydrolases, as has been reported before.31, 32 However, this does not preclude their use in combination with analyses that utilize a separation technique or a specific detection method. Here, we have used 1D SDS-PAGE, but analogous to other studies, methods such as LC-MS/MS,33 microarray34-36 or activity-based ELISA37 may also be applied. Highly selective inhibitors are still a future challenge, but design based on co-crystal structures and screening may improve inhibitor properties. In this light, it will be interesting to synthesize -lactams that not only have substituents on the nitrogen and the 4-position, but also at the 3-position. Docking within the EcGlpG active site suggests that such an arrangement may lead to a combination of the interactions that are observed for the -lactam L29 and the -lactone 48 (Supplementary Figure 5), which might result in a higher affinity and selectivity. Rhomboid inhibitors reveal autoprocessing. During our labeling experiments we observed that the rhomboids PsAarA and VcROM appeared on gels as multiple bands that ran close together. We have previously reported a similar observation for the rhomboid EcGlpG. When EcGlpG was reacted with an isocoumarin ABP, this caused a cross-link between two TMs, resulting in a double band in the gel.15 To test whether the multiple bands for PsAarA and VcROM were caused by reaction with the FP-Rh probe, the rhomboids were incubated with DMSO or FP-Rh for 2h and visualized by fluorescence (for activity) and Western Blot or Coomassie stain (for protein abundance). For PsAarA, a clear double band was observed with



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molecular weights of approximately 33 and 30 kDa, while for VcROM, multiple bands were visible, with the strongest species at approximately 31 kDa, and several species below (Figure 6a). The multiple rhomboid species were also visible in the DMSO control, indicating that they were not caused by reaction with the FP-Rh probe. Without any incubation time, the VcROM sample almost exclusively showed a protein species at 31 kDa, which corresponds to the molecular weight of the full length protein, and only upon incubation at 37 °C, lower species started to occur (Figure 6a, Supplementary Figure 6). PsAarA was already isolated as a double band, but on incubation, the top band became less intense, whereas the lower species increased in intensity (Figure 6b). We reasoned that the different species could occur through autoprocessing. In order to exclude that the observed bands were caused through proteolysis during expression or by a protease that might have been co-purified during purification, we performed a time-course experiment with different inhibitors: the Roche “Complete” cocktail of protease inhibitors, which does not inhibit rhomboids,9 compound 19, which inhibits both VcROM and PsAarA, or DMSO as a control. Strikingly, the formation of the lower VcROM protein species was prevented by addition of rhomboid inhibitor 19, but not by addition of “Complete” (Figure 6c). As shown by FPRh labeling, complete does not influence the VcROM activity, whereas compound 19 does. These results indicate that processing is indeed dependent on VcROM activity. Also for PsAarA incubation with rhomboid inhibitor 19, but not with “Complete” prevented the conversion of the top species into the lower one (Figure 6c). We also expressed and purified the active site PsAarA mutant S150A. In contrast to the wild type species, this catalytically inactive form only showed a single band on gel, both directly after purification and after incubation at 37 °C (Figure 6c). This band corresponds to the upper band in the WT sample. Hence, these experiments demonstrate that PsAarA is already being processed during expression, and that the active form of PsAarA is responsible for this process.


