Analytical Biochemistry 485 (2015) 97–101
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Development of a fluorescence anisotropy-based assay for Dop, the first enzyme in the pupylation pathway Nir Hecht, Eyal Gur ⇑ The Department of Life Sciences & the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel
a r t i c l e
i n f o
Article history: Received 20 April 2015 Received in revised form 8 June 2015 Accepted 11 June 2015 Available online 18 June 2015 Keywords: Dop Mycobacteria Proteasome Proteolysis Pup
a b s t r a c t The Pup-proteasome system (PPS) carries out regulated tagging and degradation of proteins in bacterial species belonging to the phyla Actinobacteria and Nitrospira. In the pathogen Mycobacterium tuberculosis, where this proteolytic pathway was initially discovered, PPS enzymes are essential for full virulence and persistence in the mammalian host. As such, PPS enzymes are potential targets for development of antituberculosis therapeutics. Such development often requires sensitive and robust assays for measurements of enzymatic activities and the effect of examined inhibitors. Here, we describe the development of an in vitro activity assay for Dop, the first enzyme in the PPS. Based on fluorescence anisotropy measurements, this assay is simple, sensitive, and compatible with a high-throughput format for screening purposes. We demonstrate how this assay can also be reliably and conveniently used for detailed kinetic measurements of Dop activity. As such, this assay is of value for basic research into Dop and the PPS. Finally, we show that the assay developed here primarily for the mycobacterial Dop can be readily employed with other Dop enzymes, using the same simple protocol. Ó 2015 Elsevier Inc. All rights reserved.
According to the World Health Organization, tuberculosis (TB)1 is second only to HIV/AIDS as the greatest single infectious agent-based killer worldwide. This, together with the rise of multidrug-resistant strains of Mycobacterium tuberculosis, reflects the urgent need for new therapies against the disease. As such, attempts are constantly being made to identify novel targets for development of anti-TB therapeutics. One such potential target is the Pup-proteasome system (PPS), a proteolytic pathway identified in M. tuberculosis and shown to be essential for full resistance of the bacteria to nitric oxide, a reactive nitrogen intermediate secreted by activated macrophages [1–4]. The PPS is also essential for the prolonged survival of M. tuberculosis cells inside host tissues and maintenance of the bacteria in a dormant state [4]. The PPS was initially discovered in M. tuberculosis [1] and shortly thereafter was found to be conserved in Actinobcteria and Nitrospira [5,6]. In these two phyla, which compose many nonpathogenic species, the PPS functions to recycle amino acids under starvation conditions [7] and, presumably, to regulate certain cellular activities. It does so by conjugating Pup (Prokaryotic ubiquitin-like protein), a 64 amino acid-long and intrinsically disordered protein, to target proteins, thereby marking them for ⇑ Corresponding author. 1
E-mail address: [email protected] (E. Gur). Abbreviations used: TB, tuberculosis; PPS, Pup-proteasome system.
http://dx.doi.org/10.1016/j.ab.2015.06.019 0003-2697/Ó 2015 Elsevier Inc. All rights reserved.
degradation by the bacterial proteasome [8,9]. A single ligase, termed PafA (Proteasome accessory factor A), catalyzes the formation of an isopeptide bond between the c-carboxylate of the Pup C-terminal glutamate side chain and e-amines of protein substrate lysines [8,10]. In M. tuberculosis and related species, Pup is translated with a glutamine, rather than a glutamate, at the C-terminal, thus requiring that a deamidation reaction catalyzed by Dop (Deamidase of Pup) convert this glutamine into a glutamate [11]. Dop can also depupylate an already pupylated substrate, namely detach Pup from the target protein [12,13]. Both Dop activities, namely deamidation and depupylation, essentially involve the hydrolysis of an amide bond at the side chain of Pup C-terminal residue (Fig. 1), and therefore rely on the same enzymatic mechanism. Notably, although Dop and PafA each require ATP for their activity, PafA hydrolyzes an ATP molecule per each reaction cycle while Dop employs ATP as a cofactor without hydrolyzing it. Since Pup deamidation is a prerequisite for conjugation by PafA, Dop activity is essential for pupylation to occur in M. tuberculosis. Accordingly, M. tuberculosis dop mutants present reduced virulence and enhanced sensitivity to nitric oxide [3]. As such, Dop represents a potential target for development of inhibitors that can function as anti-TB medicine. The search for such inhibitors would benefit from the availability of a Dop activity assay that is simple, sensitive, and compatible with a high-throughput format for
98
Anisotropy-based Dop assay / N. Hecht, E. Gur / Anal. Biochem. 485 (2015) 97–101
Depupylation
Deamidation
PupQ
COO
O
O
H N
H N
glutamine side-chain
H
Dop
Dop
PupE
lysine side-chain
Target protein
tein pro
COO-
d
pupy lat e -
Pup
PupE COOO-
O
COOO-
NH2
O
NH3
Target protein
Fig.1. The Dop-catalyzed reactions deamidation and depupylation. The two activities of Dop employ an identical mechanism. PupQ and PupE denote Pup forms varying at their C-terminal residue. Q denotes a C-terminal glutamine whereas E denotes glutamate at that position.
screening purposes. Indeed, similar motivation has already led to the recent development of a Dop assay [14]. However, as this assay relies on chemical synthesis of a fluorogenic version of Pup, it is fairly complicated to establish. Accordingly, we describe in this paper the development of a simpler Dop assay based on fluorescence anisotropy and consider its potential use. Rather than employing chemical synthesis, our novel assay relies on the ability of PafA to catalyze Pup conjugation to fluorescein-labeled lysine residues. Specifically, we used fluorescein-5-carboxamide lysine (5-FAM Lys) to generate pupylated 5-FAM Lys (hereafter, Pup-Fl), as previously reported [15]. Following purification of Pup-Fl to homogeneity, its depupylation can be readily measured in a simple, continuous, and sensitive manner by monitoring changes in fluorescence anisotropy. Finally, the assay established here will facilitate mechanistic analysis of Dop activity, as it is general and can be easily adapted for use with Dop orthologs that are phylogenetically distant from the M. tuberculosis protein. Materials and methods Protein expression and purification Recombinant Mycobacterium smegmatis PafA and Pup were purified as previously described [16]. All Dop variants carried C-terminal polyhistidine tags and were expressed in Escherichia coli strain ER2566 from plasmid pET11a. Following induction with IPTG, the cultures were incubated overnight at 18 °C. Cells were lysed by sonication, and purification using Ni2+–NTA–agarose (Qiagen) was carried out according to a standard protocol, except that for purification of M. smegmatis Dop, buffers contained 10% glycerol (v/v). A second size exclusion chromatography purification step relied on a Superdex 75 column (GE Healthcare). For the M. smegmatis Dop variants, the buffer used for purification contained 50 mM Hepes, pH 8.0, 150 mM NaCl, 20 mM MgCl2, and 10% (v/v) glycerol. For purification of A. cellulolyticus Dop, the buffer contained 50 mM Hepes, pH 8.0, 300 mM NaCl, and 1 mM DTT. The protein was further purified by anion-exchange chromatography using a MonoQ column (GE Healthcare) and a linear 0 to 1 M NaCl gradient.
