Báo cáo khoa học: Functional role of the linker region in purified human P-glycoprotein pot

Functional role of the linker region in purified human P-glycoprotein Tomomi Sato 1 , Atsushi Kodan 2 , Yasuhisa Kimura 3 , Kazumitsu Ueda 2,3 , Toru Nakatsu 1 and Hiroaki Kato 1 1 Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan 2 Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, Japan 3 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan Human P-glycoprotein (P-gp, ABCB1), which conveys multidrug resistance, is a drug efflux pump that trans- ports a wide variety of structurally unrelated com- pounds out of cells [1–4]. The transport by P-gp is driven by energy from ATP hydrolysis, and P-gp is classified as a member of the ATP-binding cassette (ABC) transporter family [5,6]. The transport of substrate by P-gp is thought to be coupled with ATP hydrolysis [7]. Without a transport substrate, P-gp has low basal ATP hydrolase (ATPase) activity, whereas with substrates P-gp exhibits high ATPase activity, which is known as substrate-stimu- lated ATPase activity [8–11]. Thus, the substrate-stim- ulated ATPase activity can be a measure of the recognition of substrate by P-gp. When titrating P-gp substrates, the activity increases up to a maximum but then decreases at high substrate concentrations. This characteristic bell-shaped activity profile has been Keywords ATPase activity; limited proteolysis; linker region; MDR1; P-glycoprotein Correspondence H. Kato, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimo-Adachi-cho, Sakyo-ku, Kyoto 606-8501, Japan Fax: +81 75 753 9272 Tel: +81 75 753 4617 E-mail: katohiro@pharm.kyoto-u.ac.jp (Received 28 December 2008, revised 19 April 2009, accepted 23 April 2009) doi:10.1111/j.1742-4658.2009.07072.x Human P-glycoprotein (P-gp), which conveys multidrug resistance, is an ATP-dependent drug efflux pump that transports a wide variety of struc- turally unrelated compounds out of cells. P-gp possesses a ‘linker region’ of $ 75 amino acids that connects two homologous halves, each of which contain a transmembrane domain followed by a nucleotide-binding domain. To investigate the role of the linker region, purified human P-gp was cleaved by proteases at the linker region and then compared with native P-gp. Based on a verapamil-stimulated ATP hydrolase assay, size- exclusion chromatography analysis and a thermo-stability assay, cleavage of the P-gp linker did not directly affect the preservation of the overall structure or the catalytic process in ATP hydrolysis. However, linker cleav- age increased the k cat values both with substrate (k sub ) and without substrate (k basal ), but decreased the k sub ⁄ k basal values of all 10 tested substrates. The former result indicates that cleaving the linker activates P-gp, while the latter result suggests that the linker region maintains the tightness of coupling between the ATP hydrolase reaction and substrate recognition. Inspection of structures of the P-gp homolog, MsbA, suggests that linker-cleaved P-gp has increased ATP hydrolase activity because the linker interferes with a conformational change that accompanies the ATP hydrolase reaction. Moreover, linker cleavage affected the specificity con- stants [k sub ⁄ K m(D) ] for some substrates (i.e. linker cleavage probably shifts the substrate specificity profile of P-gp). Thus, this result also suggests that the linker region regulates the inherent substrate specificity of P-gp. Abbreviations ABC, ATP-binding cassette; ATPase, ATP hydrolase; calcein-AM, 3¢,6¢-di(O-acetyl)-4¢,5¢-bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein tetraacetoxymethyl ester; NBD, nucleotide-binding domain; P-gp, P-glycoprotein; PIPES, piperazine-N,N’-bis(2-ethanesulfonic acid); TM, transmembrane; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; b-UDM, n-undecyl-b- D-maltopyranoside. 3504 FEBS Journal 276 (2009) 3504–3516 ª 2009 The Authors Journal compilation ª 2009 FEBS analyzed using modified Michaelis–Menten kinetics [12–15]. The kinetic models used to evaluate bell- shaped activity curves take into account an activating substrate-binding step at low substrate concentrations and an inhibitory drug-binding step at high substrate concentrations, followed by a catalytic ATP-hydrolysis step. P-gp is a 1280-amino acid polypeptide divided into two highly homologous halves: the N-half and the C-half [16]. Each half contains a transmembrane (TM) domain consisting of six TM helices followed by a nucleotide-binding domain (NBD) [16]. These two halves are connected by a ‘linker region’ of $ 75 amino acids that spans the region from approximately Glu633 to Tyr709 [16–18]. It is fascinating and still unknown why the two halves of P-gp are connected by a linker region, while the two halves of bacterial [19] and some mammalian ABC transporters, such as ABCG family members [20], are not connected by a linker region and act as homodimers or heterodimers. Recently, limited prote- olysis of the linker region has shed light on its involve- ment in the ATPase activity of P-gp. The linker region has been shown to be highly susceptible to different proteases [21–24], and the trypsin and chymotrypsin cleavage sites were identified as Arg680 and Leu682, respectively [22]. Trypsin cleavage increased the basal, verapamil-stimulated and vinblastine-stimulated ATPase activities, suggesting that cleavage of the linker activates P-gp [21]. By contrast, cleavage with chymotrypsin