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Interestingly, a gel analysis with a higher resolving power showed that some of the processed VcROM species were not reactive towards FP-Rh, indicating that they lack an active catalytic site (marked by arrowheads in Figure 6d). Edman degradation allowed the identification of the two slowest migrating species (Figure 6e). The first species corresponds to the full length protein with an eight amino acid N-terminal extension originating from the GST-Tag and Precission protease cleavage site.22 The second species starts at amino acid 16, resulting from proteolytic processing between Ala15 and Phe16 (Figure 6e), and appears to be inactive (Figure 6d). The cleavage site itself is found in the cytoplasmic domain of the VcROM according to the predicted topology (Figure 6f). The calculated molecular weights of these two species are 31.6 and 29.4 kDa, respectively, and correspond well with the observed molecular weights in the gel analysis. For the other cleavages, it seems likely that they also occur from the N-terminal side, because the C-terminus of the full length rhomboid is located close to TM6, which contains the active site histidine. Hence, any processing at the C-terminal side would compromise the active site dyad. This would be in contradiction with the observed reactivity of the lower processed forms towards FP-Rh, which suggests that the active site remains intact. For PsAarA, the occurrence of only two different forms indicates that the processing takes place at one specific site. We wondered why the full length PsAarA was isolated together with a processed form and VcROM as single species. An explanation was found in the location of the purification tags. PsAarA carries a C-terminal His-tag. Processing at the N-terminal side will therefore yield two species upon purification. VcROM, however, was expressed with an Nterminal GST tag followed by a Precission cleavage site, which was used for elution. Any prior proteolytic processing would therefore cleave off the GST tag and not result in isolation of these processed forms. Although we were not able to map the exact PsAarA cleavage site, the presence of the C-terminal His-tag in both forms and its 3 kDa lower molecular weight indicate that the processing occurs at the N-terminal side, in the predicted first TMD.



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For the sequence of labeling and cleavage of the faster migrating protein species, we considered two possibilities: (1) labeling prior to cleavage: in this case only the full length rhomboid is labeled and then truncated into smaller, fluorescently labeled fragments; or (2) first truncation into smaller species which are then labeled. In a time course, where VcROM was first incubated at 37 °C for 0 or 24 h and subsequently labeled by FP-Rh for 2 h, it can be seen that the full length species has completely disappeared after 24 h and that there is no active, full length species left (Figure 6c). Despite this, the lower bands are still being labeled, indicating that they possess an intact active site dyad with reactivity against FP-Rh. In the past, one report has suggested that processing of the cytosolic domain of human PARL depends on PARL activity, but no direct autoprocessing could be shown.38 Since we here have used purified proteins in combination with rhomboid protease inhibitors, the cleavage must occur by autoprocessing. At this point, it is unclear what the biochemical reason for this proteolytic processing is. For EcGlpG, the cytoplasmic domain is involved in dimerization,39 which is in turn implicated in exosite-mediated substrate recognition.40 Whether this is also true for other rhomboids, remains to be elucidated, but it is tempting to speculate that proteolytic processing may affect dimerization or substrate recognition. In conclusion, we have here shown that rhomboid ABPP can be more generally applied than on the previously used EcGlpG: the FP-Rh probe labels rhomboids from three different phylogenetic domains and can therefore be considered a universal ABP for rhomboid proteases. Because of the lack of a universal substrate for rhomboid activity read-out, competitive ABPP represents a powerful tool for inhibitor screening against rhomboid proteases. The here described inhibitor fingerprint represents the largest amount of rhomboid proteases from different organisms screened against a set of the same small molecule inhibitors. In addition to two ‘pan’ rhomboid inhibitors (the known DCI and isocoumarin 21), we found that the tested lactones display a higher degree of selectivity than the isocoumarins. Interestingly, cluster analyses of rhomboids based on sequence showed a different pattern than based on inhibition.


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Sequence similarities do apparently not imply susceptibility for the same inhibitor structures. All in all, these results underline the importance of a deeper structural understanding for efficient inhibitor development, but suggest that selective inhibition of related rhomboids is feasible.

METHODS Chemicals. Dodecyl--D-maltoside (Carl Roth) and FP-Rhodamine (Thermo Fisher) were used as received. All library compounds have been described in the literature31,

32, 41

, or were

purchased if commercially available. Protein expression and purification. All rhomboids were expressed and purified according to literature procedures9, 22 with minor modifications as detailed in the supporting information. Gel-based ABPP. 200 ng of rhomboid sample was diluted into 20 L reaction buffer (50 mM HEPES pH 7.4 with 10% (v/v) glycerol and 0.0125% (w/v) DDM), and incubated for 30 min at 37 °C with either 100 M compound or an equal volume of DMSO as vehicle control. Next, FP-Rh was added to a final concentration of 1 M and incubated for 2 h at 37 °C in the dark. The reaction was stopped by addition of 4x Laemmli buffer. 10 L were applied to 15% SDS-PAGE. Gels were scanned on a Typhoon Trio+ and analyzed using ImageJ.6 Silver staining was performed using an ammoniacal silvers staining protocol.7 For Western blot analysis of PsAarA, proteins were transferred onto nitrocellulose with a semi-dry blotter. The membrane was blocked with 3% milk powder in PBST (PBS with 0.1% (v/v) Tween). The proteins were visualized using anti-His6-peroxidase (Roche) and visualized on a Typhoon Trio+ scanner.

