Molecular Tension-Indexed Bioluminescent Probe for Determining

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Bioconjugate Chem. 2009, 20, 2324–2330

Molecular Tension-Indexed Bioluminescent Probe for Determining Protein-Protein Interactions Sung Bae Kim,† Moritoshi Sato,‡ and Hiroaki Tao†,* Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan. Received July 28, 2009; Revised Manuscript Received October 25, 2009

This study demonstrates a unique, nontranscriptional assay system based on molecular tension of a luciferase artificially appended by protein-protein binding. We hypothesized that an artificially appended molecular tension to a full-length luciferase may diversify the enzymatic activity through a modification of the active site. For the basic probe design, a full-length luciferase was sandwiched between two component proteins of interest. The length of flexible linkers between the components was minimized to exert an efficient molecular tension to the sandwiched luciferase. When N- and C-terminal ends of Renilla luciferase 8 were flanked by the ligandbinding domain of human estrogen receptor R (ER LBD) and SH2 domain of Src, named ERS, this simple probe was surprisingly sensitive to estrogens. The luminescence spectra by ERS were largely enhanced by an addition of 4-hydroxytamoxifen (OHT), 17β-estradiol, and genistein. The detection limit of ERS reached 1 nM OHT. Quantum yield (QY) and Michaelis-Menten constant of ERS were found to be 6.3% and 94.3 µM, respectively. The enzymatic activities of ERS are also governed by different types of coelenterazine (CTZ) variants. The two hydroxy groups in CTZ are critical for the enzymatic activities of ERS. This study is the first example that an artificially appended molecular tension to a full-length luciferase can be taken as an optical signature upon molecular imaging. This study also provides new insight into the construction of a new lineage of bioluminescent probes for estimating protein-protein interactions.

INTRODUCTION Recent revolutionary advances in enzyme manipulation technologies now allow researchers to carry out quantitative examination of molecular dynamics and cell signaling in living subjects (1). Luciferases are nearly ideal reporters for bioanalysis and molecular imaging: (i) The assays are potentially very simple, sensitive, and do not require an external light source or cofactors. (ii) The assay time is usually less than one min, which enables a high throughput analysis. (iii) The assay is cheap, nonhazardous, and broadly applicable to living organisms. To date, various luciferases have been engineered for determining protein-protein interactions: (i) Bioluminescence resonance energy transfer (BRET) is a potential technique for determining protein-protein interactions on the basis of energy transfer between bioluminescent donor and acceptor proteins (2) (3) (4). (ii) Two-hybrid assay allows a simple determination of protein-protein interactions in living mammalian cells and animals (5). (ii) Protein-fragment complementation assay (PCA) is another strategy for determining protein-protein interactions in cell lines, where monomeric luciferases are split into two fragments with resulting temporally inactive portions (6) (7) (8) (9) (10). The luciferase activity is reconstituted only upon protein-protein interactions. (iii) Recently, a circular permutation (CP) of a dissected luciferase was utilized to determine protein-protein interactions. Original N- and C-terminals of a luciferase are directly linked with a flexible linker, whereas new N- and C-terminals are created in the middle of the luciferase sequence, and modified with proteins of interest (11) (12). This * Corresponding author: [email protected]. † AIST. ‡ The University of Tokyo.

manipulation exerts a conditional reconstitution of the luciferase activity in response to ligand-activated protein-protein interactions. Many of the previous studies dissect the luciferases into two fragments for a temporal loss and conditional recovery of luciferase activities. However, this methodology inevitably invades the intrinsic enzymatic property of the luciferase and recovers merely 0.5-5% of the original luciferase activity (13) (14). The poor absolute luminescence intensities are especially problematic upon application to living animals. Furthermore, the luciferase-dissection strategy requires a sophisticated probe design and a tedious optimization step for deciding a suitable splitting site for the luciferase. These procedures consequently consume time and resources. In our review examinations, many of the previous dissection sites in luciferases are frequently inadequate in other protein-protein interaction cases (15). To address such drawbacks of the conventional methods, we have investigated a simple but efficient bioluminescent probe for determining protein-protein interactions. We initially hypothesized that a luciferase activity may vary according to the molecular tension artificially appended to the full-length luciferase. Renilla luciferase 8 (RLuc8) was selected to examine the probability. N- and C-terminal ends of full-length RLuc8 were fused with the ligand-binding domain of the human estrogen receptor (ER LBD)1 and the Src homology domain 2 of V-Src (SH2), respectively. Upon ligand activation, Tyr537 in ER LBD is phosphorylated, and consequently recognized by the adjacent SH2 domain. The ligand-activated ER LBD–SH2 binding appends a molecular tension to the sandwiched fulllength RLuc8. The consequent variation in the luciferase activity was surprisingly large enough to be indexed as an optical signature. This probe was named ERS from the consecutive component initials, that is, ER LBD-RLuc8-SH2.