For purification of A. cellulolyticus Pup, the encoding gene was cloned into plasmid pSH21 under the transcriptional control of the T7 promoter in fusion to a DNA sequence encoding an N-terminal polyhistidine tag, the human titin I27 domain, and a TEV protease recognition sequence. The polyhistidine–I27–TEV–Pup chimera was expressed at 30 °C, and Ni2+–NTA purification was carried out as above. Following TEV cleavage, a buffer exchange step was carried out, and the polyhistidine–I27–TEV portion of the chimera was removed by loading the solution onto a Ni2+–NTA column. The flowthrough was collected, and Pup was further purified by anion-exchange chromatography using a MonoQ column (GE Healthcare) and a linear 0 to 1 M NaCl gradient. Pupylation of 5-FAM Lys Pupylation of 5-FAM Lys (AnaSpec) was carried out in pupylation buffer (50 mM Hepes, pH 7.5, 100 mM NaCl, 20 mM MgCl2, and 10% glycerol (v/v)) containing 2 mM ATP, 200 lM Pup, and 5 lM PafA for 3 h at 30 °C. After a buffer exchange step, Ni2+– NTA purification was carried out as above and the resulting Pup-Fl was loaded onto a Superdex 75 size exclusion column (GE Healthcare) equilibrated with 25 mM Hepes, pH 8.0. The concentration of Pup-Fl following all purification steps was measured spectroscopically using a 1 cm path-length cuvette and the extinction coefficients of fluorescein at 280 and 490 nm (24,000 and 82,000 M 1 cm 1, respectively) and of Pup at 280 nm (1490 M 1 cm 1). The following expression yielded Pup-Fl concentration: (A280–0.29A490)/1490. The degree of labeling was assessed by comparing the concentration of Pup-Fl to that of fluorescein. The latter was determined spectroscopically at 490 nm. The degree of labeling typically exceeded 90%. Fluorescence anisotropy assay Fluorescence anisotropy reactions contained Dop and Pup-Fl in pupylation buffer in a final volume of 50 ll. Fluorescence at the parallel and perpendicular polarizations was measured in 384-well plates using a Synergy 2 plate reader (Biotek). The excitation and emission wavelengths were 485 and 528 nm, respectively.
99
Anisotropy-based Dop assay / N. Hecht, E. Gur / Anal. Biochem. 485 (2015) 97–101
Fluorescent gels
such, PafA can conjugate the lysine side chain of 5-FAM Lys to Pup in a typical pupylation reaction to generate Pup-Fl. Following 5-FAM Lys pupylation, the remaining unconjugated dye was removed by a buffer exchange step. The polyhistidine-tagged PafA was then removed via passage of the solution through a Ni2+–NTA column. Finally, the flowthrough solution was subjected to size exclusion chromatography, which yielded highly pure Pup-Fl (Fig. 2C). To test whether Dop can depupylate Pup-Fl, namely hydrolyze the isopeptide bond in Pup-Fl and release the 5-FAM Lys moiety, a depupylation reaction was established using Pup-Fl and purified M. smegmatis Dop. Samples were removed before and 1 h after the onset of the reaction for SDS-PAGE analysis. A fluorescence scan of the gel followed by Coomassie brilliant blue staining revealed that Pup-Fl is indeed a Dop substrate and that the 5-FAM Lys moiety is released by Dop (Fig. 2D).
Pup-Fl was observed by in-gel fluorescence emission at 521 nm using a Typhoon FLA 9500 scanner (GE Healthcare). Following fluorescence imaging, the gels were stained by Coomassie brilliant blue. Results Assay design and generation of a fluorescent Pup derivative As a first step in creating a novel in vitro assay for Dop activity, we sought a model system that would be a good mimic for the M. tuberculosis system, and allow for easy experimental manipulations throughout the design process. To this end, we used purified M. smegmatis proteins, as these present high similarity to their M. tuberculosis orthologs and are readily available in our laboratory. In general, M. smegmatis, a fast-growing and nonpathogenic mycobacterium, is an accepted model organism for studying pathogenic mycobacteria. The Pup orthologs from the two species share 92% identity and 98% similarity, while their Dop proteins share 94% identity and 97% similarity. Such extensive degree of identity extends to components of the PPS in the two species. For instance, like Dop, M. tuberculosis and M. smegmatis PafA proteins share 94% identity and 97% similarity. Therefore, information gained from studies of the M. smegmatis PPS is often relevant to the M. tuberculosis system. Our assay is based on the use of a fluorescent Pup variant. Essentially, if the Dop-catalyzed reaction would detach the dye from Pup, it would be accompanied by a change in fluorescence anisotropy, since the freedom of this dye to rotate in solution will greatly increase following its release from Pup (Fig. 2A). To prepare fluorescently labeled Pup, we followed Smirnov et al. [15] and used a recombinant polyhistidine-tagged version of M. smegmatis PafA to conjugate Pup, also obtained from M. smegmatis, to 5-FAM Lys in an enzymatic reaction (Fig. 2B). 5-FAM Lys is essentially a fluorescein molecule covalently bonded to the a amine of a lysine. As