Associated content Supporting information Experimental details, Supplementary Figures and Tables. This material is available free of charge via the Internet at http://pubs.acs.org.



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Author information Corresponding author *E-mail: [email protected], [email protected] Author contributions E.V.W designed research, performed research, analyzed data and wrote the paper. A.Z. performed research. S.H.L.V. designed research, performed research, analyzed data and wrote the paper. All authors approved of the final version of the paper. Notes The authors declare no competing financial interest.

Acknowledgements This work was supported by funding of the Deutsche Forschungsgemeinschaft (to S.H.L.V.) and the Elite Network of Bavaria (to E.V.W). We thank M. Freeman, M. Lemberg, M. Lemieux and S. Urban for rhomboid vectors, S. Sieber and R. Liskamp for -lactones and sulfonyl fluoride compounds, K. Strisovsky for arranging Edman degradation, M. Teese for help with structural alignment and phylogenetic analysis, and D. Langosch for general support.

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Figure legends Figure 1. Rhomboid proteases and inhibitors. a) Rhomboid proteases of the ‘secretase’ type.2 The rhomboid 6 TM core structure (left) and 7 TM rhomboid with an additional C-terminal TM helix. b) The different scaffolds reported as rhomboid inhibitors. Dichloroisocoumarin (DCI) as the first discovered inhibitor and the related 4-chloro-isocoumarin structures (ICs). Fluorophosphonates (FPs), such as the general serine hydrolase probe FP-biotin, are highly reactive electrophiles and may react with most serine hydrolases.42 Other inhibitor scaffolds include -lactones, -lactams and chloromethyl ketones (CMKs). Note that all of these inhibitors contain electrophiles and undergo a mechanism-based reaction with the active site serine of the rhomboid protease.

Figure 2. Competitive rhomboid activity-based protein profiling. a) Schematic representation of competitive ABPP. A purified enzyme or cell lysate is treated with a potential inhibitor. Subsequently, the residual active enzyme(s) are labeled by means of an ABP. b) The ABP FPRh and its mechanism of covalent labeling of a serine protease. c) Active WT EcGlpG, but not an inhibited wild type or active site mutant, can be efficiently labeled by FP-Rh and visualized by in-gel fluorescent scanning (Mut = EcGlpG S201A). Figure 3. An inhibitor fingerprint for rhomboid proteases. a) In duplicate, EcGlpG was incubated with 50 small molecules (100 M) or DMSO, and the residual active protease was then labeled by FP-Rh and analyzed on gel. Quantification was performed by gel band densitometry and the values converted into a heat map. b) A heat map representation of inhibition of 13 rhomboid proteases. Cluster analysis was performed on compounds as well as rhomboids. c) Left: phylogenetic analysis of rhomboids sequences (see Figure S3 for their alignment). The numbers at the nodes represent the percentage or replicate trees in which the associated taxa


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clustered together in the bootstrap test for all values greater 70%. Right: Clustering of rhomboid proteases according to their inhibition profile. The numbers at the nodes represent the correlation of the members within the node.

Figure 4. Covalent modification and fluorescent labeling of rhomboid proteases from different species. a) Both -lactones and isocoumarins display a covalent inhibition mechanism. The active site S201 attacks the electrophilic carbonyl group, leading to opening of the lactone or isocoumarin ring structure. In a second step, the alkyne of the inhibitor can be functionalized with an azide-conjugated fluorophore by copper-catalyzed azide-alkyne cycloaddition. b) Rhomboids from seven different species were incubated with the indicated compounds (100 M) and detected by conjugation with a fluorophore. All tested compounds, except for compound 14, gave a fluorescent signal, indicating the covalent nature of inhibition. Labeling of the AaROM by compound 14 may have been too weak or instable for visualization.