10.1021/bc900330w CCC: $40.75  2009 American Chemical Society Published on Web 11/25/2009

Molecular Tension-Indexed Bioluminescent Probe

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Figure 1. (A) Crystal structure of Renilla luciferase 8 (RLuc8; pdb 2PSJ) and firefly luciferase (FLuc). Red and yellow arrows indicate N- and C-terminal ends, respectively. The red chemical in RLuc8 shows coelenterazine (CTZ). The rectangular active site embraces CTZ. An external molecular tension may distort this rectangular frame. (B) Schematic structures of cDNA constructs. Abbrevations: ER LBD, ligand binding domain of human estrogen receptor; RLuc8, Renilla luciferase 8; CBLuc, click beetle luciferase red; GLuc, Gaussia luciferase; SH2, SH2 domain of Src; Kz, kozak sequence. (C) Variance in the bioluminescence emission spectra before and after addition of 1 µM 4-hydroxytamoxifen (OHT), obtained by a spectrophotometer (F-7000, Hitachi). Inset shows the relative luminescence intensities before and after addition of 1 µΜ OHT, obtained by a luminometer (Berthold). ER LBD-RLuc8-SH2 (ERS) showed a considerable enhancement in the bioluminescence in response to OHT, whereas ER LBD-GLuc-SH2 (EGS) and ER LBD-CBLuc-SH2 (ECS) did not enhance the luminescence intensities. (D) The chemical structure shows coelenterazine (CTZ) interacting with the active site of RLuc8 (left: a modification of a reference (18)). Dotted circles highlight amino acids close to the C-terminal end of RLuc8. An illustration of the working mechanism of ERS in response to a ligand (right). A ligand activates an interaction between proteins A and B, which consequently transforms the sandwiched luciferase. This transformation is not valid in ER+RS combination.

The sensitivity and selectivity of ERS to various ligands are initially demonstrated with respect to the sensorial properties. The spectral properties of ERS according to various coelenterazine (CTZ) derivatives are also discussed. Quantum yields

(QYs), turnover rates, and Michaelis-Menten constants (KM) of ERS are reported. Finally, we propose the molecular mechanism of ERS upon sensing estrogens based on a series of control studies.

1 Abbreviations: ER LBD, the ligand-binding domain of the human estrogen receptor (305-550 AA); SH2, the Src homology domain 2 of a proto-oncogene tyrosine-protein kinase, V-Src; ERS, a bioluminescent probe custom-made and named from the consecutive component initials, that is, ER LBD-RLuc8-SH2; pCold I vector, A Coldshock expression vector (Takara) for E. coli; pErs, a custom-made plasmid encoding ERS (ER LBD-RLuc8-SH2); pEcs, a custom-made plasmid encoding ECS (ER LBD-CBLuc-SH2); pEgs, a custom-made plasmid encoding EGS (ER LBD-GLuc-SH2); pEr, a custom-made plasmid encoding ER (ER LBD-RLuc8); pRs, a custom-made plasmid encoding RS (RLuc8SH2); COS-7, a mammalian culture cell line derived from African green monkey kidney fibroblast; OHT, a known antagonist of estrogen receptors called 4-hydroxytamoxifen; CTZ, a native luciferin for various luciferases derived from marine animals called coelenterazine; CTZ hcp, a synthetic derivative of coelenterazine carrying hydrogen (h) at C2 and cyclopentyl (cp) group at C8 position; CTZ cp, a synthetic derivative of coelenterazine carrying a cyclopentyl (cp) group at C8 position; CTZ fcp, a synthetic derivative of coelenterazine carrying fluorine (f) at C2 and cyclopentyl (cp) group at C8 position; CTZ ip, a synthetic derivative of coelenterazine carrying iodine (i) at C2 and 2-propionyl (p) group at C8 position; E2, an agonist of estrogen receptors called 17β-estradiol; CTZ 400A, a synthetic derivative of coelenterazine carrying no side chains at C2, C6, and C8 positions, also called DeepBlueC.

