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A Tryptophan Residue Located at the Middle of Putative Transmembrane Domain 11 is Critical for the Function of Organic Anion Transporting Polypeptide 2B1 Jialin Bian, Meng Jin, Mei Yue, Meiyu Wang, Hongjian Zhang, and Chunshan Gui Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00648 • Publication Date (Web): 31 Aug 2016 Downloaded from http://pubs.acs.org on September 3, 2016

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Molecular Pharmaceutics

A Tryptophan Residue Located at the Middle of Putative Transmembrane Domain 11 is Critical for the Function of Organic Anion Transporting Polypeptide 2B1 Jialin Bian, Meng Jin, Mei Yue, Meiyu Wang, Hongjian Zhang, and Chunshan Gui*

Department of Pharmaceutical Analysis, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China

*

Corresponding author: Department of Pharmaceutical Analysis, College of

Pharmaceutical Sciences, Soochow University, 199 Renai Road, Suzhou Industrial Park, Suzhou 215123, China. Phone:

+86

512

65882089.

Fax:

+86

512

[email protected].

ACS Paragon Plus Environment

65882089.

E-mail:


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ABSTRACT: Organic anion transporting polypeptide 2B1 (OATP2B1) which is highly expressed in enterocytes and hepatocytes could be a key determinant for the intestinal absorption and hepatic uptake of its substrates, most of which are amphipathic organic anions. Tryptophan residues may possess a multitude of functions for a transport protein through aromatic interactions, such as maintaining the proper protein structure, guiding the depth of membrane insertion, or interacting directly with substrates. There are totally six tryptophan residues in OATP2B1. However, little is known about their role in the function and expression of OATP2B1. Our results show that while W272, W276, and W277 located at the border of extracellular loop 3 and transmembrane domain 6 exhibit a moderate effect on the surface expression of OATP2B1, W611 located at the middle of transmembrane domain

11

plays

a

critical

role

in

the

function

of

OATP2B1.

The

tryptophan-to-alanine mutation of W611 changes the kinetic characteristics of OATP2B1-mediated estrone-3-sulfate (E3S) transport radically, from a monophasic saturation curve (with Km and Vmax values being of 7.1 ± 1.1 µM and 182 ± 7 pmol/normalized mg/min, respectively) to a linear curve. Replacing alanine with a phenylalanine will rescue most of OATP2B1’s function, suggesting that the aromatic side chain of residue 611 is very important. However, hydrogen-bond forming and positively charged groups at this position are not favorable. The important role of W611 is not substrate-dependent. Molecular modeling indicates that the side chain of W611 faces towards the substrate translocation pathway and might interact with


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substrates directly. Taken together, our findings reveal that W611 is critical for the function of OATP2B1.

KEYWORDS: OATP2B1, transporter, transmembrane domain, tryptophan residue, molecular modeling

ABBREVIATIONS: BSP, bromosulfophthalein; DHEAS, dehydroepiandrosterone sulfate; DMEM, Dulbecco’s Modified Eagle’s medium; E3S, estrone-3-sulfate; ESI, electrospray ionization; FBS, fetal bovine serum; HEK293, human embryonic kidney cells; IS, internal standard; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MRM, multiple reaction monitoring; OATP, organic anion transporting polypeptide; PBS, phosphate-buffered saline; TM, transmembrane domain



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INTRODUCTION The organic anion transporting polypeptides (OATPs for humans; Oatps for other species) form a superfamily of sodium-independent transport systems that mediate the transmembrane transport of a wide range of amphipathic endogenous and exogenous organic substances.1,2 So far 11 human OATPs have been identified and most of them are expressed in multiple tissues throughout the body.3-5 OATP2B1, which is localized at the apical membrane of enterocytes6 and sinusoidal membrane of hepatocytes7 as well as in various other tissues,8-10 could be a key determinant for the intestinal absorption and hepatic uptake of its substrates. Therefore, OATP2B1 might have great importance to the absorption and disposition of its substrate drugs such as aliskiren, fexofenadine, glibenclamide, and statins.11-15 Hydropathy analysis and experimental evidence indicate that all OATPs comprise 12 putative transmembrane domains (TMs).16-19 Common structural features among OATPs include a large extracellular loop 5 with conserved cysteine residues between TMs 9 and 10, and an OATP superfamily signature with the sequence D-X-RW-(I,V)-GAWWX-G-(F,L)-L located at the border of extracellular loop 3 and TM 6.1,3 The three tryptophan residues in the superfamily signature are highly conserved among human OATPs and it was reported that two of them, namely W258 and W259 (corresponding to W276 and W277 in OATP2B1), are critical for the function of OATP1B1.20 However, little is known about the role of tryptophan residues in the function of OATP2B1.


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In OATP2B1 there are totally six tryptophan residues, namely W272, W276, W277, W523, W611, and W629. To determine their locations in the structure of OATP2B1, first of all we constructed a topological model for OATP2B1. Previously, based on the crystal structure of EmrD (PDB entry 2GFP) and fold recognition method, we developed a three-dimensional structure model for OATP1B3.18 Based on the 3D model of OATP1B3 and the sequence alignment pattern between OATP2B1 and 1B3 which is determined by a multiple sequence alignment of all 11 human OATPs, we constructed a topological model for OATP2B1 (Figure 1A). OATP2B1 consists of 709 amino acids with N- and C-terminals located intracellularly. The starting and ending residues for each of these 12 transmembrane domains were determined and labeled in the model. As shown in Figure 1A, W272, W276, and W277 are located at the border of extracellular loop 3 and TM 6; W523 is located at the extracellular loop 5 between TMs 9 and 10; W611 is located at the middle of TM 11; and W629 is located at the extracellular loop 6 between TMs 11 and 12. Interestingly, five of these six tryptophan residues are located at the extracellular side of OATP2B1, while only W611 is located at the middle of a transmembrane α-helical segment. Sequence alignment of all human OATPs shows that besides the three conserved tryptophan residues (W272, W276, and W277) in the superfamily signature, W629 is also conserved in human OATPs; however, W523 is not conserved at all. W611 is conserved between OATP2 family members, but it is not conserved with all other OATP family members (Figure 1B). (Figure 1)



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In the present study, the role of these six tryptophan residues in the function and expression of OATP2B1 has been examined. Our findings reveal that W272, W276, and W277 have a moderate effect on the surface expression of OATP2B1, and W611 plays a critical role in the function of OATP2B1. However, W523 and W629 do not show any significant effect on both the surface expression and function of OATP2B1.