Figure 5. Comparison of -lactone and -lactam structures covalently bound to EcGlpG. a) The four-membered ring scaffold of -lactones and -lactams. b) The reaction of -lactone 48 and lactam L29 with the active site serine of EcGlpG, leading to a covalent bond between S201 and the ring-opened inhibitor structure. c) Docked structure of ring-opened 48 (yellow; oxygen atoms in red) in the active site of EcGlpG. The EcGlpG is depicted as a surface model, with basic residues in dark blue, acidic residues in red and polar residues in light blue. H150 and H254 are displayed as stick models in green. d) Covalently bound -lactam inhibitor L29 (purple; oxygen atoms in red; PDB: 3ZMI). e) Overlay of the bound forms of 48 and L29.

Figure 6. Autoprocessing of two bacterial rhomboids. a) Lower running protein species were detected for VcROM and PsAarA. Both rhomboid proteases were reacted with DMSO or FP-Rh



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(1 M for 2h) and samples were visualized by a fluorescent scanner. For PsAarA, protein abundance was detected by Western Blot. For VcROM Coomassie stain was used. Catalytically active bands were detected that ran lower than the full length rhomboid protease. b) Lower molecular weight species of VcROM and PsAarA are formed over time. c) In a time course, VcROM and PsAarA show an increased formation of the lower molecular weight species, but not when rhomboid inhibitor 19 is used. The S150A mutant of PsAarA does not show processing. Note that truncated VcROM species are still labeled by FP-Rh, even when the full length VcROM is absent. d) Some of the truncated VcROM species do not show reactivity against FP-Rh, indicated with stars. e) Edman degradation revealed the nature of the full length and the slightly lower running species of VcROM, as indicated with a and b in Figure 6d. f) The topology of recombinantly expressed VcROM as predicted by Phobius.43


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Table 1. Apparent IC50 values (M) for two isocoumarin pan rhomboid inhibitors Rhomboid

AaROM

1.1  0.2

1.7  0.2

AfROM

8.1  2.0

N.D.

BsYqgP

4.1  0.9

25  6

EcGlpG

19  4.5

N.D.*

HiGlpG

0.66  0.10

4.6  1.0

MjROM

3.1  0.5

9.7  1.6

MmRHBDL1

28  9

39  6

MmRHBDL3

70  13

24  3

PhROM

16  5

25  10

PsAarA

10  2

10.0  0.8

TmROM

1.0  0.4

28  7

VcROM

27  9

76  28

Table 2. Apparent IC50 values (M) for selective rhomboid inhibitors. Compound

Rhomboid

IC50

AfROM

5.0  0.9

HiGlpG

65  9

TmROM

27  6



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TmROM

48  10

EcGlpG

44  6


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Figure 1. Rhomboid proteases and inhibitors. (A) Rhomboid proteases of the ‘secretase’ type.2 The rhomboid 6 TM core structure (left) and 7 TM rhomboid with an additional C-terminal TM helix. (B) The different scaffolds reported as rhomboid inhibitors. Dichloroisocoumarin (DCI) as the first discovered inhibitor and the related 4-chloro-isocoumarin structures (ICs). Fluorophosphonates (FPs), such as the general serine hydrolase probe FP-biotin, are highly reactive electrophiles and may react with most serine hydrolases.42 Other inhibitor scaffolds include β-lactones, β-lactams and chloromethyl ketones (CMKs). Note that all of these inhibitors contain electrophiles and undergo a mechanism-based reaction with the active site serine of the rhomboid protease. 66x102mm (300 x 300 DPI)