EXPERIMENTAL PROCEDURES Construction of Plasmids. As a template for polymerase chain reaction (PCR), cDNAs encoding the following luciferases were obtained from the corresponding providers: Renilla luciferase 8 (RLuc8) was kindly presented by Professor Gambhir (Stanford University); click beetle luciferase red (CBLuc) was purchased from Promega; and Gaussia luciferase (GLuc) from Nanolight. cDNAs encoding the human estrogen receptor (hER) and full-length V-Src were presented by Professor Umezawa (Tokyo University). The cDNAs encoding ER LBD and SH2 domain of V-Src were generated by PCR using corresponding primers to introduce unique restriction sites, HindIII/ BamHI and KpnI/ XhoI, respectively. Both ends of cDNAs encoding RLuc8, CBLuc, and GLuc were also modified by PCR to introduce specific restriction sites, BamHI and KpnI. The cDNA blocks were ligated to make a series of chimera constructs as shown in Figure 1 and subcloned into a mammalian expression vector pcDNA 3.1(+) vector (Invitrogen). A plasmid for Escherichia coli expression was constructed as follows: The cDNA encoding ERS was modified by PCR to

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Kim et al.

Figure 2. (A) Dose-response curves of ER LBD-RLuc8-SH2 (ERS) in response to steroid hormones. The luminescence intensity reflects the enzymatic activity of transformed RLuc8 in ERS in response to varying concentrations of OHT. Inset shows ligand sensitivity of a series of control probes. EmRS shows a point-mutated form of ERS at Tyr537, while ER+RS means coexistence of the two truncated forms of ERS, that is, ER and RS. Values were measured in triplicate. The working mechanism of ERS and ER+RS was illustrated in the middle column. (B) Ligand selectivity of ERS. The luminescence intensities were compared after activation of ERS with various ligands. Abbrevations: 4-hydroxytamoxifen (OHT), 17β-estradiol (E2), 5R-dihydrotestosterone (DHT), testosterone (T), genistein (genis).

introduce unique restriction sites, SacI/ XhoI, at the 5′- and 3′ends. The chimera DNA restricted by SacI and XhoI was subcloned into the E. coli Coldshock expression vector pCold I vector (Takara). The sequences of the cDNAs were confirmed with a BigDye Terminators v1.1 cycle sequencing kit and a genetic sequencer (ABI PRISM 310 Genetic Analyzer, Applied Biosystems). The consequent plasmids were named pErs, pEcs, pEgs, pEr, and pRs according to the consecutive component initials. The corresponding probes after expression were named ERS, ECS, EGS, ER, and RS. The pCold I vector for expressing ERS was named pCold-Ers. Bioluminescence Spectra of COS-7 Cells Carrying pErs. Variance in the bioluminescence spectra from mammalian COS-7 cells carrying pErs was examined in the presence or absence of 4-hydroxytamoxifen (OHT) (Figure 1C). For the present experiment, COS-7 cells were cultured in a Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/ streptomycin (P/S; Gibco) at 37 °C in 5% CO2. The cells were seeded in 12-well culture plates, transiently transfected with 2 µg of pErs per well using a transfection reagent, TransIT-LT1 (Mirus), and incubated for 16 h at 37 °C in 5% CO2 before experiments. The cells on the 12-well plates were stimulated for 20 min with vehicle (0.1% DMSO) or 1 µM 4-hydroxytamoxifen (OHT). The cells were washed once with phosphate buffered saline (PBS; pH 7.4, 0.01 M), and lysed with a lysis buffer (Promega). The lysates were transferred into a quartz cuvette for monitoring the spectral variance with a spectrophotometer (F-7000, Hitachi) in the presence of coelenterazine native (CTZ). Ligand Sensitivity of COS-7 Cells Carrying pErs, pEcs, or pEgs. Relative ligand sensitivities among ERS, ECS, and EGS were examined in the presence or absence of OHT (Figure 1C Inset). COS-7 cells carrying pErs, pEcs, or pEgs were stimulated with vehicle (0.1% dimethyl sulfoxide (DMSO)) or OHT for 20 min, washed once with PBS, and finally lysed with a lysis buffer for 15 min. The cell lysates were transferred to test tubes, and the subsequent bioluminescence intensities were determined with a luminometer (MiniLumat LB9506; Berthold) in the presence of coelenterazine (CTZ). The brief procedure for the normalization of bioluminescence was as follows: The RLuc luminescence intensities were expressed as a ratio of relative luminescence unit (RLU), that

