Communication Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Stable and Functional Rhomboid Proteases in Lipid Nanodiscs by Using Diisobutylene/Maleic Acid Copolymers Marta Barniol-Xicota† and Steven H. L. Verhelst*,†,‡ †
Laboratory of Chemical Biology, Department of Cellular and Molecular Medicine, KU Leuven − University of Leuven, Herestraat 49 box 802, 3000 Leuven, Belgium ‡ AG Chemical Proteomics, Leibniz Institute for Analytical Sciences − ISAS, Otto-Hahn-Str. 6b, 44227 Dortmund, Germany
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ABSTRACT: Rhomboid proteases form a paradigm for intramembrane proteolysis and have been implicated in several human diseases. However, their study is hampered by difficulties in solubilization and purification. We here report on the use of polymers composed of maleic acid and either diisobutylene or styrene for solubilization of rhomboid proteases in lipid nanodiscs, which proceeds with up to 48% efficiency. We show that the activity of rhomboids in lipid nanodiscs is closer to that in the native membrane than rhomboids in detergent. Moreover, a rhomboid that was proteolytically unstable in detergent turned out to be stable in lipid nanodiscs, underlining the benefit of using these polymer-stabilized nanodiscs. The systems are also compatible with the use of activity-based probes and can be used for small molecule inhibitor screening, allowing several downstream applications. Figure 1. (A) Crystal structure of the E. coli rhomboid protease GlpG (PDB code: 2IC8) with the protein in cartoon depiction and the active site residues S201 and H254, in TM4 and TM6, respectively, drawn as sticks. (B) Chemical formulas of SMA and DIBMA. For SMA 3:1, x ≈ 3 and n ≈ 9, for DIBMA, m ≈ 37. (C) Schematic representation of a rhomboid protease in a membrane and its solubilization by lipid nanodisc formation with a SMA or DIBMA polymer, or by micelle formation upon addition of a detergent.
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homboid proteases are among the most widespread intramembrane proteases (IMPs) and have been found in virtually all sequenced organisms.1,2 The active site of rhomboids is located in the plane of the membrane and consists of a serine and a histidine residue located in two different transmembrane helices (Figure 1A).3 The natural substrates, which are transmembrane proteins themselves, are cleaved within their transmembrane helix or in a juxtamembrane region. The biological roles of rhomboid proteases are diverse and range from EGF signaling4−6 to quorum sensing.7 Rhomboid proteases have also been implied in human disease, including Parkinson’s disease8,9 and malaria.10,11 Structurally, they are the best characterized IMPs,3,12,13 and a variety of different rhomboid inhibitor scaffolds have been reported.14−18 Virtually all information on the structure and inhibition of rhomboids was gathered by using the Escherichia coli model rhomboid GlpG (EcGlpG). Production and purification of rhomboid proteases from other species remains a very challenging task. Unfortunately, this impedes further structural studies and inhibitor development for biomedically relevant rhomboid proteases. The lipid bilayer of the membrane is key for the structure and function of many integral membrane proteins, including rhomboids. Unfolding experiments on EcGlpG have shown that rhomboid proteases display low intrinsic thermodynamic stability, caused by various weak transmembrane packing interactions.19 The membrane environment is therefore © XXXX American Chemical Society
thought to have a stabilizing effect on the structure of rhomboid proteases. 20 Although the reconstitution of rhomboids in liposomes has been described, it requires prior solubilization and purification in a detergent environment.21−23 The usage of lipid nanoparticles is a recent strategy to prevent removing the lipid bilayer environment around membrane proteins.24 The application of amphipathic polymers composed of maleic acid and styrene or diisobutylene (SMA25,26 or DIBMA,27,28 respectively; collectively xMA; Figure 1B) is a rapidly emerging solubilization method. These polymers can be applied directly to membranes, resulting in the formation of particles containing membrane proteins in a native-like lipid bilayer stabilized by an xMA polymer belt (Figure 1C). In this paper, we report the direct solubilization of rhomboid proteases into lipid nanodiscs by xMA polymers. We show that xMA lipid particles (xMALPs) result in only slightly lower solubilization efficiency compared with the Received: August 7, 2018 Published: October 22, 2018 A