or proteinase K decreased the verapa- mil-stimulated and vinblastine-stimulated ATPase activities, even though the basal and colchicine-stimu- lated ATPase activities increased upon cleavage [22]. This disproportional alteration between basal and substrate-stimulated ATPase activity upon cleavage suggests that ATP hydrolysis and transport are proba- bly uncoupled by cleavage of the linker [22]. Although these results provided valuable suggestions for the role of the P-gp linker region, the molecular details, such as the involvement of the linker region in substrate recognition, are still unclear. In addition, it is still unknown why different proteases caused various changes in the ATPase activity of P-gp. As all previous studies used crude membrane fractions containing P-gp, the results could be affected by other proteins. In fact, it was reported that the linker region of P-gp interacts with other proteins such as RING finger protein 2 (RNF2) [25] and both alpha-tubulin and beta-tubulin [26]. Therefore, in order to investigate how the ATPase activity of P-gp is modulated by alter- ations in the linker region, it would be preferable to use a highly purified preparation of P-gp. Purified P-gp will not only exclude the effects of interacting proteins but can also be used to perform kinetic analy- ses of the native and linker-cleaved P-gp. In the present study, we investigated the functional role of the linker region of human P-gp using a highly purified and properly folded protein preparation [27]. We performed limited proteolysis experiments on P-gp and confirmed that the linker region was the site most susceptible to protease digestion; we also identified five cleavage sites, four of which were novel. Using a P-gp preparation in which the linker had been cleaved by trypsin and chymotrypsin, we measured the basal and substrate-stimulated ATPase activities for 10 transport substrates. This kinetic analysis provided new insight into the role of the linker region. In addition, we fur- ther analyzed the functional role of the linker region based on the crystal structures of a P-gp homolog, MsbA [28]. Results Protease treatment of purified P-gp in detergent micelles To investigate the role of the linker region of P-gp, a linker-cleaved P-gp was generated by protease cleavage because the linker region is highly susceptible to prote- ase cleavage [21–24]. Highly purified P-gp in detergent micelles was incubated with trypsin, chymotrypsin, V8 protease, or subtilisin, as indicated in the Materials and methods. Despite the different substrate specifici- ties of these proteases, all cleaved P-gp in a similar pattern and generated two fragments, with molecular masses of 67–69 and 60–65 kDa, as determined using SDS–PAGE (Fig. 1). Trypsin cleavage kinetics of reconstituted P-gp and its residual ATPase activity The relationship between the degree of trypsin cleav- age of P-gp and its residual verapamil-stimulated ATPase activity was investigated. Reconstituted P-gp was readily cleaved into two fragments, of 69 and 60 kDa, by SDS–PAGE (Fig. 2A). Further cleavage generated a 56 kDa fragment. As P-gp cleavage pro- ceeded, the verapamil-stimulated ATPase activity gradually increased to a maximum of 340% within 105 min. The increase in verapamil-stimulated ATPase activity correlated with a decrease in the residual amount of native P-gp (Fig. 2B). This increase in verapamil-stimulated ATPase activity was also observed following cleavage with chymotrypsin, as described below. T. Sato et al. Functional role of the linker region in P-gp FEBS Journal 276 (2009) 3504–3516 ª 2009 The Authors Journal compilation ª 2009 FEBS 3505 Identification of the cleavage sites by N-terminal amino acid sequence analysis To determine the protease cleavage sites, we performed N-terminal amino acid sequence analysis by Edman degradation. The results are summarized in Table 1, and the corresponding fragments on SDS–PAGE are shown in Figs 1 and 2A and in Fig. S1 as (A–E). These data identified fragments A, B, C and D as the C-terminal side fragments from Arg680, Leu662, Glu652 and Lys685, respectively. All of these cleavage sites are located in the P-gp linker region from Glu633 to Tyr709. Thus, the two fragments shown in Fig. 1 occurred upon cleavage of the linker region and were thereby identified as the N-half and C-half fragments of P-gp. Fragment E had a molecular mass of 37 kDa and was also generated by extensive trypsin digestion (shown in Fig. S1). The N-terminal amino acid sequence analysis identified fragment E as the C-termi- nal fragment from Arg933, which was predicted to be located on the cytoplasmic side of the TM 11 helix in a Sav1866-based homology model of P-gp [29]. Size-exclusion chromatography analysis To investigate the structural properties of trypsin- cleaved P-gp, we performed size-exclusion chromato- graphy. As shown by the arrows in lane 2 of Fig. 3A, all P-gp molecules were cleaved by trypsin into two fragments that migrated at 60 and 69 kDa when ana- lyzed using SDS–PAGE. The trypsin-cleaved P-gp and the native P-gp were subjected to size-exclusion chro- matography (Fig. 3B). The trypsin-cleaved P-gp eluted as a sharp peak at the same retention volume as the native P-gp (Fig. 3B). To further corroborate this result, the peak fractions of trypsin-cleaved P-gp were collected and rechromatographed, resulting in elution at the same retention volume as that of native P-gp (data not shown). These