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Figure 2. Competitive rhomboid activity-based protein profiling. a) Schematic representation of competitive ABPP. A purified enzyme or cell lysate is treated with a potential inhibitor. Subsequently, the residual active enzyme(s) are labeled by means of an ABP. b) The ABP FP-Rh and its mechanism of covalent labeling of a serine protease. c) Active WT EcGlpG, but not an inhibited wild type or active site mutant, can be efficiently labeled by FP-Rh and visualized by in-gel fluorescent scanning (Mut = EcGlpG S201A). 65x58mm (300 x 300 DPI)


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Figure 3. An inhibitor fingerprint for rhomboid proteases. a) In duplicate, EcGlpG was incubated with 50 small molecules (100 µM) or DMSO, and the residual active protease was then labeled by FP-Rh and analyzed on gel. Quantification was performed by gel band densitometry and the values converted into a heat map. b) A heat map representation of inhibition of 13 rhomboid proteases. Cluster analysis was performed on compounds as well as rhomboids. c) Left: phylogenetic analysis of rhomboids sequences (see Figure S3 for their alignment). The numbers at the nodes represent the percentage or replicate trees in which the associated taxa clustered together in the bootstrap test for all values greater 70%. Right: Clustering of rhomboid proteases according to their inhibition profile. The numbers at the nodes represent the correlation of the members within the node. 137x154mm (300 x 300 DPI)



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Figure 4. Covalent modification and fluorescent labeling of rhomboid proteases from different species. a) Both β-lactones and isocoumarins display a covalent inhibition mechanism. The active site S201 attacks the electrophilic carbonyl group, leading to opening of the lactone or isocoumarin ring structure. In a second step, the alkyne of the inhibitor can be functionalized with an azide-conjugated fluorophore by coppercatalyzed azide-alkyne cycloaddition. b) Rhomboids from seven different species were incubated with the indicated compounds (100 µM) and detected by conjugation with a fluorophore. All tested compounds, except for compound 14, gave a fluorescent signal, indicating the covalent nature of inhibition. Labeling of the AaROM by compound 14 may have been too weak or instable for visualization. 66x147mm (300 x 300 DPI)


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Figure 5. Comparison of β-lactone and β-lactam structures covalently bound to EcGlpG. (A) The fourmembered ring scaffold of β-lactones and β-lactams. (B) The reaction of β-lactone 48 and β-lactam L29 with the active site serine of EcGlpG, leading to a covalent bond between S201 and the ring-opened inhibitor structure. (C) Docked structure of ring-opened 48 (yellow; oxygen atoms in red) in the active site of EcGlpG. The EcGlpG is depicted as a surface model, with basic residues in dark blue, acidic residues in red and polar residues in light blue. H150 and H254 are displayed as stick models in green. (D) Covalently bound β-lactam inhibitor L29 (purple; oxygen atoms in red; PDB: 3ZMI). (E) Overlay of the bound forms of 48 and L29. 139x91mm (300 x 300 DPI)



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Figure 6. Autoprocessing of two bacterial rhomboids. a) Lower running protein species were detected for VcROM and PsAarA. Both rhomboid proteases were reacted with DMSO or FP-Rh (1 µM for 2h) and samples were visualized by a fluorescent scanner. For PsAarA, protein abundance was detected by Western Blot. For VcROM Coomassie stain was used. Catalytically active bands were detected that ran lower than the full length rhomboid protease. b) Lower molecular weight species of VcROM and PsAarA are formed over time. c) In a time course, VcROM and PsAarA show an increased formation of the lower molecular weight species, but not when rhomboid inhibitor 19 is used. The S150A mutant of PsAarA does not show processing. Note that truncated VcROM species are still labeled by FP-Rh, even when the full length VcROM is absent. d) Some of the truncated VcROM species do not show reactivity against FP-Rh, indicated with stars. e) Edman degradation revealed the nature of the full length and the slightly lower running species of VcROM, as indicated with a and b in Figure 6d. f) The topology of recombinantly expressed VcROM as predicted by Phobius.43 140x120mm (300 x 300 DPI)


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30x8mm (300 x 300 DPI)



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39x9mm (300 x 300 DPI)


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Inhibitor Fingerprinting of Rhomboid Proteases by Activity-Based

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