A Fl
O OH
fast tumbling
PupE anisotropy
O
+
O
HN
Pup-Fl
OH
O
PafA
HOOC HOOC
O
O
5-FAM Lys Fl
Dop
slow tumbling
Using Pup-Fl, depupylation can be assessed by monitoring changes in fluorescence anisotropy. As such, a large decrease in fluorescence anisotropy was observed when Pup-Fl was mixed with Dop and ATP. In contrast, no significant decrease in fluorescence anisotropy was observed in the absence of ATP, or when an inactive Dop mutant, Dop D95A [17], was used instead of the wild type enzyme (Fig. 3A). These controls indicated that the decrease in fluorescence anisotropy resulted from the enzymatic reaction catalyzed by Dop, rather than by a potential contamination in the Dop preparation that does indeed exist, as obvious from the SDS-PAGE in Fig. 2D. Notably, some mild decrease in anisotropy was observed in the absence of ATP. However, longer incubations without ATP indicated that this decrease is transient (Fig. 3A, inset). It is possible, therefore, that Dop was copurified with some residual ATP contamination bound to it. Following dilution into the reaction mixture, such Dop-bound ATP allowed for several depupylation cycles to occur before its dissociation from the enzyme. Importantly, the Pup-Fl depupylation measurements reported here were performed in 384-well plates, while for fluorescence
B
Pup Pup
A continuous fluorescence anisotropy-based assay
OH
HOOC HOOC
N H
N H
O
O
N H
anisotropy
D
C 1
2
3
4
1
2
3
64 51
-Dop 0 1
4 Polyhis-PafA
39
+Dop -Dop 0 1h 0 1
+Dop 0 1h
64 51
Polyhis-Dop
39
28 19
28
Pup-Fl
14 6
5-FAM Lys
3
CBB scan
19
Pup-Fl
14 6
5-FAM Lys CBB scan
Fig.2. Assay design and generation of Pup-Fl. (A) The basic principle of the assay. (B) Schematic illustration of the PafA-mediated conjugation of 5-FAM Lys to Pup. (C) Samples were collected throughout Pup-Fl purification following the end of the labeling reaction (1), buffer exchange of the reaction mixture (2) and from the flowthrough of a Ni–NTA column (3). In addition, samples were collected from a concentrated eluate of size exclusion chromatography (4). The samples were analyzed by SDS-PAGE visualized by a fluorescent scan (left) and Coomassie brilliant blue staining (right) of the gel. (D) Aliquots collected before and 1 h after Dop cleavage of Pup-Fl were analyzed and visualized as in C.
100
Anisotropy-based Dop assay / N. Hecht, E. Gur / Anal. Biochem. 485 (2015) 97–101
0.11
0.10
no ATP
0.09 anisotropy
fluorescence anisotropy
0.11
0.08
0.11
0.09 0
10
20
30
40
50
60
time (min)
0.07
w.t. Dop + ATP
0.06 0
5
10
15
20
25
fluorescence anisotropy
A
AcDop, AcPup-Fl MsDop, MsPup-Fl MsDop, AcPup-Fl
0.10
AcDop, MsPup-Fl
0.09 0.08 0.07 0.06 0
time (min)
B
10
20
30
40
depupylation rate (min-1 per Dop)
time (min) 0.6
Fig.4. Expansion of the assay to orthologous Dop-Pup pairs. Reactions using the indicated combinations of the M. smegmatis (Ms) and A. cellulolyticus (Ac) orthologous proteins were performed as described in the legend to Fig. 3A.
0.4
Km = 1.0 + 0.1 µM 0.2
Vmax = 0.62 + 0.02 min
-1
0.0 0
2
4
6
8
10
[Pup-(5-FAM lys)] (µM)
depupylation rate (min-1 per Dop)
C 0.6
0.4
Kapp = 8.9 + 1.6 µM
0.2
0.0 0
20
40
60
80
was exemplified by adapting the assay for use with Dop and Pup orthologs from Acidothermus cellulolyticus. Indeed, by combining the results of such an assay with structural information available for A. cellulolyticus Dop [17], the only Dop ortholog whose structure has been deciphered, improved mechanistic understanding of this enzyme could be obtained. Recombinant A. cellulolyticus Dop and Pup were purified to homogeneity. Purified Pup was labeled with 5-FAM Lys using PafA. Since Pup orthologs share extremely high degrees of similarity in the C-terminal region necessary for interaction with Dop and PafA [17], M. smegmatis PafA efficiently conjugated A. cellulolyticus Pup to 5-FAM Lys (data not shown). Depupylation of A. cellulolyticus Pup-Fl, prepared like M. smegmatis Pup-Fl above, was examined. We found that A. cellulolyticus Dop catalyzed depupylation of its cognate Pup-Fl as well as of M. smegmatis Pup-Fl (Fig. 4). In fact, A. cellulolyticus Dop catalyzed depupylation of M. smegmatis Pup-Fl faster than M. smegmatis Dop. In contrast, A. cellulolyticus Pup-Fl was a poor substrate of M. smegmatis Dop (Fig. 4).
100
[ATP] (µM) Fig.3. A fluorescence anisotropy-based assay for Dop activity. (A) Analysis of Dop (0.5 lM) activity using Pup-Fl (4 lM). The decrease in fluorescence anisotropy following Dop addition was monitored. The inset shows the results of a ‘‘no ATP’’ control on extended incubation time. (B) Steady-state rates of Dop depupylation as a function of increasing Pup-Fl concentration. The data were fitted to the HenriMichaelis-Menten equation. Averages and standard deviations (error bars) of three experiments are presented. (C) As in B, except rates were measured at increasing ATP concentrations.