results strongly indicate that the N-half and C-half fragments of P-gp retain a stable interaction, even after the linker is cleaved. A B Fig. 2. Trypsin treatment and residual ATPase activity of reconsti- tuted P-gp. (A) Time course of trypsin treatment analyzed using SDS–PAGE followed by silver staining. (B) The verapamil-stimulated ATPase activity of P-gp with (d) or without ( ) trypsin treatment, and the remaining amount of P-gp (s). The verapamil-stimulated ATPase activity before protease digestion was set to a value of 100%. The remaining amounts of native P-gp (%) were quantified using IMAGE J software (National Institutes of Health). In ATPase activity measurement, all data points represent the means ± SD from three independent assays. Error bars are shown unless they are smaller than the symbol. Fig. 1. SDS–PAGE of P-gp cleaved by various proteases. The SDS– PAGE lanes are as follows: lane 1, native P-gp; lane 2, trypsin- cleaved P-gp (5-min incubation); lane 3, chymotrypsin-cleaved P-gp (30-min incubation); lane 4, V8 protease-cleaved P-gp (15-min incubation); lane 5, subtilisin-cleaved P-gp (5-min incubation). The fragment to the right of each asterisk was identified by N-terminal amino acid sequence analysis. Functional role of the linker region in P-gp T. Sato et al. 3506 FEBS Journal 276 (2009) 3504–3516 ª 2009 The Authors Journal compilation ª 2009 FEBS Comparison of the thermostability of native and trypsin-cleaved P-gp To examine the effect of protease cleavage on the thermo- stability of P-gp, the residual ATPase activities of native and trypsin-cleaved P-gp were examined after incubation at various temperatures. Reconstituted P-gp (0.01 mgÆmL )1 ) was treated with trypsin at a P-gp : trypsin ratio of 40 : 1 (w ⁄ w) at 20 °C for 3 h, which resulted in no residual native P-gp. After incubation of the native P-gp and the trypsin-cleaved P-gp at 40, 45, or 50 °C, the verapamil-stimulated ATPase activities were measured. As shown in Fig. 4, the residual ATPase activities of trypsin-cleaved P-gp and of native P-gp were similar after the heat treatments. This result indicates that there is no effect of trypsin cleavage on the thermo- stability of P-gp. Comparison of MgATP affinity between the native P-gp and the trypsin-cleaved P-gp To examine the effect of trypsin cleavage on the affin- ity for MgATP, we compared the K m values for MgATP, designated K m(MgATP) . The reconstituted P-gp was treated with trypsin at a P-gp : trypsin ratio of 200 : 1 (w ⁄ w), at 20 °C for 1.5 h. The ATPase activi- ties were measured in the presence of 50 lm verapamil and various concentrations of MgATP. There were no considerable differences in the K m(MgATP) values between the native P-gp and the trypsin-cleaved P-gp, which were 0.59 ± 0.33 and 0.89 ± 0.37 mm, respec- tively. Thus, the linker-cleaved P-gp and the native P-gp have similar affinities for MgATP. Kinetic properties of the native P-gp and the protease-cleaved P-gp with respect to several transport substrates To investigate the effect of linker cleavage on the recognition of various transport substrates, we deter- mined the kinetic parameters of P-gp ATPase activity with respect to 10 transport substrates. The chemical structures of the transport substrates tested in this study are shown in Fig. S2. Figure 5A shows the initial rates of ATP hydrolysis as a function of the verapamil concentration. The Table 1. Results of N-terminal amino acid sequence analysis. Corresponding fragments a Cleavage conditions b Protease N-terminal sequence obtained Sequence surrounding cleavage site c Position within structure A I Trypsin KLSTK 677 AQDRflKLSTK Linker region B I Chymotrypsin IRKRS 659 RSSLflIRKRS Linker region C I V8 protease M(S ⁄ L)XM(D ⁄ E) 649 DALEflMSSND Linker region D II Trypsin EALDE 682 LSTKflEALDE Linker region E II Trypsin KAHIF 930 NSLRflKAHIF Membrane surface d a Fragment A is shown in Figs 1 and 2A, fragments B and C are shown in Fig. 1, fragment D is shown in Figs 2A and S1, and fragment E is shown in Fig. S1. b Detailed cleavage conditions are described in the Materials and methods. c The identified cleavage sites are denoted by arrows (fl). d The cleavage site of fragment E was located on the cytoplasmic side of the TM 11 helix. A B Fig. 3. Size-exclusion chromatography. Purified P-gp was incubated with trypsin at a P-gp : trypsin ratio of 200 : 1 (w ⁄ w) at 20 °C for 30 min. The cleaved P-gp was analyzed using SDS–PAGE and size- exclusion chromatography. (A) SDS–PAGE of injected samples. Lane 1, native P-gp; lane 2, trypsin-treated P-gp. (B) Size- exclusion chromatography profiles of native (dotted line) and trypsin-treated (solid line) P-gp. T. Sato et al. Functional role of the linker region in P-gp FEBS Journal 276 (2009) 3504–3516 ª 2009 The Authors Journal compilation ª 2009 FEBS 3507 ATPase profiles of trypsin-cleaved P-gp and of chymo- trypsin-cleaved P-gp showed a characteristic pattern [30,31], similar to that of native P-gp. ATPase activity was stimulated in the presence of low substrate concen- trations but was inhibited in the presence of high substrate concentrations. These three curves were fitted by the modified Michaelis–Menten equation (Eqn 1) [13], and kinetic parameters were calculated as listed in Table 2. Similar curves were obtained with rhodamine 123, colchicine, nicardipine, rhodamine B and vinblas- tine, and the curves were fitted by Eqn (1) (data not shown). With the other substrates [nicardipine, digoxin, paclitaxel, 6¢-di(O-acetyl)-4¢,5¢-bis[N,N-bis(carboxym- ethyl)aminomethyl]fluorescein tetraacetoxymethyl ester (calcein-AM) and valinomycin], the simple Michaelis– Menten equation (Eqn 2) was used to calculate kinetic parameters because the curves did not obey Eqn (1) (Fig. 5B). The kinetic parameters for various structurally unrelated transport substrates obtained with native, trypsin-cleaved and chymotrypsin-cleaved P-gp are summarized in Table 2. The k basal values obtained with native, trypsin-cleaved and chymotrypsin-cleaved P-gp were 0.189, 2.38 and 2.95 s )1 , respectively. Thus, linker cleavage increased the k basal value, as reported previously [21,22]. Likewise, for each substrate, the k sub values obtained with protease-cleaved P-gp were higher than those obtained with native P-gp. Thus, linker cleavage also increased the k sub value. This result is inconsistent with a previous report that chymotrypsin cleavage decreased the verapamil-stimulated and vinblastine-stimulated ATPase activities [21,22]. However, the crude membrane used in these previous Residual ATPase activity (%) 0 20 40 60 80 100 0 5 10 15 20 Time of incubation (min) 40 °C 45 °C 50 °C Trypsin-cleaved P-gp Native P-gp Fig. 4. Residual ATPase activity after heating at 40, 45 and 50 °C. The residual ATPase activity profile for native P-gp incubated at 40 °C( ), 45 °C( ) and 50 °C(d), and that for trypsin-cleaved P-gp incubated at 40 °C(h), 45 °C(4) and 50 °C(s), are shown. The ATPase activity was measured in the presence of 50 l M verap- amil. All data points represent the means ± SD from three indepen- dent assays. Error bars are shown unless they are smaller than the symbol. 0 6 4 2 8 1 Verapamil (µ M) ATPase activity (s –1 ) 9 10 –1 10 1 10 2 10 3 10 4 1 3 5 7 Native Trypsin Chymotrypsin 10 A B ATPase activity (s –1 ) Valinomycin (µM) 0 1 2 3 4 5 6 0 4 2 1 3 5 6 7 8 9 10 11 12 Native Trypsin Chymotrypsin Fig. 5. Substrate concentration dependence of native and protease-cleaved P-gp ATPase activity. Purified and reconstituted P-gp was incu- bated with trypsin, chymotrypsin, or no protease at 20 °C for 1.5 h. The ratio of P-gp : trypsin and P-gp : chymotrypsin was 200 : 1 (w ⁄ w) and 100 : 1 (w ⁄ w), respectively. The ATPase activities of native (s), trypsin-cleaved ( ) and chymotrypsin-cleaved ( ) P-gp were measured in the presence of various concentrations of transport substrates. All data points represent the means ± SD from three independent assays. Error bars are shown unless they are smaller than the symbol. (A) The ATPase profiles with various concentrations of verapamil. Solid lines are fits to the modified Michaelis–Menten equation (Eqn. 1). (B) The ATPase profiles with various concentrations of valinomycin. Solid lines are fits to the simple Michaelis–Menten equation (Eqn. 2) Functional role of the linker region in P-gp T. Sato et al. 3508 FEBS Journal 276 (2009) 3504–3516 ª 2009 The Authors Journal compilation ª 2009 FEBS studies may have contained contaminating proteins that interact with P-gp [25,26] and cause this inconsistency. The k sub values ranged from 0.410 to 3.48 s )1 , while the k basal value was 0.189 s )1 for native P-gp. Thus, substrate-stimulated ATPase activities were clearly observed for native P-gp, as previously described [8–10,31]. For trypsin-cleaved P-gp and chymotrypsin- cleaved P-gp, the k sub values ranged from 3.44 to 11.1 s )1 and from 3.69 to 11.7 s )1 , while the k basal values were 2.38 and 2.95 s )1 , respectively. Thus, substrate- stimulated ATPase activities were also observed for protease-cleaved P-gp. The fold stimulation of the ATPase activity for each substrate is represented as k sub ⁄ k basal values. For each substrate, the k sub ⁄ k basal Table 2. Kinetic parameters of native and protease-cleaved P-gp. Reconstituted P-gp (0.01–0.03 mgÆmL )1 ) was incubated with either trypsin or chymotrypsin at 20 °C for 1.5 h. The P-gp : trypsin and P-gp : chymotrypsin ratios were 200 : 1 and 100 : 1, respectively. Native P-gp was prepared by incubating 0.01–0.03 mgÆmL )1 of reconstituted protein without proteases at 20 °C for 1.5 h. The cleavage was stopped and ATPase activity was measured as indicated in the Materials and methods. All data represent the means ± SD from three independent assays. Transport substrates a Protease treatment k basal b (s )1 ) k sub c (s )1 ) k sub ⁄ k basal d (-fold) K m(D) (lM) k sub ⁄ K m(D) (s )1 ÆmM )1 ) No substrate Native 0.189 ± 0.093 Trypsin 2.38 ± 0.33 Chymotrypsin 2.95 ± 0.41 Rhodamine 123 (342) Native 1.84 ± 0.01 9.8 ± 4.8 35.4 ± 1.2 52.0 ± 1.8 Trypsin 8.19 ± 0.24 3.4 ± 0.5 11.8 ± 3.5 692 ± 203 Chymotrypsin 8.56 ± 0.25 2.9 ± 0.4 17.5 ± 5.7 489 ± 158 Colchicine (399) Native 1.25 ± 0.03 6.2 ± 3.1 720 ± 48 1.66 ± 0.12 Trypsin 7.81 ± 0.02 2.9 ± 0.5 278 ± 24 28.2 ± 2.4 Chymotrypsin 6.40 ± 0.21 2.5 ± 0.5 280 ± 72 22.9 ± 5.9 Verapamil (440) Native 3.28 ± 0.13 17 ± 9 4.23 ± 0.84 775 ± 156 Trypsin 8.27 ± 0.12 3.5 ± 0.5 0.614 ± 0.139 13 500 ± 3100 Chymotrypsin 7.71 ± 0.18 2.6 ± 0.4 0.637 ± 0.276 12 100 ± 5300 Nicardipine (479) Native 3.48 ± 0.14 18 ± 9 1.91 ± 0.21 1820 ± 210 Trypsin 8.44 ± 0.46 3.6 ± 0.5 0.652 ± 0.442 12 900 ± 8800 Chymotrypsin 7.80 ± 0.30 2.6 ± 0.4 0.383 ± 0.151 20 400 ± 8100 Rhodamine B (479) Native 2.23 ± 0.07 12 ± 6 29.8 ± 6.0 74.9 ± 15.1 Trypsin 6.99 ± 0.33 2.9 ± 0.4 10.9 ± 1.2 641 ± 76 Chymotrypsin 7.22 ± 0.26 2.5 ± 0.4 14.2 ± 1.2 509 ± 47 Digoxin (781) Native 0.791 ± 0.033 4.2 ± 2.1 187 ± 16 4.23 ± 0.41 Trypsin 5.65 ± 0.08 2.4 ± 0.3 119 ± 21 47.3 ± 8.3 Chymotrypsin 5.99 ± 0.07 2.0 ± 0.3 101 ± 16 59.2 ± 9.5 Vinblastine (808) Native 1.90 ± 0.07 10 ± 5 1.21 ± 0.12 1560 ± 168 Trypsin 6.39 ± 0.31 2.7 ± 0.4 2.01 ± 0.23 3170 ± 389 Chymotrypsin 6.54 ± 0.23 2.2 ± 0.3 2.36 ± 0.92 2770 ± 1090 Paclitaxel (849) Native 0.410 ± 0.017 2.2 ± 1.1 0.874 ± 0.039 469 ± 29 Trypsin 3.44 ± 0.11 1.5 ± 0.2 0.775 ± 0.232 4440 ± 1340 Chymotrypsin 3.69 ± 0.05 1.3 ± 0.2 0.665 ± 0.272 5630 ± 2340 Calcein-AM (996) Native 3.19 ± 0.05 17 ± 8 2.03 ± 0.70 1570 ± 550 Trypsin 11.2 ± 0.7 4.7 ± 0.7 2.43 ± 0.41 4590 ± 830 Chymotrypsin 11.2 ± 0.2 3.8 ± 0.5 3.35 ± 0.37 3360 ± 380 Valinomycin (1,111) Native 2.95 ± 0.17 16 ± 8 0.395 ± 0.082 7460 ± 1610 Trypsin 11.1 ± 0.3 4.7 ± 0.7 1.11 ± 0.25 10 000 ± 2300 Chymotrypsin 11.7 ± 0.5 4.0 ± 0.6 1.51 ± 0.48 7770 ± 2470 a Values in parentheses indicate the relative molecular mass of each transport substrate. b k basal is the k cat value for basal ATPase activity with no transport substrates. c k sub is the k cat value for substrate-stimulated ATPase activity with each transport substrate. d k sub ⁄ k basal represents the fold stimulation by each transport substrate. T. Sato et al. Functional role of the linker region in P-gp FEBS Journal 276 (2009) 3504–3516 ª 2009 The Authors Journal compilation ª 2009 FEBS 3509 values of trypsin-cleaved P-gp and chymotrypsin- cleaved P-gp were lower than those of native P-gp. Thus, the fold stimulation by the transport substrate decreased with linker cleavage. There were some differences in the K m(D) values between the protease-cleaved P-gp and native P-gp. With digoxin, vinblastine, paclitaxel and calcein-AM, the K m(D) values for each substrate obtained with protease-cleaved P-gp were similar to those obtained with the native P-gp, and the differences in the K m(D) values between protease-cleaved P-gp and native P-gp were within twofold. With rhodamine 123, colchicine, verapamil, nicardipine and rhodamine B, the K m(D) values obtained with protease-cleaved P-gp were two to sevenfold lower than those obtained with native P-gp. With valinomycin, the K m(D) values obtained with protease-cleaved P-gp were three to fourfold higher than those obtained with native P-gp. The k sub ⁄ K m(D) values obtained with protease-cleaved P-gp were 1 to 17-fold higher than those obtained with native P-gp. The degree of increase in the k sub ⁄ K m(D) values with linker-cleaved P-gp differed for each substrate. The k sub ⁄ K m(D) value is a measure of substrate specificity [32]. Thus, the k sub ⁄ K m(D) value for ATPase activity can be assumed to represent the transport substrate specificity, and the shifts in substrate specificity with the linker-cleaved P-gp can be represented as the relative ratio of the k sub ⁄ K m(D) value between the native P-gp and the protease-cleaved P-gp. The relative ratio of the k sub ⁄ K m(D) value between the native P-gp and the protease-cleaved P-gp for each transport substrate is shown in Fig. 6. The relative ratios of the k sub ⁄ K m(D) values are < 100% because the k sub ⁄ K m(D) values of native P-gp are less than those of protease-cleaved P-gp. With vinblastine, calcein-AM and valinomycin, the relative ratios of the k sub ⁄ K m(D) values between the native P-gp and the protease-cleaved P-gp were relatively high, with values ranging from 34% to 96%, whereas with the other substrates, the relative ratios were low and the values ranged from 6% to 15%. For V8 protease-cleaved P-gp, the kinetic tendency described above was similar to that of trypsin-cleaved P-gp and chymotrypsin-cleaved P-gp (data not shown). Discussion We investigated the role of the linker region in human P-gp that spans from approximately Glu633 to Tyr709. As previously reported [21–24], the linker region appears to be the most flexible part of the P-gp structure (Fig. 1). We identified the cleavage sites of trypsin, chymotrypsin and V8 protease as Arg680, Leu662 and Glu652, respectively (Table 1). Nuti et al. [22] identified the same trypsin cleavage site at Arg680, but a different chymotrypsin cleavage site at Leu682. This difference may be a result of different P-gp prepa- rations; while our study used a purified preparation in detergent micelles, Nuti et al. used crude membrane fractions [22]. A comparison between native P-gp and linker- cleaved P-gp indicated that the linker region of P-gp seems to participate in neither the preservation of the overall structure nor the ATPase reaction itself. This is supported by the following findings. (i) Cleaving the linker did not inactivate the ATPase activity of P-gp; rather, linker-cleaved P-gp exhibited higher basal and substrate-stimulated ATPase activity than native P-gp (Fig. 2 and Table 2). These results indicate that, as for the native P-gp, the N-half and the C-half fragments of the linker-cleaved P-gp interact with each other during ATP hydrolysis. In addition, when recombinant N-half and C-half P-gp fragments were expressed alone, they did not exhibit substrate-stimulated ATPase activity [33]. (ii) Size-exclusion chromatography analysis indi- cated that the N-half and C-half fragments of P-gp are neither aggregated nor dissociated by cleavage of the linker region (Fig. 3). This finding is further supported by co-immunoprecipitation studies [21] and a pull- down assay [34]. (iii) Cleavage of the linker did not affect the thermostability of P-gp (Fig. 4). Increases in the verapamil-stimulated