is, RLU (+)/RLU (-), where RLU (+) and RLU (-) are the luminescence intensities with 1 µg of cell lysate after the cells were incubated with and without a ligand, respectively; the RLU is an amplified value of photon counts generated from the luminometer (arbitrary unit). Sensorial Properties of ERS to Varying Concentrations of Ligands. Sensorial properties of ERS to ligands were estimated with different concentrations of ligands (Figure 2A). COS-7 cells were transfected with pErs as described in Figure 1C. The cells cultured on 24-well plates were stimulated with vehicle (0.1% DMSO) or varying concentrations of OHT or testosterone (T) for 20 min. The cells were washed once with PBS and lysed. Ligand-activated ER LBD–SH2 interactions were estimated on the basis of the bioluminescence variance using a spectrophotometer (F-7000, Hitachi). The ∆RLU was calculated as shown in a previous study (16): the bioluminescence intensities developed by varying concentrations of a ligand were subtracted by the bioluminescence intensities developed by vehicle (0.1% DMSO). Values were measured in triplicate. A series of control studies was parallely conducted with COS-7 cells carrying pErs, pEmRS, or both pEr and pRs (Figure 2A Inset). The cells were stimulated with vehicle (0.1% DMSO) or 1 µM OHT for 20 min. The subsequent luminescence variances were determined with a luminometer (MiniLumat LB9506; Berthold). Substrate Dependence of OHT-Activated ERS. Contribution of coelenterazine derivatives to the enzymatic activities of ERS was examined on the basis of the bioluminescence spectra (Figure 3A). COS-7 cells transfected with pErs were cultured on 24-well plates. The cells were stimulated with 1 µM OHT for 20 min, washed once with PBS, and lysed. The lysates were transferred into a quartz cuvette for monitoring the spectral variance with a spectrophotometer (F-7000, Hitachi) in the presence of coelenterazine native (CTZ), coelenterazine hcp (CTZ hcp), coelenterazine cp (CTZ cp), coelenterazine fcp (CTZ fcp), or coelenterazine ip (CTZ ip). Values were measured in duplicate. Enzymatic Properties of ERS in the Presence or Absence of OHT. ERS was affinity-column purified for determining quantum yields (QYs) and turnover rates according to ligand stimulations and variation of CTZ derivatives (Table 1). pCold-Ers was transformed into BL21 (DE3) pLysS competent cells (Novagen). The seed culture of the bacterial strain carrying pCold-Ers was initially grown in 10 mL of Luria-Bertani



Figure 3. (A) Bioluminescence emission spectra before and after stimulation of 1 µM 4-hydroxytamoxifein (OHT). The spectra generated by ER LBD-RLuc8-SH2 (ERS) were recorded using a spectrophotometer (F-7000, Hitachi). The gray arrow shows the spectrum variance before and after supplementation of 1 µM OHT. Inset shows the peak heights of the spectra before and after addition of 1 µM OHT. Maximal bioluminescence intensities were obtained in the presence of native coelenterazine (CTZ), while coelenterazine fcp (CTZ fcp) exhibited the largest signal-to-background ratios. Values were averaged after duplicate determination. The marks “+” and “-” show the presence or absence of 1 µM OHT. (B) Comparison of the bioluminescence emission spectra of ERS and net Renilla luciferase 8 (RLuc8) alone before and after stimulation of 1 µM OHT. All of the luminescence spectra were recorded in the presence of CTZ fcp. Intact RLuc8 alone was insensitive to OHT, that is, the spectra are almost superimposed (red lines), whereas ERS varied with the luminescence spectra in response to OHT (black lines). The dotted circles show critical side chains of CTZ for interacting with ERS. Dotted circles 1, 2, and 3 highlight (i) the p-OH of the phenol attached at C6 of native CTZ, (ii) the p-OH group on the benzyl substituent at C2 of native CTZ, and (iii) the benzyl group attached at C8 of native CTZ, respectively. Table 1. Bioluminescence Activity Variance of ER LBD-RLuc8-SH2 (ERS) According to Ligands and Substratesa probe RLuc8 ERS

substrateb CTZ CTZ CTZ CTZ CTZ CTZ CTZ CTZ CTZ CTZ CTZ CTZ

cp fcp hcp ip 400A f i

stimulator vehicle OHT e E2 e -

quantum yield (%) 6.9 (0.1 6.3 ( 1.1 5.6 ( 0.9 5.0 ( 0.7 5.0 ( 0.6 1.3 ( 0.1 1.8 ( 0.2 1.9 ( 0.2 1.3 ( 0.1 1.4 ( 0.0 1.7 ( 0.2 1.5 ( 0.2 f

d

KMc(µM)

turnover rate (photons/s/mol)

1.6 ( 0.2 94.3 ( 37.1 f

4.3 ( 0.2 × 1022 6.1 ( 0.5 × 1019 -

a Values were measured in quadruplicate, and standard errors of the mean are given. As the internal standards, quantum yields (QYs) of RLuc and RLuc8 were parallelly estimated with our instrument (Mithras LB940; Berthold). b CTZ indicates coelenterazine dissolved in a phosphate buffered saline (PBS; 0.01 M, pH 7.4). c KM means Michaelis-Menten constant. d Vehicle represents 0.1% dimethyl sulfoxide (DMSO). e OHT and E2 were dissolved in 0.1% DMSO to make 1 µM (final concentration). f Values are from the literature (21).

