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

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.. Trypsin cleavage increased the basal,

Tomomi Sato1, Atsushi Kodan2, Yasuhisa Kimura3, Kazumitsu Ueda2,3, Toru Nakatsu1and

Hiroaki Kato1

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 kcat values both with substrate (ksub) and without substrate (kbasal), but decreased the ksub⁄ kbasal 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 [ksub⁄ Km(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.

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

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-trypsin-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.

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 50C, 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 Km values for

MgATP, designated Km(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 Km(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

fragmentsa

Cleavage conditionsb Protease

N-terminal sequence obtained

Sequence surrounding cleavage sitec

Position within structure

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.bDetailed cleavage conditions are described in the Materials and methods.cThe 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.

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.

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 kbasalvalues 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 kbasal value, as reported previously [21,22] Likewise, for each substrate, the ksub values obtained with protease-cleaved P-gp were higher than those obtained with native P-gp Thus, linker cleavage also increased the ksub 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

0

20

40

60

80

100

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

9

1 3 5 7 Native

Trypsin Chymotrypsin

10

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)

studies may have contained contaminating proteins that

interact with P-gp [25,26] and cause this inconsistency

The ksubvalues ranged from 0.410 to 3.48 s)1, while the

kbasal 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 ksub values ranged from 3.44 to 11.1 s)1and from 3.69 to 11.7 s)1, while the kbasalvalues 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

ksub⁄ kbasal values For each substrate, the ksub⁄ kbasal

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)1of 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 kbasalb (s)1) ksubc (s)1)

k sub ⁄ k basald

(-fold) Km(D)(l M )

k sub ⁄ K m(D)

(s)1Æm M )1)

Chymotrypsin 2.95 ± 0.41

a

Values in parentheses indicate the relative molecular mass of each transport substrate.bk basal is the k cat value for basal ATPase activity with no transport substrates c ksub is the kcatvalue for substrate-stimulated ATPase activity with each transport substrate d ksub⁄ k basal

represents the fold stimulation by each transport substrate.

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

Km(D) values between the protease-cleaved P-gp and

native P-gp With digoxin, vinblastine, paclitaxel and

calcein-AM, the Km(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 Km(D)values between protease-cleaved P-gp and

native P-gp were within twofold With rhodamine 123,

colchicine, verapamil, nicardipine and rhodamine B, the

Km(D)values obtained with protease-cleaved P-gp were

two to sevenfold lower than those obtained with native

P-gp With valinomycin, the Km(D)values obtained with

protease-cleaved P-gp were three to fourfold higher than

those obtained with native P-gp The ksub⁄ Km(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 ksub⁄ Km(D)values with linker-cleaved

P-gp differed for each substrate The ksub⁄ Km(D)value is

a measure of substrate specificity [32] Thus, the

ksub⁄ Km(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 ksub⁄ Km(D) value between the native P-gp and the

protease-cleaved P-gp The relative ratio of the

ksub⁄ Km(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 ksub⁄ Km(D) values are < 100% because the ksub⁄ Km(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 ksub⁄ Km(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

ksub

Fig 6 Ratios of the ksub⁄ K m(D) values between native and

prote-ase-cleaved P-gp The ksub⁄ 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.

of native P-gp (Fig 2), revealing that cleavage of the

P-gp linker region increased P-gp ATPase activity

The ksub 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 ksub⁄ kbasal 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 ksub⁄ kbasal 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

ksub⁄ kbasal values The ksub⁄ kbasal value is a ratio of

coupled to uncoupled ATPase activity with substrate

recognition, suggesting that the ksub⁄ kbasal 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 Pi⁄ 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 Km(D) values and a few dozen-fold differences in the ksub⁄ Km(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 ksub⁄ Km(D)

of ATPase activity can be assumed to represent trans-port substrate specificity Thus, we evaluated the rela-tive ratio of the ksub⁄ Km(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 ksub⁄ Km(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

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 4C 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–

*

*

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

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.

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 23C for 20 min

Protease treatment of purified P-gp in detergent

micelles

Purified P-gp (2 mgÆmL)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)1of asolectin] was treated with trypsin,

chymotrypsin, V8 protease, or subtilisin at 37C 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 mgÆmL)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 37C 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 4C The running buffer consisted of

20 mm PIPES (pH 6.5), 200 mm NaCl, 10% glycerol,

5 mm 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 lL of 40 mm Tris–HCl (pH 7.4) 0.1 mm EGTA, 2 mm dithiothreitol, 5 mm MgATP and various concentrations of transport substrates at 37C 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 50C for

15 min before electrophoresis SDS–PAGE was performed