ATPase activ- ity correlated with a decrease in the residual amount Rhodamine 123 Colchicine Verapamil Nicardipine Rhodamine B Digoxin Vinblastine Paclitaxel Calcein-AM Valinomycin 0 10 60 50 40 30 20 70 80 130 120 110 100 90 140 The relative ratios of k sub /K m(D) values between native and protease cleaved P-gp (% protease cleaved P-gp) Fig. 6. Ratios of the k sub ⁄ K m(D) values between native and prote- ase-cleaved P-gp. The k sub ⁄ K m(D) value of native P-gp was divided by that of trypsin-cleaved (filled columns) or chymotrypsin-cleaved (open columns) P-gp. The quotients are shown as a percentage. Functional role of the linker region in P-gp T. Sato et al. 3510 FEBS Journal 276 (2009) 3504–3516 ª 2009 The Authors Journal compilation ª 2009 FEBS of native P-gp (Fig. 2), revealing that cleavage of the P-gp linker region increased P-gp ATPase activity. The k sub values for all of the tested substrates were increased by linker cleavage (Table 2). Thus, the increased ATPase activity with linker cleavage was common for all the substrates tested. Moreover, the substrate-stimulated ATPase activity was observed in both protease-cleaved P-gp and native P-gp (Table 2), indicating that linker-cleaved P-gp can recognize trans- port substrates. Taken together, these data suggest that the linker region regulates the ATP hydrolysis rate. Thus, one possible role for the linker region is that it serves as a cleavage activation site, as described previously [21]. However, increased ATPase activity also indicates that the linker region has another role as a suppressor of ATPase activity in the native P-gp. The k sub ⁄ k basal values of native P-gp were higher and ranged from 2.2 to 18, whereas those of linker-cleaved P-gp were lower and ranged from 1.3 to 4.7 (Table 2). Thus, the linker region appears to suppress the basal ATPase activity rather than the substrate-stimulated ATPase activity. The k sub ⁄ k basal values of the half-size ABC transporters, MsbA and Sav1866, ranged from 2 to 5, which are more similar to the linker-cleaved P-gp values than to those of native P-gp [35–38]. Thus, the linker region seems to be necessary to achieve high k sub ⁄ k basal values. The k sub ⁄ k basal value is a ratio of coupled to uncoupled ATPase activity with substrate recognition, suggesting that the k sub ⁄ k basal value repre- sents the tightness of coupling between ATP hydro- lysis and substrate recognition. Therefore, the linker region of P-gp may increase the tightness of coupling between ATP hydrolysis and substrate recognition and contribute to efficient substrate recognition. The involvement of the linker region in the coupling of ATP hydrolysis with transport was suggested previ- ously by Nuti et al. [22]. To investigate how cleavage of the P-gp linker increased ATPase activity, we examined the crystal structures of the inward-facing (closed apo) state and outward-facing (nucleotide bound) state of MsbA [28], a bacterial homolog of P-gp. The conformational change between these two states is thought to regulate the rate of ATP hydrolysis. This is because the forma- tion of a canonical ATP dimer sandwich of the NBDs and subsequent ATP hydrolysis occur in the outward- facing state, and P i ⁄ ADP release restores the inward- facing state. The linker region of P-gp can be assumed to connect the C-terminal helix of subunit A (shown in red in Fig. 7) with the N-terminal elbow helix of sub- unit B (shown in purple in Fig. 7) in the MsbA dimer. In the inward-facing state, there appears to be less interaction between the linker region and each subunit because both ends of these two helices are exposed to solvent. However, in the outward-facing state, the N-terminal elbow helix is in closer proximity to the plasma membrane, and the C-terminal a-helix moves to the bottom center of the NBD dimer (Fig. 7B). Thus, the linker region should pass around the NBD surface, and there appears to be more interactions between the linker region and subunit B because the C-terminal a-helix comes into close proximity to the NBD of subunit B. Therefore, during a conformational change between the two states, the linker region might interact with subunit B and cause steric hindrance. Moreover, some interactions within the linker region in the inward-facing state may need to be broken in order to change the linker from a contracted to an extended structure (Fig. 7A). Taken together, this analysis indicates that because of interference of the linker region, native P-gp cannot easily change its conformation. However, in the absence of the linker interference, the linker-cleaved P-gp can change conformation more easily and exhibit increased ATPase activity. The analysis of the kinetic parameters for 10 trans- port substrates indicated that linker cleavage modu- lated the ATPase activity differently for each substrate. With some transport substrates, several-fold differences in the K m(D) values and a few dozen-fold differences in the k sub ⁄ K m(D) values were observed between the native P-gp and the protease-cleaved P-gp (Table 2). Thus, these data indicate that the linker region affects P-gp substrate recognition, although there seems to be no direct interaction between the linker region and transport substrates. The k sub ⁄ K m(D) of ATPase activity can be assumed to represent trans- port substrate specificity. Thus, we evaluated the rela- tive ratio of the k sub ⁄ K m(D) values between the native P-gp and the