(LB) broth supplemented with ampicillin (0.14 mM) at 37 °C for 16 h. The seed culture was transferred into 400 mL of LB broth in a flask, extensively incubated in a cooled incubator (Taitac) at 15 °C for 24 h after addition of isopropyl β-D-1thiogalactopyranoside (IPTG; a final concentration of 1 mM). The cells harvested by centrifugation at 5000 g were dissolved in 10 mL of Tris-HCl (20 mM, pH 7.6) and crushed with a tip sonicator for cell homogenation. His-tag-fused ERS in the homogenate was purified with a TALON metal affinity resin (Clontech). The eluted ERS solution was dialyzed overnight at 4 °C against phosphate buffered saline (PBS; 0.01 M, pH 7.4). Concentration of the purified ERS was determined using a Bradford reagent (Bio-Rad). In the presence of an excess level of enzymes, quantum yields (QYs) are defined by Ntot/Stot, where Ntot and Stot mean the total amounts of emitted photons and consumed substrate, respectively (17). If a luciferase level is excess compared to luciferins (E . S), all of the luciferins should be consumed. In this case, the initial level of luciferin is equivalent to the total consumed luciferin. For measurements of quantum yields (QYs), ERS was first mock-stimulated or stimulated with vehicle (0.1% DMSO), 1

µM OHT (final concentration), or 1 µM 17β-estradiol (E2; final concentration). A quantity of 60 µL of ERS (24 pmol) was set on each well of a 96-well plate, which was then loaded in a microplate reader (Mithras LB940; Berthold). At the same time, the injector of the microplate reader was filled with CTZ solution dissolved in 0.01 M PBS (pH 7.4). Immediately after automatic injection of 10 µL of CTZ (0.59 pmol) into each well, the total light emission was integrated until the reaction approached completion (ca. 10 min) at room temperature (22.5 °C). QYs according to CTZ variants were estimated as follows: 10 µL of CTZ or its derivatives (0.59 pmol per well) was set in each well of the 96-well plate, while the ERS solution was loaded in the injector. After automatic injection of 30 µL of the ERS solution (12 pmol per well) to each well, the total light emission was integrated until the reaction approached completion (ca. 10 min). As internal standards, QYs of RLuc and RLuc8 were estimated in the same instrumental setup for fidelity. Turnover rate is defined by Vmax/Etot, where Vmax and Etot are the maximal reaction rate and the total enzyme amount applied. For the measurement, 40 µL of native CTZ (ranging from 1.2 to 0.075 nmol) was set in each well of a 96-well plate, while PBS-buffered ERS solution was loaded in the injector before


the experiments. After automatic injection of 10 µL of ERS (1 pmol) to each well, the kinetics were immediately recorded every 0.05 s. The initial rates were plotted against the CTZ concentrations applied. The kinetic data were processed using a kinetic curve fitting software GraFit6 (Erithacus Software).