DOI: 10.1021/jacs.8b08441 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Communication
Journal of the American Chemical Society
Figure 2. Solubilization of EcGlpG. (A) Application of DIBMA polymer solubilizes crude membranes: a turbid membrane suspension turns clear upon addition of the DIBMA polymer (upper panel) and increasing concentrations of DIBMA polymer lead to better solubilization, having its max at 2.5% v/v concentration after which an improvement cannot be observed as judged from the residual membrane pellet upon ultracentrifugation (lower panel). (B) DDM detergent as well as DIBMA solubilized recombinant EcGlpG can be purified by Ni-NTA mediated chromatography. E1− E5 corresponds to the different elution fractions. (C) Solubilization of GlpG by DDM detergent, DIBMA or SMA25 all yield active protease, as judged by labeling with the activity-based probe FP-Rh, which is diminished upon addition of an inhibitor. (D) Relative activities of GlpG in different environments, as judged by FP-Rh labeling, measured in triplicates. ** denotes p-value in t test < 0.01.
tion yields the xMAs as white powders (Figure S1 in Supporting Information), which were made into ready-to-use 6% stock solutions (v/v). The direct addition of the xMA stock solutions to membrane pellets containing overexpressed, His6-tagged EcGlpG led to solubilization (Figure 2A). Compared with the standard solubilization by dodecyl-maltoside (DDM), which is usually carried out at 4 °C, the xMA solubilization was most effective at 37 °C, without hampering the protease stability (see also later). Purification of GlpG by Ni2+-NTA affinity chromatography led to comparable purity (Figure 2B) albeit with somewhat lower yields than for detergent solubilization (Table S1 in Supporting Information). In order to assess the activity state of the solubilized EcGlpG, we applied labeling with the activity-based probe (ABP) FP-Rh, a general serine hydrolase probe29 that is also pan-reactive against rhomboid proteases.30 Satisfyingly, all solubilized rhomboids were labeled with FP-Rh, indicating an intact active site (Figure 2C). The labeling was diminished by pretreatment with the isocoumarin inhibitor S016,31 illustrating that xMALPs are compatible with screening for small molecule inhibitors. Comparison with labeling of a membrane pellet, in which EcGlpG resides in its native membrane, revealed similar activity as the xMALPs, with DIBMALPs showing activity closest to the native lipid bilayer (Figure 2D). Strikingly, DDM-solubilized EcGlpG displayed approximately 8 times higher activity compared with the membrane pellet, which may be due the increased mobility or active site accessibility. A recent study on membrane protein conformational dynamics that used EcGlpG also revealed a much higher activity after solubilization in DDM compared with SMALPs, which is in accordance with our findings here.20 We characterized EcGlpG-containing DIBMALPs by different biophysical experiments. Dynamic light scattering (DLS) revealed a narrow size distribution (Figure 3A) of the lipid nanodiscs with an average diameter of 10.8 and 12.7 nm for SMA30 and DIBMA, respectively, which is in accordance with literature values.24 Transmission electron microscopy allowed