anisotropy detection, a plate reader equipped with light polarizers was used. This setup is well suited for a high-throughput format necessary for screening potential Dop inhibitors. The assay can also be used to collect kinetic measurements. For instance, a classical Michaelis-Menten experiment was performed by measuring initial rates at increasing Pup-Fl concentrations. Fitting the Michaelis-Menten rate equation to the data collected revealed that Dop depupylates Pup-Fl with a Km of 1.0 ± 0.1 lM and a Vmax of 0.62 ± 0.02 min 1 per Dop molecule (Fig. 3B). We, moreover, extracted the apparent affinity of Dop for ATP (Kapp = 8.9 ± 1.6 lM) via measurements of initial rates at increasing ATP concentration (Fig. 3C). Expansion of the assay to orthologous enzymes The methodology described here can be applied to Dop–Pup pairs other than those of M. smegmatis or M. tuberculosis. This
Discussion This study reports the establishment of a robust fluorescence anisotropy-based Dop activity assay. The assay has several advantages over the existing method [14] developed to measure Dop activity. In the novel assay, there is no need for a chemical synthesis of a fluorogenic Pup, a relatively laborious process, which requires chemical expertise, equipment, and reagents. Instead, all that is required is Pup, PafA, and a commercially available reagent (i.e., 5-FAM Lys). Since lysine residues are natural sites for PafA-mediated pupylation, the attachment of 5-FAM Lys to Pup proceeds through a simple in vitro pupylation reaction. Highly purified Pup-Fl can be subsequently obtained following a number of routine purification procedures. In the second phase of the assay, the fluorescent group is released from Pup-Fl by the depupylation actions of Dop. Although Pup-Fl is not a natural depupylation substrate, the isopeptide bond between the Pup carboxyl glutamate and the 5-FAM Lys is mediated through the lysine side chain and is identical to that recognized in a natural Dop substrate. We further demonstrated that this assay is compatible with a high-throughput format and, therefore, can be utilized for screening purposes. For such applications, the use of a fluorescence anisotropy-based assay is highly advantageous, owing to low background noise, as compared with fluorescence intensity-based screens. We also showed that this assay can be used for detailed kinetic measurements of Dop activity. In addition, given that PafA requires only the conserved C-terminal half of Pup for binding
Anisotropy-based Dop assay / N. Hecht, E. Gur / Anal. Biochem. 485 (2015) 97–101
and ligation, the methodology described here can be easily expanded for use with Dop-Pup pairs from different species, as exemplified using the Dop-Pup pair of A. cellulolyticus. In summary, we have developed a continuous fluorescencebased Dop assay that can be easily established, and can be employed for both screening purposes and detailed biochemical analysis of Dop activity. Acknowledgments We thank Maayan Korman and Shai Schlüssel for purified proteins and fruitful discussion, and Guy Adler for help with fluorescent scans. This work was supported by the Israel Science Foundation (ISF) Grant 588/14. References [1] K.H. Darwin, S. Ehrt, J.C. Gutierrez-Ramos, N. Weich, C.F. Nathan, The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide, Science 302 (2003) 1963–1966. [2] K.H. Darwin, Prokaryotic ubiquitin-like protein (Pup) proteasomes and pathogenesis, Nat. Rev. Microbiol. 7 (2009) 485–491. [3] F.A. Cerda-Maira, M.J. Pearce, M. Fuortes, W.R. Bishai, S.R. Hubbard, K.H. Darwin, Molecular analysis of the prokaryotic ubiquitin-like protein (Pup) conjugation pathway in Mycobacterium tuberculosis, Mol. Microbiol. 77 (2010) 1123–1135. [4] S. Gandotra, M.B. Lebron, S. Ehrt, The Mycobacterium tuberculosis proteasome active site threonine is essential for persistence yet dispensable for replication and resistance to nitric oxide, PLoS Pathog. 6 (2010) e1001040. [5] R. De Mot, Actinomycete-like proteasomes in a Gram-negative bacterium, Trends Microbiol. 15 (2007) 335–338.
101
[6] R.E. Valas, P.E. Bourne, Rethinking proteasome evolution: two novel bacterial proteasomes, J. Mol. Evol. 66 (2008) 494–504. [7] Elharar et al., Survival of mycobacteria depends on proteasome-mediated amino acid recycling under nutrient limitation, EMBO J. 33 (2014) 1802–1814. [8] M.J. Pearce, J. Mintseris, J. Ferreyra, S.P. Gygi, K.H. Darwin, Ubiquitin-like protein involved in the proteasome pathway of Mycobacterium tuberculosis, Science 322 (2008) 1104–1107. [9] K.E. Burns, W.T. Liu, H.I. Boshoff, P.C. Dorrestein, C.E. Barry III, Proteasomal protein degradation in Mycobacteria is dependent upon a prokaryotic ubiquitin-like protein, J. Biol. Chem. 284 (2009) 3069–3075. [10] E. Guth, M. Thommen, E. Weber-Ban, Mycobacterial ubiquitin-like protein ligase PafA follows a two-step reaction pathway with a phosphorylated Pup intermediate, J. Biol. Chem. 286 (2011) 4412–4419. [11] F. Striebel, F. Imkamp, M. Sutter, M. Steiner, A. Mamedov, E. Weber-Ban, Bacterial ubiquitin-like modifier Pup is deamidated and conjugated to substrates by distinct but homologous enzymes, Nat. Struct. Mol. Biol. 16 (2009) 647–651. [12] K.E. Burns, F.A. Cerda-Maira, T. Wang, H. Li, W.R. Bishai, K.H. Darwin, Depupylation’’ of prokaryotic ubiquitin-like protein from mycobacterial proteasome substrates, Mol. Cell 39 (2010) 821–827. [13] F. Imkamp, F. Striebel, M. Sutter, D. Ozcelik, N. Zimmermann, P. Ser, E. WeberBan, Dop functions as a depupylase in the prokaryotic ubiquitin-like modification pathway, EMBO Rep. 11 (2010) 791–797. [14] R. Merkx, K.E. Burns, P. Slobbe, F. El Oualid, D. El Atmioui, K.H. Darwin, H. Ovaa, Synthesis and evaluation of a selective fluorogenic Pup derived assay reagent for Dop, a potential drug target in Mycobacterium tuberculosis, ChemBioChem 13 (2012) 2056–2060. [15] D. Smirnov, A. Dhall, K. Sivanesam, J.R. Sharar, C. Chatterjee, J. Am. Chem. Soc. 135 (2013) 2887–2890. [16] N. Ofer, N. Forer, M. Korman, M. Vishkautzan, I. Khalaila, E. Gur, Allosteric transitions direct protein tagging by PafA the prokaryotic ubiquitin-like protein (Pup) ligase, J. Biol. Chem. 288 (2013) 11287–11293. [17] D. Özcelik, J. Barandun, N. Schmitz, M. Sutter, E. Guth, F.F. Damberger, F.H. Allain, N. Ban, E. Weber-Ban, Structures of Pup ligase PafA and depupylase Dop of the prokaryotic ubiquitin-like modification pathway, Nat. Commun. 3 (2012) 1014.