RESULTS Correlation between Molecular Tension and Bioluminescence Intensity of RLuc8. For an initial evaluation of molecular tension-bioluminescence correlation, a series of luciferases were sandwiched between ER LBD and Src SH2 domain (Figure 1). The examined luciferases include a full-length Renilla luciferase 8 (RLuc8; 36 kDa), Gaussia luciferase (GLuc; 20 kDa), and click beetle luciferase red (CBLuc; 60 kDa). Bioluminescence spectra of ERS largely varied before and after supplementation of 4-hydroxytamoxifen (OHT). The peak height (λmax: 486 nm) was surprisingly 3.5 times enhanced by a stimulation of 1 µM OHT. In contrast, both EGS and ECS did not exhibit a considerable variance in the luminescence intensities in the same experimental setup. The results are interpreted as follows: (i) According to the crystal structural analysis on RLuc8, the active site of RLuc8 consists of amino acids ranging from Asn53 to Phe286 (18). A ligand-activated ER LBD-SH2 binding should append a tension to the active site of the sandwiched RLuc8 (Figure 1D). (ii) Molecular structural comparison of ERS with EGS and ECS reveals that globular RLuc8 is appropriate to receive molecular tension, and thus advantageous for determining protein-protein interactions compared to other luciferases. Verification of Phosphorylation of ER LBD and the Subsequent ER LBD-SH2 Binding. It was previously demonstrated with a Western blot analysis that Tyr537 of ER LBD is phosphorylated by an addition of E2 and recognized by the SH2 domain of V-Src (9) (19) (20). Additionally, we verified the ligand-activated ER LBD-SH2 binding with a point mutant of ERS at Tyr537 to phenylalanine (Y537F), named EmRS (Figure 2A Inset). EmRS exhibited a largely diminished signalto-background (S/B) ratio (ca. 1.7 times) compared to intact ERS. As another control study, ERS was split into ER and RS for exerting a tension-free experimental setup, where ER and RS mean ER LBD-linked RLuc8 and RLuc8-linked SH2 (Figure 2A Inset), respectively. COS-7 cells carrying both ER and RS exhibited no variance in the luminescence intensities in response to 1 µM OHT, whereas ERS efficiently enhanced the bioluminescence intensities in response to the same stimulation. These results in Figure 2A Inset demonstrate that (i) a ER LBD-SH2 binding appends a molecular tension to the sandwiched RLuc8, resulting in an enhancement of the enzymatic activity; (ii) the ER LBD-SH2 binding occurs through a ligandactivated conformation change and phosphorylation of Tyr537 of ER LBD; and (iii) the luminescence intensities enhanced by ER LBD-SH2 binding can be taken as a measure of the estrogenicity of ligands. Ligand Sensitivity and Selectivity of ERS. Ligand sensitivity and selectivity of ERS were estimated in varying concentrations and types of ligands (Figure 2A,B). First, COS-7 cells carrying ERS were stimulated with varying concentrations of OHT or testosterone (T) for 20 min, and the consequent elevation of bioluminescence intensities was determined with a luminometer (Minilumat LB9507; Berthold). The luminescence intensities were quickly enhanced from 1 nM OHT and reached a plateau at 1 µM OHT. Fifty percent effective concentration (EC50) appeared at 6 nM OHT. In addition, we estimated the ligand selectivity of ERS in the presence of various steroids and chemicals. COS-7 cells carrying ERS were stimulated for 20 min with vehicle (0.1%

Kim et al.

DMSO) or 1 µM of E2, OHT, 5R-dihydrotestosterone (DHT), testosterone (T), genistein, or cortisol. The consequent bioluminescence intensites by ligands were subtracted by those from COS-7 cells stimulated with vehicle (0.1% DMSO). The results show that E2 elevates the bioluminescence intensities, which are almost equivalent with OHT, while genistein called a phytoestrogen also exhibited significant luminescence intensities under the same conditions. On the other hand, the other steroids such as DHT, T, and cortisol did not exhibit any considerable activity for ER LBD-SH2 binding. Genistein is one of several known isoflavones to interact with animal and human ER, that is, phytoestrogen or dietary estrogens. E2 is a natural, steroidal agonist of ER, whereas OHT is known as the synthetic antagonist. The luminescence intensities by ligands decreased in the following order: E2 g OHT > genistein . DHT ) T ) cortisol. The results represent that (i) the estrogenicities of E2, OHT, and genistein are efficiently converted by ERS to bioluminescence intensities through an intramolecular ER LBD-SH2 binding and (ii) ERS exerts ligand selectivity. Substrate Specificity of ERS. We fabricated a bioluminescent probe, where an intramolecular ER LBD-SH2 binding appends a molecular tension to the active site of RLuc8. Thus, we speculated that this transformation of the active site may influence substrate specificity. On the basis of this consideration, we examined the substrate specificity of ERS in the presence or absence of OHT (Figure 3). According to the results, the native coelenterazine, CTZ, was superior to other derivatives in the absolute bioluminescence intensities, whereas CTZ fcp was beneficial in the S/B ratio. The absolute luminescence intensities in the presence of native CTZ were ca. four times stronger than those in the presence of the other CTZ derivatives. The peak ratio before and after supplementation of OHT was as large as six in the presence of CTZ fcp (Figure 3A Inset). The distinctive luminescence intensities of native CTZ compared to other derivatives corresponded to the results in Table 1. Enzymatic Properties of ERS. To examine the enzymatic properties of ERS, the quantum yields (QYs), Michaelis-Menten constant (KM), and turnover rates were estimated by altering the ligands and CTZ variants (Table 1). The measurements were conducted using a luminescence microplate reader (Mithras LB940; Berthold) equipped with an automatic injector. QY, KM, and turnover rate of column-purified ERS were 6.3 ( 1.1%, 94.3 ( 37.1 µM, and 6.05 ( 0.51 × 1019 photons/s/ mol, respectively. This QY of ERS is approximately equivalent to a previous calculation, that is, 6.9 ( 0.1%, with intact RLuc8 (21). On the other hand, the present KM value of ERS is 58 times higher than a reference evaluation, that is, 1.6 ( 0.2 µM with native RLuc8. The turnover rate of ERS is 705 times slower than a previous calculation with intact RLuc8 (Table 1) (22). These comparisons demonstrate that enzymatic properties of RLuc8 except the QY are largely impaired by modification with ER LBD (28 kDa) and SH2 (12 kDa). QYs were similarly estimated after stimulation of vehicle-, OHT-, or E2-stimulated ERS (Table 1). QYs were found to be invariant by stimulation of the ligands. It is because E2 or OHT may alter the conformation of ER LBD in the purified ERS. However, ER LBD in purified ERS is not phosporylated because of the deficiency of endogenous protein kinases. This is additional evidence of phosphorylation-mediated ER LBD-SH2 binding. In addition, QYs of ERS were determined in the presence of a different kind of coelenterazine (CTZ) variant (Table 1). The results exhibited that ERS with native CTZ exhibited approximately four times higher QYs than the other CTZ derivatives, implicating substrate selectivity of ERS to native CTZ. This substrate preference was not significant in a previous study with intact RLuc8 (21).