standard detergent used for rhomboid proteases. The activity of rhomboids in xMALPs resembles the lipid membrane environment better than when in micelles. Moreover, xMALPs are able to stabilize rhomboid proteases that process themselves in micelles. Hence, they are an attractive choice for solubilization of IMPs as well as for a more accurate study in their native environment. In order to compare detergent solubilization of rhomboid proteases with solubilization in xMALPs, we selected three different polymers: two types of SMA copolymers (XIRAN SL25010 S25 and XIRAN SL30010 S30, which differ in their styrene: maleic acid ratio; 3:1 and 2.3:1 respectively) and DIBMA (Figure 1B). To date, more than 30 examples of membrane proteins solubilized in SMALPs have been reported. Contrastingly, only few examples have been described using DIBMA as the solubilizing polymer of choice, despite its apparent advantages over the styrene containing analogs. These advantages include its lower disturbance of the lipid acyl chain order, hence providing a better native state condition, and its superior versatility (e.g., it does not precipitate in the presence of divalent cations28). Initial experiments with commercial SMA and DIBMA solutions led to poorly reproducible solubilization. Although in some research reports the xMA polymer is pretreated by dialysis25 or synthesized by hydrolysis of the maleic anhydride precursor followed by precipitation,26 the majority of solubilizations have been performed with the untreated polymer solution available from commercial suppliers. This prevents exact control over the polymer concentration, contaminating substances that may be present, and the pH, which is critical for the correct solubilizing function of the polymer; all in all adding several variability factors that can compromise the reproducibility of solubilization. We therefore isolated the polymer from the commercial solutions by the simple and straightforward precipitation protocol: addition of hydrochloric acid reverts the maleic acid from its soluble sodium salt form to its protonated state, leading to rapid precipitation. Further washing and lyophilizaB
DOI: 10.1021/jacs.8b08441 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Communication
Journal of the American Chemical Society
Figure 4. Stabilization of VcROM in DIBMALPs. (A) Incubation at 20 °C in DDM micelles leads to rapid degradation of the full length VcROM (full arrow) to a lower molecular weight species (open arrow), whereas it is stable in DIBMALPs. (B) Percentage intact full length VcROM over time when solubilized with DDM or DIBMA. During expression, all VcROM appears as full-length protein species (Figure S3C).
Figure 3. Characterization of EcGlpG containing xMALPs. (A) DLS revealed narrow size distribution of SMALPs and DIBMALPs with embedded EcGlpG. (B) Transmission electron microscopy pictures of DIBMALPs with EcGlpG. (C) FT-IR data (1460−1720 cm−1) for EcGlpG in DDM (red), DIBMALPs (blue) or SMALPs (green). Theoretical peak maxima for α-helix, random coil and β-sheet are indicated in the amide-I region. (D) TLC analysis of E. coli crude membranes (C.M.) and DIBMALPs. Upper panel shows separation of neutral lipids from phospholipids, in the lower panel, the different phospholipids were separated (PE, phosphatidylethanolamine; CL, cardiolipin; PG, phoshatidylglycerol).
protein band during the entire process of purification and prolonged incubation at different temperatures (Figures 4 and S3). These results illustrate that detergent solubilization may lead to rhomboid degradation, that the membrane environment can lead to protection against undesired proteolytic processing and generally lead to more stable purified rhomboids. Overall, this work describes the solubilization of rhomboid proteases in xMALPs. We have presented a straightforward protocol for the isolation of xMAs from solutions provided by commercial suppliers. Solubilization of rhomboids using these isolated polymers proceeded with satisfying yields in comparison with detergent. The xMALPs lead to a higher stability of rhomboids, as exemplified by solubilization of full length VcROM without autoprocessing. Notably, xMALPs tolerate the use of ABPs and can therefore be used in screening for inhibitors. We expect that xMALPs will aid the future solubilization and purification of challenging IMPs, such as eukaryotic rhomboids or other IMPs that may undergo autoprocessing or unfolding upon detergent solubilization. This will enable further structural studies and inhibitor development for biomedically relevant IMPs.