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Development of a fluorescence anisotropy-based assay for Dop, the first enzyme in the pupylation pathway Nir Hecht, Eyal Gur ⇑ The Department of Life Sciences & the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel
a r t i c l e
i n f o
Article history: Received 20 April 2015 Received in revised form 8 June 2015 Accepted 11 June 2015 Available online 18 June 2015 Keywords: Dop Mycobacteria Proteasome Proteolysis Pup
a b s t r a c t The Pup-proteasome system (PPS) carries out regulated tagging and degradation of proteins in bacterial species belonging to the phyla Actinobacteria and Nitrospira. In the pathogen Mycobacterium tuberculosis, where this proteolytic pathway was initially discovered, PPS enzymes are essential for full virulence and persistence in the mammalian host. As such, PPS enzymes are potential targets for development of antituberculosis therapeutics. Such development often requires sensitive and robust assays for measurements of enzymatic activities and the effect of examined inhibitors. Here, we describe the development of an in vitro activity assay for Dop, the first enzyme in the PPS. Based on fluorescence anisotropy measurements, this assay is simple, sensitive, and compatible with a high-throughput format for screening purposes. We demonstrate how this assay can also be reliably and conveniently used for detailed kinetic measurements of Dop activity. As such, this assay is of value for basic research into Dop and the PPS. Finally, we show that the assay developed here primarily for the mycobacterial Dop can be readily employed with other Dop enzymes, using the same simple protocol. Ó 2015 Elsevier Inc. All rights reserved.
According to the World Health Organization, tuberculosis (TB)1 is second only to HIV/AIDS as the greatest single infectious agent-based killer worldwide. This, together with the rise of multidrug-resistant strains of Mycobacterium tuberculosis, reflects the urgent need for new therapies against the disease. As such, attempts are constantly being made to identify novel targets for development of anti-TB therapeutics. One such potential target is the Pup-proteasome system (PPS), a proteolytic pathway identified in M. tuberculosis and shown to be essential for full resistance of the bacteria to nitric oxide, a reactive nitrogen intermediate secreted by activated macrophages [1–4]. The PPS is also essential for the prolonged survival of M. tuberculosis cells inside host tissues and maintenance of the bacteria in a dormant state [4]. The PPS was initially discovered in M. tuberculosis [1] and shortly thereafter was found to be conserved in Actinobcteria and Nitrospira [5,6]. In these two phyla, which compose many nonpathogenic species, the PPS functions to recycle amino acids under starvation conditions [7] and, presumably, to regulate certain cellular activities. It does so by conjugating Pup (Prokaryotic ubiquitin-like protein), a 64 amino acid-long and intrinsically disordered protein, to target proteins, thereby marking them for ⇑ Corresponding author. 1
E-mail address: [email protected] (E. Gur). Abbreviations used: TB, tuberculosis; PPS, Pup-proteasome system.
http://dx.doi.org/10.1016/j.ab.2015.06.019 0003-2697/Ó 2015 Elsevier Inc. All rights reserved.
degradation by the bacterial proteasome [8,9]. A single ligase, termed PafA (Proteasome accessory factor A), catalyzes the formation of an isopeptide bond between the c-carboxylate of the Pup C-terminal glutamate side chain and e-amines of protein substrate lysines [8,10]. In M. tuberculosis and related species, Pup is translated with a glutamine, rather than a glutamate, at the C-terminal, thus requiring that a deamidation reaction catalyzed by Dop (Deamidase of Pup) convert this glutamine into a glutamate [11]. Dop can also depupylate an already pupylated substrate, namely detach Pup from the target protein [12,13]. Both Dop activities, namely deamidation and depupylation, essentially involve the hydrolysis of an amide bond at the side chain of Pup C-terminal residue (Fig. 1), and therefore rely on the same enzymatic mechanism. Notably, although Dop and PafA each require ATP for their activity, PafA hydrolyzes an ATP molecule per each reaction cycle while Dop employs ATP as a cofactor without hydrolyzing it. Since Pup deamidation is a prerequisite for conjugation by PafA, Dop activity is essential for pupylation to occur in M. tuberculosis. Accordingly, M. tuberculosis dop mutants present reduced virulence and enhanced sensitivity to nitric oxide [3]. As such, Dop represents a potential target for development of inhibitors that can function as anti-TB medicine. The search for such inhibitors would benefit from the availability of a Dop activity assay that is simple, sensitive, and compatible with a high-throughput format for
98
Anisotropy-based Dop assay / N. Hecht, E. Gur / Anal. Biochem. 485 (2015) 97–101
Depupylation
Deamidation
PupQ
COO
O
O
H N
H N
glutamine side-chain
H
Dop
Dop
PupE
lysine side-chain
Target protein
tein pro
COO-
d
pupy lat e -
Pup
PupE COOO-
O
COOO-
NH2
O
NH3
Target protein
Fig.1. The Dop-catalyzed reactions deamidation and depupylation. The two activities of Dop employ an identical mechanism. PupQ and PupE denote Pup forms varying at their C-terminal residue. Q denotes a C-terminal glutamine whereas E denotes glutamate at that position.
screening purposes. Indeed, similar motivation has already led to the recent development of a Dop assay [14]. However, as this assay relies on chemical synthesis of a fluorogenic version of Pup, it is fairly complicated to establish. Accordingly, we describe in this paper the development of a simpler Dop assay based on fluorescence anisotropy and consider its potential use. Rather than employing chemical synthesis, our novel assay relies on the ability of PafA to catalyze Pup conjugation to fluorescein-labeled lysine residues. Specifically, we used fluorescein-5-carboxamide lysine (5-FAM Lys) to generate pupylated 5-FAM Lys (hereafter, Pup-Fl), as previously reported [15]. Following purification of Pup-Fl to homogeneity, its depupylation can be readily measured in a simple, continuous, and sensitive manner by monitoring changes in fluorescence anisotropy. Finally, the assay established here will facilitate mechanistic analysis of Dop activity, as it is general and can be easily adapted for use with Dop orthologs that are phylogenetically distant from the M. tuberculosis protein. Materials and methods Protein expression and purification Recombinant Mycobacterium smegmatis PafA and Pup were purified as previously described [16]. All Dop variants carried C-terminal polyhistidine tags and were expressed in Escherichia coli strain ER2566 from plasmid pET11a. Following induction with IPTG, the cultures were incubated overnight at 18 °C. Cells were lysed by sonication, and purification using Ni2+–NTA–agarose (Qiagen) was carried out according to a standard protocol, except that for purification of M. smegmatis Dop, buffers contained 10% glycerol (v/v). A second size exclusion chromatography purification step relied on a Superdex 75 column (GE Healthcare). For the M. smegmatis Dop variants, the buffer used for purification contained 50 mM Hepes, pH 8.0, 150 mM NaCl, 20 mM MgCl2, and 10% (v/v) glycerol. For purification of A. cellulolyticus Dop, the buffer contained 50 mM Hepes, pH 8.0, 300 mM NaCl, and 1 mM DTT. The protein was further purified by anion-exchange chromatography using a MonoQ column (GE Healthcare) and a linear 0 to 1 M NaCl gradient.