Considering that the chemical backbone of CTZ is similar to that of the variants, the side chains of CTZ should play a critical role in the substrate selectivity of ERS. The basic structure of CTZ is called imidazo[1,2-a]pyrazin-3(7H)-one. CTZ 400A is deficient in both (i) the p-OH group of the phenol attached at C6 of native CTZ and (ii) the p-OH group on the benzyl substituent at C2 of native CTZ. The importance of these functional groups in CTZ was proven with the poorest luminescence intensity and S/B ratio in the presence of CTZ 400A as shown in Figure 3A. CTZ cp lacks the benzyl group attached at C8 of native CTZ. In practice, CTZ cp showed a diminished luminescence intensity and S/B ratio compared to native CTZ. Taken together, the present structural comparison between native CTZ and its variants revealed that three side chains are critical for the substrate activity: (i) the p-OH of the phenol attached at C6 of CTZ, (ii) the benzyl group attached at C8 of CTZ, and (iii) the p-OH group on the benzyl substituent at C2 of CTZ. Their contributions to enzymatic reactions of ERS were significant in this decreasing order: (i) > (ii) > (iii).

DISCUSSION Molecular Mechanism of the Present Molecular Distortion-Indexed Probe. The phosphorylation of estrogen receptor (ER) at Tyr537 is recognized by the Src homology domain 2 of V-Src (SH2), known as a nongenomic pathway of ER (19) (20). We previously demonstrated that both 17β-estradiol (E2) and 4-hydroxytamoxifen (OHT) phosphorylate Tyr537 of ER LBD using both our custom-made probes and Western blot analysis (9). In the present study, when RLuc8 is sandwiched between ER LBD and SH2 with a tight, minimal linker, the enzymatic activity surprisingly depended on the estrogenicity of the stimulators (Figures 1 and 2). Our control studies using both EmRS and ER+RS revealed that this activity variance is mediated by ligand-activated ER LBD-SH2 binding and the subsequent molecular distortion of the active site of RLuc8. Thus, the molecular mechanism may be explained as follows: (i) A specific ligand phosphorylates ER LBD at Tyr537; (ii) the phosphorylation is recognized by SH2, initiating ER LBD-SH2 binding; (iii) this binding should append a molecular tension to the sandwiched, globular RLuc8. In practice, Asn53, His285, and Phe286 in the active site of RLuc8 are in proximity of N- or C-terminal end of RLuc8 (1-311 AA) (18); and consequently, (iv) the molecular tension artificially appended by ER LBD–SH2 binding should vary the enzymatic activities. In contrast to RLuc8, beetle luciferases such as FLuc consist of two main domains linked with a long flexible hinge (23) (Figure 1A). The flexible hinge should relieve the molecular tension appended by ER LBD–SH2 binding. This relaxation of molecular tensions in CBLuc results in poor variance in the bioluminescence intensities of ECS by ligand activations. EGS carrying a full-length GLuc also enhanced little bioluminescence in response to OHT (Figure 1C). The sandwiched GLuc in EGS is the smallest luciferase among those discovered, which may sterically relieve the molecular tension to GLuc. Interpretation of Enzymatic Properties of ERS. According to a previous measurement, quantum yield (QY) and turnover rate of native Renilla luciferase 8 (RLuc8) are ca. 6.9 ( 0.1% and (4.4 ( 0.2) × 1022 photons/s/mol enzyme (21). This QY approximately corresponds with our calculation with ERS, whereas the literature turnover rate of intact RLuc8 is 7.1 × 102 times higher than that of ERS. A fundamental difference between native RLuc8 and ERS is the modification of RLuc8 with ER LBD (28 kDa) and SH2 (12 kDa). Considering that some amino acid membering the active sites are close to the Nand C-terminals of RLuc8 (18), the modification of RLuc8 with ER LBD and SH2 is considered to influence the turnover rate rather than QY. In addition, both ER LBD and SH2 inside ERS were