visualization of the nanodiscs and confirmed the size as determined by DLS (Figure 3B). FT-IR experiments revealed that EcGlpG in DIBMALPs is structurally similar to GlpG in micelles, which has been shown to be mainly α-helical (Figure 3C).3 EcGlpG in DIBMALPs has a higher α-helical content compared with DDM and SMALPs, which may be due to the lower perturbation of lipid acyl chain order and an overall more stable protein structure. Naturally, the IR spectrum of SMALPs also contains aborption bands of the styrene moiety, whereas these are absent in DIBMALPs (Figure 3C). We confirmed the presence of phospholipids in the EcGlpG containing DIBMALPs by using a phosphorus assay. An average of 10 phospholipids was associated per rhomboid molecule, which, although low, is in accord with previous findings of SMALPs25 and with a crystal structure of EcGlpG in a lipid environment that showed association of 14 lipids as a partial annulus surrounding the protein.32 TLC analysis of crude membranes and DIBMALP-solubilized membranes showed that DIBMALPs contain the main lipid classes form the E. coli membrane, but are enriched in neutral lipids (Figure 3D and Figure S2), which may explain the relatively low amount of phospholipids found in the phosphorus assay. Next, we turned our attention to the rhomboid protease of Vibrio cholerae (VcROM), which has previously been shown to self-process.30 Following the same procedure as for EcGlpG, VcROM was isolated in DIBMALPs as well as in DDM micelles. Whereas VcROM in DDM rapidly self-processed to a lower form, VcROM in DIBMALPs appeared as a full length
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.8b08441. Supporting figures and experimental procedures (PDF)
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AUTHOR INFORMATION
Corresponding Author
*
[email protected];
[email protected] ORCID
Marta Barniol-Xicota: 0000-0001-7957-2199 Steven H. L. Verhelst: 0000-0002-7400-1319 C
DOI: 10.1021/jacs.8b08441 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Communication
Journal of the American Chemical Society Notes
(15) Ticha, A.; Stanchev, S.; Vinothkumar, K. R.; Mikles, D. C.; Pachl, P.; Began, J.; Skerle, J.; Svehlova, K.; Nguyen, M. T. N.; Verhelst, S. H. L.; Johnson, D. C.; Bachovchin, D. A.; Lepsik, M.; Majer, P.; Strisovsky, K. General and Modular Strategy for Designing Potent, Selective, and Pharmacologically Compliant Inhibitors of Rhomboid Proteases. Cell Chem. Biol. 2017, 24 (12), 1523−1536. (16) Goel, P.; Jumpertz, T.; Mikles, D. C.; Ticha, A.; Nguyen, M. T. N.; Verhelst, S.; Hubalek, M.; Johnson, D. C.; Bachovchin, D. A.; Ogorek, I.; Pietrzik, C. U.; Strisovsky, K.; Schmidt, B.; Weggen, S. Discovery and Biological Evaluation of Potent and Selective NMethylene Saccharin-Derived Inhibitors for Rhomboid Intramembrane Proteases. Biochemistry 2017, 56 (51), 6713−6725. (17) Yang, J.; Barniol-Xicota, M.; Nguyen, M. T. N.; Ticha, A.; Strisovsky, K.; Verhelst, S. H. L. Benzoxazin-4-ones as novel, easily accessible inhibitors for rhomboid proteases. Bioorg. Med. Chem. Lett. 2018, 28 (8), 1423−1427. (18) Goel, P.; Jumpertz, T.; Ticha, A.; Ogorek, I.; Mikles, D. C.; Hubalek, M.; Pietrzik, C. U.; Strisovsky, K.; Schmidt, B.; Weggen, S. Discovery and validation of 2-styryl substituted benzoxazin-4-ones as a novel scaffold for rhomboid protease inhibitors. Bioorg. Med. Chem. Lett. 2018, 28 (8), 1417−1422. (19) Baker, R. P.; Urban, S. Architectural and thermodynamic principles underlying intramembrane protease function. Nat. Chem. Biol. 2012, 8 (9), 759−768. (20) Reading, E.; Hall, Z.; Martens, C.; Haghighi, T.; Findlay, H.; Ahdash, Z.; Politis, A.; Booth, P. J. Interrogating Membrane