For purification of A. cellulolyticus Pup, the encoding gene was cloned into plasmid pSH21 under the transcriptional control of the T7 promoter in fusion to a DNA sequence encoding an N-terminal polyhistidine tag, the human titin I27 domain, and a TEV protease recognition sequence. The polyhistidine–I27–TEV–Pup chimera was expressed at 30 °C, and Ni2+–NTA purification was carried out as above. Following TEV cleavage, a buffer exchange step was carried out, and the polyhistidine–I27–TEV portion of the chimera was removed by loading the solution onto a Ni2+–NTA column. The flowthrough was collected, and Pup was further purified by anion-exchange chromatography using a MonoQ column (GE Healthcare) and a linear 0 to 1 M NaCl gradient. Pupylation of 5-FAM Lys Pupylation of 5-FAM Lys (AnaSpec) was carried out in pupylation buffer (50 mM Hepes, pH 7.5, 100 mM NaCl, 20 mM MgCl2, and 10% glycerol (v/v)) containing 2 mM ATP, 200 lM Pup, and 5 lM PafA for 3 h at 30 °C. After a buffer exchange step, Ni2+– NTA purification was carried out as above and the resulting Pup-Fl was loaded onto a Superdex 75 size exclusion column (GE Healthcare) equilibrated with 25 mM Hepes, pH 8.0. The concentration of Pup-Fl following all purification steps was measured spectroscopically using a 1 cm path-length cuvette and the extinction coefficients of fluorescein at 280 and 490 nm (24,000 and 82,000 M 1 cm 1, respectively) and of Pup at 280 nm (1490 M 1 cm 1). The following expression yielded Pup-Fl concentration: (A280–0.29A490)/1490. The degree of labeling was assessed by comparing the concentration of Pup-Fl to that of fluorescein. The latter was determined spectroscopically at 490 nm. The degree of labeling typically exceeded 90%. Fluorescence anisotropy assay Fluorescence anisotropy reactions contained Dop and Pup-Fl in pupylation buffer in a final volume of 50 ll. Fluorescence at the parallel and perpendicular polarizations was measured in 384-well plates using a Synergy 2 plate reader (Biotek). The excitation and emission wavelengths were 485 and 528 nm, respectively.
99
Anisotropy-based Dop assay / N. Hecht, E. Gur / Anal. Biochem. 485 (2015) 97–101
Fluorescent gels
such, PafA can conjugate the lysine side chain of 5-FAM Lys to Pup in a typical pupylation reaction to generate Pup-Fl. Following 5-FAM Lys pupylation, the remaining unconjugated dye was removed by a buffer exchange step. The polyhistidine-tagged PafA was then removed via passage of the solution through a Ni2+–NTA column. Finally, the flowthrough solution was subjected to size exclusion chromatography, which yielded highly pure Pup-Fl (Fig. 2C). To test whether Dop can depupylate Pup-Fl, namely hydrolyze the isopeptide bond in Pup-Fl and release the 5-FAM Lys moiety, a depupylation reaction was established using Pup-Fl and purified M. smegmatis Dop. Samples were removed before and 1 h after the onset of the reaction for SDS-PAGE analysis. A fluorescence scan of the gel followed by Coomassie brilliant blue staining revealed that Pup-Fl is indeed a Dop substrate and that the 5-FAM Lys moiety is released by Dop (Fig. 2D).
Pup-Fl was observed by in-gel fluorescence emission at 521 nm using a Typhoon FLA 9500 scanner (GE Healthcare). Following fluorescence imaging, the gels were stained by Coomassie brilliant blue. Results Assay design and generation of a fluorescent Pup derivative As a first step in creating a novel in vitro assay for Dop activity, we sought a model system that would be a good mimic for the M. tuberculosis system, and allow for easy experimental manipulations throughout the design process. To this end, we used purified M. smegmatis proteins, as these present high similarity to their M. tuberculosis orthologs and are readily available in our laboratory. In general, M. smegmatis, a fast-growing and nonpathogenic mycobacterium, is an accepted model organism for studying pathogenic mycobacteria. The Pup orthologs from the two species share 92% identity and 98% similarity, while their Dop proteins share 94% identity and 97% similarity. Such extensive degree of identity extends to components of the PPS in the two species. For instance, like Dop, M. tuberculosis and M. smegmatis PafA proteins share 94% identity and 97% similarity. Therefore, information gained from studies of the M. smegmatis PPS is often relevant to the M. tuberculosis system. Our assay is based on the use of a fluorescent Pup variant. Essentially, if the Dop-catalyzed reaction would detach the dye from Pup, it would be accompanied by a change in fluorescence anisotropy, since the freedom of this dye to rotate in solution will greatly increase following its release from Pup (Fig. 2A). To prepare fluorescently labeled Pup, we followed Smirnov et al. [15] and used a recombinant polyhistidine-tagged version of M. smegmatis PafA to conjugate Pup, also obtained from M. smegmatis, to 5-FAM Lys in an enzymatic reaction (Fig. 2B). 5-FAM Lys is essentially a fluorescein molecule covalently bonded to the a amine of a lysine. As