prepared by genetic truncation from a full-length ER and Src. The hydrophobic cross sections may have been exposed to the cytosol and would have been easy to impair, aggregate, or decompose. QYs before and after stimulation of purified ERS with 1 µM OHT or E2 were almost invariant (Table 1). These results are interpreted as follows: It is known that both OHT- and E2-bound ER LBDs expose the hydrophobic site to the cytosol and recruit phosphorylation (24) (25). However, the present in Vitro system with purified ERS lacks tyrosine kinases. Ligand-activated ER LBD cannot be phosphorylated and thus are not recognized by SH2. This is indirect evidence that ER LBD-SH2 binding occurs via phosphorylation of ER LBD. The other control studies in Figure 2A using EmRS (“m” abbreviates the mutation in Tyr537) and ER+RS and literature comprising a Western blot analysis (9) (19) (20) also support this view. Determination of Protein Phosphorylation with ERS. Phosphorylation-mediated protein-protein interactions prevail among intracellular signal transduction pathways in mammalian cells (26) (27). The phosphorylation of Tyr537 is a prerequisite for pivotal actions of ER including (i) estrogen-dependent hyperphosphorylation of the serine residues, (ii) dimerization, (iii) nuclear retention, and (iv) DNA binding (20). In particular, a number of effects of estrogens such as kinase activation in the cytosol are so rapid that they cannot be related to direct gene expression. These actions are known as nongenomic actions and mediated through phosphorylation of membrane-associated ERs (28). The phosphorylation is conducted by cytoplasmic Src (19) (20) and thus can be an index of nongenomic actions of ER mediated by Src. On the basis of this knowledge, ERS was designed as a molecular distortion-indexed bioluminescent probe, in which ER LBD-SH2 binding was fabricated to evoke a molecular tension to the sandwiched RLuc8. Conventional approaches to determine protein phosphorylation heavily depend on the Western blot analysis. However, by using such a conventional method, it is impossible to trace the ligand-activated, rapid dynamics of intracellular protein-protein interactions. The present study represents a competent methodology for tracing ligand-activated protein-protein interactions via molecular distortion. Sensorial Properties of ERS. Cell-based assays may be categorized into (i) genetic, transcriptional, and (ii) nontranscriptional assays. A genetic, transcriptional assay such as a reporter gene assay requires a long ligand stimulation time until the reporter protein is sufficiently accumulated. In contrast, the present method is characterized as a nontranscriptional assay using an intramolecular transformation of a luciferase. ERS is expressed earlier and localized in adequate intracellular compartments of interest. The luminescence intensity is ready to be developed once the cell is stimulated by a signal. The present data acquisition time, 20 min, is largely shortened in comparison with those of the reporter gene and protein-splicing schemes, which require from 2 to 24 h (29). Thus, the present method is applicable to a high-throughput analysis of the activities of bioactive small molecules, which activate temporal, short-term molecular events. The detection limits of precedent nontranscriptional assays such as FRET-based and single-chain probes to E2 ranged from 0.1 nM to 10 nM (9) (30) (31). Considering that the detection limit of ERS to OHT is close to 0.1 nM, the present scheme is competent for a sensitive determination of estrogen activities. Taken together, we have demonstrated a unique, nontranscriptional assay system based on the molecular tension of a luciferase appended by ER LBD-SH2 binding. Full-length RLuc8 was sandwiched between ER LBD and Src SH2 for constructing an integrated molecule-format probe. The working mechanism is supposed as follows: (i) estrogens first activate a phosphorylation of Tyr537 of ER LBD and subsequent intramolecular interaction between ER LBD and Src SH2. (ii) This


binding triggers a distortion of the active site of full-length RLuc8. (iii) This molecular tension results in an enhancement of the luciferase activity. Modification of RLuc8 with ER LBD and Src SH2 interestingly decrease the turnover rate rather than the QY. ERS exhibited substrate selectivity to native CTZ, and was as sensitive as 1 nM OHT. The present strategy for determining phosphorylation and the consequent molecular distortion may be utilized in evaluating a broad range of ligandmediated protein-protein interactions. Future studies should be directed for investigating an optimal combination of luciferases and a pair of interacting proteins, where distortion of the active site of luciferases may exert significant color changes and sensitive intensity variances.

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Molecular Tension-Indexed Bioluminescent Probe for Determining

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