Protein Conformational Dynamics within Native Lipid Compositions. Angew. Chem., Int. Ed. 2017, 56 (49), 15654−15657. (21) Urban, S.; Wolfe, M. S. Reconstitution of intramembrane proteolysis in vitro reveals that pure rhomboid is sufficient for catalysis and specificity. Proc. Natl. Acad. Sci. U. S. A. 2005, 102 (6), 1883−1888. (22) Dickey, S. W.; Baker, R. P.; Cho, S.; Urban, S. Proteolysis inside the membrane is a rate-governed reaction not driven by substrate affinity. Cell 2013, 155 (6), 1270−81. (23) Wolf, E. V.; Seybold, M.; Hadravova, R.; Strisovsky, K.; Verhelst, S. H. L. Activity-based protein profiling of rhomboid proteases in liposomes. ChemBioChem 2015, 16, 1616−1621. (24) Lee, S. C.; Khalid, S.; Pollock, N. L.; Knowles, T. J.; Edler, K.; Rothnie, A. J.; Thomas, O. R. T.; Dafforn, T. R. Encapsulated membrane proteins: A simplified system for molecular simulation. Biochim. Biophys. Acta, Biomembr. 2016, 1858 (10), 2549−2557. (25) Knowles, T. J.; Finka, R.; Smith, C.; Lin, Y. P.; Dafforn, T.; Overduin, M. Membrane proteins solubilized intact in lipid containing nanoparticles bounded by styrene maleic acid copolymer. J. Am. Chem. Soc. 2009, 131 (22), 7484−5. (26) Lee, S. C.; Knowles, T. J.; Postis, V. L.; Jamshad, M.; Parslow, R. A.; Lin, Y. P.; Goldman, A.; Sridhar, P.; Overduin, M.; Muench, S. P.; Dafforn, T. R. A method for detergent-free isolation of membrane proteins in their local lipid environment. Nat. Protoc. 2016, 11 (7), 1149−62. (27) Oluwole, A. O.; Klingler, J.; Danielczak, B.; Babalola, J. O.; Vargas, C.; Pabst, G.; Keller, S. Formation of Lipid-Bilayer Nanodiscs by Diisobutylene/Maleic Acid (DIBMA) Copolymer. Langmuir 2017, 33 (50), 14378−14388. (28) Oluwole, A. O.; Danielczak, B.; Meister, A.; Babalola, J. O.; Vargas, C.; Keller, S. Solubilization of Membrane Proteins into Functional Lipid-Bilayer Nanodiscs Using a Diisobutylene/Maleic Acid Copolymer. Angew. Chem., Int. Ed. 2017, 56 (7), 1919−1924. (29) Patricelli, M. P.; Giang, D. K.; Stamp, L. M.; Burbaum, J. J. Direct visualization of serine hydrolase activities in complex proteomes using fluorescent active site-directed probes. Proteomics 2001, 1 (9), 1067−71. (30) Wolf, E. V.; Zeissler, A.; Verhelst, S. H. Inhibitor fingerprinting of rhomboid proteases by activity-based protein profiling reveals inhibitor selectivity and rhomboid autoprocessing. ACS Chem. Biol. 2015, 10 (10), 2325−2333. (31) Vosyka, O.; Vinothkumar, K. R.; Wolf, E. V.; Brouwer, A. J.; Liskamp, R. M.; Verhelst, S. H. L. Activity-based probes for rhomboid
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the Laboratory of Bacteriology at KU Leuven for usage of their French Press, Dr. Rodrigo Gallardo for help with DSL measurements, Dr. Pieter Baatsen for recording TEM data, Prof. Dr. Paul Van Veldhoven for help with TLC analysis, and Clàudia Perpiña ̀ for subcloning VcROM. This work was financially supported by a Marie Curie Individual Fellowship (to M.B.-X.), the Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen, the Senatsverwaltung für Wirtschaft, Technologie und Forschung des Landes Berlin and the Bundesministerium für Bildung und Forschung.
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DOI: 10.1021/jacs.8b08441 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Journal of the American Chemical Society proteases discovered in a mass spectrometry-based assay. Proc. Natl. Acad. Sci. U. S. A. 2013, 110 (7), 2472−7. (32) Vinothkumar, K. R. Structure of rhomboid protease in a lipid environment. J. Mol. Biol. 2011, 407 (2), 232−47.
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DOI: 10.1021/jacs.8b08441 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