linker-cleaved P-gp to investigate the effect of the linker region on P-gp substrate specificity. With vinblastine, calcein-AM and valinomycin, the relative ratios of the k sub ⁄ K m(D) values between the native P-gp and the linker-cleaved P-gp were higher than those with the other substrates (Fig. 6). This result indicates that native P-gp has relatively higher substrate specificity for these three substrates than the linker-cleaved P-gp. Therefore, the relative substrate specificity is shifted by linker cleavage, suggesting that the linker region enhances the inherent substrate specificity of P-gp. Transport measurement is needed to elucidate the role of the linker in substrate export. A relatively hydrophilic substrate, such as the peptide DAMGO (Tyr-d-Ala-Gly-N-Methyl-Phe-Gly-ol) [39], would be suitable for this measurement. T. Sato et al. Functional role of the linker region in P-gp FEBS Journal 276 (2009) 3504–3516 ª 2009 The Authors Journal compilation ª 2009 FEBS 3511 Materials and methods Materials L-1-Tosylamido-2-phenylethyl chloromethyl ketone (TPCK)- treated trypsin was purchased from Promega (Madison, WI, USA). Chymotrypsin and V8 proteinase were purchased from Roche Diagnostics (Mannheim, Germany). Subtilisin, digoxin, nicardipine and calcein-AM were purchased from Sigma (St Louis, MO, USA). Rhodamine 123, colchicine, verapamil, rhodamine B, vinblastine and pacritaxel were purchased from Wako (Osaka, Japan). Valinomycin was purchased from Fluka (Buchs, Switzerland). n-Undecyl-b-d- maltopyranoside (b-UDM) was purchased from Anatrace (Maumee, OH, USA). Escherichia coli total lipid extract was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Expression and purification of P-gp Histidine-tagged wild-type human P-gp was expressed using the baculovirus ⁄ expressSF+ insect cell system and purified as described previously [27], with slight modifications. Briefly, the expressSF+ membranes containing overexpres- sed P-gp were solubilized with buffer containing 1.0% b-UDM, and insoluble materials were removed by centrifu- gation at 100 000 g for 1 h. The P-gp was purified by one- step affinity chromatography using Talon Superflow Metal Affinity Resin (Clontech, Mountain View, CA, USA) with buffer containing 0.087% b-UDM. When necessary, the purified P-gp was concentrated using an Amicon-Ultra device with a molecular mass cut-off of 50 k (Millipore, Bedford, MA, USA). The P-gp preparation had high pur- ity, as shown in lane 1 of Fig. 1. All purification steps were performed at 4 °C. The purified P-gp was stored at )80 °C until further use. Reconstitution into liposomes To prepare liposomes, 50 mg of E. coli total lipid extract dissolved in chloroform was dried and hydrated with 2.5 mL of ATPase reaction buffer (40 mm Tris–HCl, pH 7.4, 0.1 mm EGTA, 2 mm dithiothreitol). The hydrated lipid suspension was subjected to five freeze–thaw cycles. Frozen stocks of lipid were stored at )80 °C. After freeze– * * C-terminal α -helix Subunit A Subunit B * * NBD TM Inward facing state A B (closed apo state) Outward facing state (nucleotide binding state) 90° Subunit A Subunit B C-terminal α -helix Elbow helix domain Fig. 7. Schematic diagrams of the P-gp linker superimposed on the MsbA structures. Two MsbA conformations in the inward-facing (closed apo state, PDB ID; 3b5x) and outward-facing (nucleotide bound state, PDB ID; 3b60) states are shown. One monomer of the MsbA (subunit A) is shown in light pink and the other (subunit B) is shown in gray. (A) Side view of the diagrams. The C-terminal a-helix of subunit A is shown in red and the N-terminal elbow helix of subunit B is shown in purple. The putative linker region of P-gp is shown as a dotted line and the start of the linker region is denoted with an asterisk. The minimum path of the linker region is shown as a blue dotted line. The arrow represents the movement of the N-terminal elbow helix of subunit B during the conformational change from the inward-facing state to the outward- facing state. (B) Bottom-up view of the NBDs: the diagrams shown in Fig. 7A were rotated 90 ° around a horizontal axis. The arrows represent the movement of each NBD during the conformational change from the inward-facing state to the outward- facing state. Functional role of the linker region in P-gp T. Sato et al. 3512 FEBS Journal 276 (2009) 3504–3516 ª 2009 The Authors Journal compilation ª 2009 FEBS thawing, the lipid suspension was sonicated in a bath soni- cator until the suspension clarified. For reconstituting P-gp into liposomes, purified P-gp (P-gp was purified using a two-step procedure: TALON metal affinity and size-exclu- sion chromatography) containing 0.06% b-UDM was diluted 10-fold in the lipid-containing ATPase reaction buffer at a protein : lipid ratio of 1 : 1 (w ⁄ w). Then, the mixture was incubated at 23 °C for 20 min. Protease treatment of purified P-gp in detergent micelles Purified P-gp (2 mgÆ mL )1 ) in detergent micelles [20 mm piperazine-N,N’-bis(2-ethanesulfonic acid) (PIPES), pH 6.5; 300 mm NaCl, 300 mm imidazole, 20% glycerol, 0.087% b-UDM, 0.1 mgÆmL )1 of asolectin] was treated with trypsin, chymotrypsin, V8 protease, or subtilisin at 37 °C for various periods of time. The P-gp : trypsin, P-gp : chymotrypsin, P-gp : V8 protease and P-gp : subtilisin ratios were 20 : 1, 20 : 1, 20 : 1 and 100 : 1 (w ⁄ w), respectively. The cleavage was stopped by adding an excess of soybean trypsin inhibitor (Roche) and 1 mm phenylmethanesulfonyl