A Fl
O OH
fast tumbling
PupE anisotropy
O
+
O
HN
Pup-Fl
OH
O
PafA
HOOC HOOC
O
O
5-FAM Lys Fl
Dop
slow tumbling
Using Pup-Fl, depupylation can be assessed by monitoring changes in fluorescence anisotropy. As such, a large decrease in fluorescence anisotropy was observed when Pup-Fl was mixed with Dop and ATP. In contrast, no significant decrease in fluorescence anisotropy was observed in the absence of ATP, or when an inactive Dop mutant, Dop D95A [17], was used instead of the wild type enzyme (Fig. 3A). These controls indicated that the decrease in fluorescence anisotropy resulted from the enzymatic reaction catalyzed by Dop, rather than by a potential contamination in the Dop preparation that does indeed exist, as obvious from the SDS-PAGE in Fig. 2D. Notably, some mild decrease in anisotropy was observed in the absence of ATP. However, longer incubations without ATP indicated that this decrease is transient (Fig. 3A, inset). It is possible, therefore, that Dop was copurified with some residual ATP contamination bound to it. Following dilution into the reaction mixture, such Dop-bound ATP allowed for several depupylation cycles to occur before its dissociation from the enzyme. Importantly, the Pup-Fl depupylation measurements reported here were performed in 384-well plates, while for fluorescence
B
Pup Pup
A continuous fluorescence anisotropy-based assay
OH
HOOC HOOC
N H
N H
O
O
N H
anisotropy
D
C 1
2
3
4
1
2
3
64 51
-Dop 0 1
4 Polyhis-PafA
39
+Dop -Dop 0 1h 0 1
+Dop 0 1h
64 51
Polyhis-Dop
39
28 19
28
Pup-Fl
14 6
5-FAM Lys
3
CBB scan
19
Pup-Fl
14 6
5-FAM Lys CBB scan
Fig.2. Assay design and generation of Pup-Fl. (A) The basic principle of the assay. (B) Schematic illustration of the PafA-mediated conjugation of 5-FAM Lys to Pup. (C) Samples were collected throughout Pup-Fl purification following the end of the labeling reaction (1), buffer exchange of the reaction mixture (2) and from the flowthrough of a Ni–NTA column (3). In addition, samples were collected from a concentrated eluate of size exclusion chromatography (4). The samples were analyzed by SDS-PAGE visualized by a fluorescent scan (left) and Coomassie brilliant blue staining (right) of the gel. (D) Aliquots collected before and 1 h after Dop cleavage of Pup-Fl were analyzed and visualized as in C.
100
Anisotropy-based Dop assay / N. Hecht, E. Gur / Anal. Biochem. 485 (2015) 97–101
0.11
0.10
no ATP
0.09 anisotropy
fluorescence anisotropy
0.11
0.08
0.11
0.09 0
10
20
30
40
50
60
time (min)
0.07
w.t. Dop + ATP
0.06 0
5
10
15
20
25
fluorescence anisotropy
A
AcDop, AcPup-Fl MsDop, MsPup-Fl MsDop, AcPup-Fl
0.10
AcDop, MsPup-Fl
0.09 0.08 0.07 0.06 0
time (min)
B
10
20
30
40
depupylation rate (min-1 per Dop)
time (min) 0.6
Fig.4. Expansion of the assay to orthologous Dop-Pup pairs. Reactions using the indicated combinations of the M. smegmatis (Ms) and A. cellulolyticus (Ac) orthologous proteins were performed as described in the legend to Fig. 3A.
0.4
Km = 1.0 + 0.1 µM 0.2
Vmax = 0.62 + 0.02 min
-1
0.0 0
2
4
6
8
10
[Pup-(5-FAM lys)] (µM)
depupylation rate (min-1 per Dop)
C 0.6
0.4
Kapp = 8.9 + 1.6 µM
0.2
0.0 0
20
40
60
80
was exemplified by adapting the assay for use with Dop and Pup orthologs from Acidothermus cellulolyticus. Indeed, by combining the results of such an assay with structural information available for A. cellulolyticus Dop [17], the only Dop ortholog whose structure has been deciphered, improved mechanistic understanding of this enzyme could be obtained. Recombinant A. cellulolyticus Dop and Pup were purified to homogeneity. Purified Pup was labeled with 5-FAM Lys using PafA. Since Pup orthologs share extremely high degrees of similarity in the C-terminal region necessary for interaction with Dop and PafA [17], M. smegmatis PafA efficiently conjugated A. cellulolyticus Pup to 5-FAM Lys (data not shown). Depupylation of A. cellulolyticus Pup-Fl, prepared like M. smegmatis Pup-Fl above, was examined. We found that A. cellulolyticus Dop catalyzed depupylation of its cognate Pup-Fl as well as of M. smegmatis Pup-Fl (Fig. 4). In fact, A. cellulolyticus Dop catalyzed depupylation of M. smegmatis Pup-Fl faster than M. smegmatis Dop. In contrast, A. cellulolyticus Pup-Fl was a poor substrate of M. smegmatis Dop (Fig. 4).
100
[ATP] (µM) Fig.3. A fluorescence anisotropy-based assay for Dop activity. (A) Analysis of Dop (0.5 lM) activity using Pup-Fl (4 lM). The decrease in fluorescence anisotropy following Dop addition was monitored. The inset shows the results of a ‘‘no ATP’’ control on extended incubation time. (B) Steady-state rates of Dop depupylation as a function of increasing Pup-Fl concentration. The data were fitted to the HenriMichaelis-Menten equation. Averages and standard deviations (error bars) of three experiments are presented. (C) As in B, except rates were measured at increasing ATP concentrations.