fluoride. The cleaved P-gp was separated by SDS–PAGE and then visua- lized with silver staining. Measurement of trypsin cleavage kinetics of reconstituted P-gp and its residual ATPase activity Purified and reconstituted P-gp (0.01 mgÆ mL )1 ) was incu- bated with TPCK-treated trypsin at a P-gp : trypsin ratio of 50 : 1 (w ⁄ w) at 20 °C for various periods of time. The cleavage was stopped by addition of excess soybean trypsin inhibitor (Roche) and 1 mm phenylmethanesulfonyl fluo- ride. The cleaved P-gp was subjected to an ATPase assay in the presence of 50 lm verapamil at 37 °C for 30 min. Then, the samples were separated by SDS–PAGE and visualized with silver staining. N-terminal amino acid sequencing of protease-cleaved P-gp N-terminal amino acid sequence analysis of protease- cleaved P-gp was performed under two conditions, as fol- lows. Condition I (mild treatment with various proteases): purified P-gp (2 mgÆmL )1 ) in buffer (20 mm PIPES, pH 6.5; 300 mm NaCl, 300 mm imidazole, 20% glycerol, 0.087% b-UDM, 0.1 mgÆmL )1 of asolectin) was incubated with trypsin, chymotrypsin, or V8 protease at a P-gp : protease ratio of 200 : 1 (w ⁄ w) at 20 °C for 30 min. Condition II (extensive trypsin treatment): purified P-gp (3 mgÆmL )1 ) in buffer (20 mm PIPES, pH 6.5; 300 mm NaCl, 300 mm imidazole, 20% glycerol, 0.087% b-UDM, 0.1 mgÆmL )1 of asolectin, 5 mm MgATP) was incubated with trypsin at a P-gp : trypsin ratio of 20 : 1 (w ⁄ w) at 37 °C for 30 min. In both conditions, 15–30 lg of the digested fragments were separated by SDS–PAGE and transferred to an Immobilon-P transfer membrane (Millipore). The fragments were stained with Coomassie Brilliant Blue R-350 (GE Healthcare, UK Ltd), excised from the membranes and analyzed using a Procise 492HT protein sequencer (Applied Biosystems, Foster City, CA, USA). Although some sub peaks were found, only the main peaks were unequivocally interpretable and recorded as valid data. Size-exclusion chromatography of native and protease-cleaved P-gp Purified P-gp (1 mgÆmL )1 ) in buffer (20 mm PIPES, pH 6.5; 300 mm NaCl, 300 mm imidazole, 20% glycerol, 0.087% b-UDM, 0.02% cholesteryl hemisuccinate) was incubated with trypsin at a P-gp : trypsin ratio of 200 : 1 (w ⁄ w) at 20 °C for 30 min. Size-exclusion chromatography was performed on a Superdex 200 10 ⁄ 300 GL column (GE Healthcare) at 4 °C. The running buffer consisted of 20 mm PIPES (pH 6.5), 200 mm NaCl, 10% glycerol, 5mm dithiothreitol, 0.06% b-UDM and 0.02% cholesteryl hemisuccinate, and the flow rate was 0.5 mL per min. Each sample (100 lL) containing 100 lg of P-gp was loaded, and the elution profiles were monitored by the absorbance at 280 nm. This experiment was performed twice. ATPase measurements The reconstituted protein (100–400 ng) was incubated in 20 lLof40mm Tris–HCl (pH 7.4) 0.1 mm EGTA, 2 mm dithiothreitol, 5 mm MgATP and various concentrations of transport substrates at 37 °C for 30 min. After the reaction, the samples (16 lL) were mixed with 15 lL of 12% SDS to stop the ATP hydrolysis reaction. The amount of released inorganic phosphate was measured using a colorimetric method [40]. All data points represent the means ± SD from three independent assays. Error bars are shown unless they are smaller than the symbol. The initial hydrolysis rate was routinely calculated using a one-point assay at 30 min because linearity in the time course was confirmed until 30 min within 37 lm per min of the initial rate (data not shown). In the present study we performed the measure- ments under conditions that restrict the initial rates below this value. SDS–PAGE analysis Samples were prepared in 1 · buffer (10 mm Tris–HCl, pH 8.0; 10% sucrose, 40 mm dithiothreitol, 1 mm EDTA, 2% SDS, 10 lgÆmL )1 pyronin Y) and incubated at 50 °C for 15 min before electrophoresis. SDS–PAGE was performed T. Sato et al. Functional role of the linker region in P-gp FEBS Journal 276 (2009) 3504–3516 ª 2009 The Authors Journal compilation ª 2009 FEBS 3513 [...]... modulation of the human P-glycoprotein involves conformational changes mimicking catalytic Functional role of the linker region in P-gp 25 26 27 28 29 30 31 32 33 34 35 36 37 38 transition intermediates Arch Biochem Biophys 450, 100–112 Rao PS, Mallya KB, Srivenugopal KS, Balaji KC & Rao US (2006) RNF2 interacts with the linker region of the human P-glycoprotein Int J Oncol 29, 1413–1419 Georges E (2007) The. . .Functional role of the linker region in P-gp T Sato et al following the method of Laemmli [41] using PAGEL (5–20%) (ATTO, Tokyo, Japan) Proteins separated in the polyacrylamide gel were visualized using a Silver Stain II Kit (Wako) Measurement of protein concentrations The protein concentration of purified P-gp was determined by measuring the absorbance at 280 nm The A0.1% value at... 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This kinetic analysis provided new insight into the role of the linker region. In addition, we fur- ther analyzed the functional role of the linker region based on the crystal structures of a. cleavage of the linker [22]. Although these results provided valuable suggestions for the role of the P-gp linker region, the molecular details, such as the involvement of the linker region in substrate recognition,. subunit B is shown in purple. The putative linker region of P-gp is shown as a dotted line and the start of the linker region is denoted with an asterisk. The minimum path of the linker region is shown