anisotropy detection, a plate reader equipped with light polarizers was used. This setup is well suited for a high-throughput format necessary for screening potential Dop inhibitors. The assay can also be used to collect kinetic measurements. For instance, a classical Michaelis-Menten experiment was performed by measuring initial rates at increasing Pup-Fl concentrations. Fitting the Michaelis-Menten rate equation to the data collected revealed that Dop depupylates Pup-Fl with a Km of 1.0 ± 0.1 lM and a Vmax of 0.62 ± 0.02 min 1 per Dop molecule (Fig. 3B). We, moreover, extracted the apparent affinity of Dop for ATP (Kapp = 8.9 ± 1.6 lM) via measurements of initial rates at increasing ATP concentration (Fig. 3C). Expansion of the assay to orthologous enzymes The methodology described here can be applied to Dop–Pup pairs other than those of M. smegmatis or M. tuberculosis. This
Discussion This study reports the establishment of a robust fluorescence anisotropy-based Dop activity assay. The assay has several advantages over the existing method [14] developed to measure Dop activity. In the novel assay, there is no need for a chemical synthesis of a fluorogenic Pup, a relatively laborious process, which requires chemical expertise, equipment, and reagents. Instead, all that is required is Pup, PafA, and a commercially available reagent (i.e., 5-FAM Lys). Since lysine residues are natural sites for PafA-mediated pupylation, the attachment of 5-FAM Lys to Pup proceeds through a simple in vitro pupylation reaction. Highly purified Pup-Fl can be subsequently obtained following a number of routine purification procedures. In the second phase of the assay, the fluorescent group is released from Pup-Fl by the depupylation actions of Dop. Although Pup-Fl is not a natural depupylation substrate, the isopeptide bond between the Pup carboxyl glutamate and the 5-FAM Lys is mediated through the lysine side chain and is identical to that recognized in a natural Dop substrate. We further demonstrated that this assay is compatible with a high-throughput format and, therefore, can be utilized for screening purposes. For such applications, the use of a fluorescence anisotropy-based assay is highly advantageous, owing to low background noise, as compared with fluorescence intensity-based screens. We also showed that this assay can be used for detailed kinetic measurements of Dop activity. In addition, given that PafA requires only the conserved C-terminal half of Pup for binding
Anisotropy-based Dop assay / N. Hecht, E. Gur / Anal. Biochem. 485 (2015) 97–101
and ligation, the methodology described here can be easily expanded for use with Dop-Pup pairs from different species, as exemplified using the Dop-Pup pair of A. cellulolyticus. In summary, we have developed a continuous fluorescencebased Dop assay that can be easily established, and can be employed for both screening purposes and detailed biochemical analysis of Dop activity. Acknowledgments We thank Maayan Korman and Shai Schlüssel for purified proteins and fruitful discussion, and Guy Adler for help with fluorescent scans. This work was supported by the Israel Science Foundation (ISF) Grant 588/14. References [1] K.H. Darwin, S. Ehrt, J.C. Gutierrez-Ramos, N. Weich, C.F. Nathan, The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide, Science 302 (2003) 1963–1966. [2] K.H. Darwin, Prokaryotic ubiquitin-like protein (Pup) proteasomes and pathogenesis, Nat. Rev. Microbiol. 7 (2009) 485–491. [3] F.A. Cerda-Maira, M.J. Pearce, M. Fuortes, W.R. Bishai, S.R. Hubbard, K.H. Darwin, Molecular analysis of the prokaryotic ubiquitin-like protein (Pup) conjugation pathway in Mycobacterium tuberculosis, Mol. Microbiol. 77 (2010) 1123–1135. [4] S. Gandotra, M.B. Lebron, S. Ehrt, The Mycobacterium tuberculosis proteasome active site threonine is essential for persistence yet dispensable for replication and resistance to nitric oxide, PLoS Pathog. 6 (2010) e1001040. [5] R. De Mot, Actinomycete-like proteasomes in a Gram-negative bacterium, Trends Microbiol. 15 (2007) 335–338.
101
[6] R.E. Valas, P.E. Bourne, Rethinking proteasome evolution: two novel bacterial proteasomes, J. Mol. Evol. 66 (2008) 494–504. [7] Elharar et al., Survival of mycobacteria depends on proteasome-mediated amino acid recycling under nutrient limitation, EMBO J. 33 (2014) 1802–1814. [8] M.J. Pearce, J. Mintseris, J. Ferreyra, S.P. Gygi, K.H. Darwin, Ubiquitin-like protein involved in the proteasome pathway of Mycobacterium tuberculosis, Science 322 (2008) 1104–1107. [9] K.E. Burns, W.T. Liu, H.I. Boshoff, P.C. Dorrestein, C.E. Barry III, Proteasomal protein degradation in Mycobacteria is dependent upon a prokaryotic ubiquitin-like protein, J. Biol. Chem. 284 (2009) 3069–3075. [10] E. Guth, M. Thommen, E. Weber-Ban, Mycobacterial ubiquitin-like protein ligase PafA follows a two-step reaction pathway with a phosphorylated Pup intermediate, J. Biol. Chem. 286 (2011) 4412–4419. [11] F. Striebel, F. Imkamp, M. Sutter, M. Steiner, A. Mamedov, E. Weber-Ban, Bacterial ubiquitin-like modifier Pup is deamidated and conjugated to substrates by distinct but homologous enzymes, Nat. Struct. Mol. Biol. 16 (2009) 647–651. [12] K.E. Burns, F.A. Cerda-Maira, T. Wang, H. Li, W.R. Bishai, K.H. Darwin, Depupylation’’ of prokaryotic ubiquitin-like protein from mycobacterial proteasome substrates, Mol. Cell 39 (2010) 821–827. [13] F. Imkamp, F. Striebel, M. Sutter, D. Ozcelik, N. Zimmermann, P. Ser, E. WeberBan, Dop functions as a depupylase in the prokaryotic ubiquitin-like modification pathway, EMBO Rep. 11 (2010) 791–797. [14] R. Merkx, K.E. Burns, P. Slobbe, F. El Oualid, D. El Atmioui, K.H. Darwin, H. Ovaa, Synthesis and evaluation of a selective fluorogenic Pup derived assay reagent for Dop, a potential drug target in Mycobacterium tuberculosis, ChemBioChem 13 (2012) 2056–2060. [15] D. Smirnov, A. Dhall, K. Sivanesam, J.R. Sharar, C. Chatterjee, J. Am. Chem. Soc. 135 (2013) 2887–2890. [16] N. Ofer, N. Forer, M. Korman, M. Vishkautzan, I. Khalaila, E. Gur, Allosteric transitions direct protein tagging by PafA the prokaryotic ubiquitin-like protein (Pup) ligase, J. Biol. Chem. 288 (2013) 11287–11293. [17] D. Özcelik, J. Barandun, N. Schmitz, M. Sutter, E. Guth, F.F. Damberger, F.H. Allain, N. Ban, E. Weber-Ban, Structures of Pup ligase PafA and depupylase Dop of the prokaryotic ubiquitin-like modification pathway, Nat. Commun. 3 (2012) 1014.