MATERIALS AND METHODS Chemicals and Reagents. Radiolabeled [3H]estrone-3-sulfate was purchased from PerkinElmer (Waltham, MA). Unlabeled estrone-3-sulfate (E3S), atorvastatin, fluvastatin, and rosuvastatin were obtained from Sigma-Aldrich (St. Louis, MO). Bromosulfophthalein (BSP) was purchased from Acros Organics (New Jersey). Fetal bovine serum (FBS), Dulbecco’s Modified Eagle’s medium (DMEM), and trypsin were from Hyclone (Logan, UT). Lipofectamine 2000 and Opti-MEM were purchased from

Invitrogen

(Carlsbad,

CA).

Sulfo-N-hydroxysuccinimide-SS-biotin,

streptavidin-agarose beads, and the BCA protein assay kit were purchased from Pierce Chemical (Rockford, IL). Antibodies for detecting the six-His tag and the Na+/K+-ATPase α subunit were purchased from Tiangen (Beijing, China) and Abcam (Boston, MA). Horseradish peroxidase-conjugated secondary antibodies were purchased from ProteinTech (Chicago, IL) and Sunshine Biotechnology (Nanjing, China). Immobilon Western blot detection kit was from Millipore (Billerica, MA). Human embryonic kidney (HEK293) cell line was from ATCC (Manassas, VA).


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Expression of OATP2B1 and Its Mutants. A six-His tag was introduced at the C-terminal end of the open reading frame of human OATP2B1*321 by PCR, and the resulting construct was cloned into the pcDNA5/FRT vector via NheI and NotI sites. Site-directed mutagenesis was performed by QuikChange method and all constructs were confirmed by DNA sequencing. HEK293 cells were cultured and transfected as described previously.18 In brief, HEK293 cells were grown at 37 °C in a humidified 5% CO2 atmosphere in DMEM medium supplemented with 10% FBS. HEK293 cells were transiently transfected with the plasmids of OATP2B1*3 and its mutants using Lipofectamine 2000 according to the manufacturer’s instruction. Transfected cells were incubated for 24 h at 37 °C and then used for surface biotinylation and transport assays. Cell Surface Biotinylation and Immunoblot Analysis.

Cell surface

biotinylation and immunoblot analysis of OATP2B1 and its mutants were carried out with the methods described previously.15 In brief, HEK293 cells were cultured and transfected in poly-D-lysine coated 6-well plates. 24 h after transfection, cells were treated with sulfo-N-hydroxysuccinimide-SS-biotin (1 mg/mL in PBS) and washed and lysed with lysis buffer (10 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, and 1% Triton X-100, pH 7.4, containing protease inhibitors). After centrifugation, the supernatants of lysates were incubated with streptavidin-agarose beads for 1 h at room temperature. After wash, cell surface proteins were recovered from the resin by incubation of the beads with 2×Laemmli buffer containing 100 mM dithiothreitol. Cell membrane proteins were then subjected to SDS-polyacrylamide gel



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electrophoresis and immunoblot analysis. OATP2B1 and its mutants were detected with a mouse anti-His antibody (Tiangen) (1:2000), followed by HRP-conjugated goat

anti-mouse

IgG

(ProteinTech)

(1:5000).

Plasma

membrane

marker

Na+/K+-ATPase was detected with a rabbit anti-Na+/K+-ATPase α subunit antibody (Abcam) (1:5000), followed by HRP-conjugated goat anti-rabbit IgG (Sunshine) (1:10000). Immunoblots were developed with chemiluminescence method and detected with X-ray film. Protein band intensities were quantitated with Quantity One software (Bio-Rad Laboratories, Hercules, CA). Uptake Assay. HEK293 cells were seeded in poly-D-lysine coated 24-well plates and transfected with Lipofectamine 2000. Transport assays were performed 24 h post-transfection. For E3S uptake assay, we used radiolabeled [3H]E3S and liquid scintillation counter to measure the amounts of E3S transported into cells. Initial experiments showed that OATP2B1-mediated uptake of 0.1 and 100 µM E3S was linear up to 1.5 min. Therefore, uptake and kinetic experiments for E3S were performed at 1 min and its procedure was the same as described in our previous paper.22 For BSP, atorvastatin, fluvastatin, and rosuvastatin uptake, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify substrates transported into cells. The uptake time and LC-MS/MS methods for atorvastatin, fluvastatin, and rosuvastatin were the same as previously described.15 In all experiments, cells transfected with empty vector served as background control. Transporter-specific uptake was calculated by subtracting the background uptake


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from OATP-transfected uptake and normalized it with protein surface expression level. The LC-MS/MS method for BSP was described as follows. Chromatographic separation was achieved with an Agela Venusil C18 column (2.1mm×50 mm, 5 µm) and the flow rate was set at 0.3 mL/min. The mobile phase consisted of 5 mM ammonium acetate aqueous solution (A) and methanol (B) with the following gradient: 0–0.5 min, 40% B; 1.5 min, 95% B; 1.5–3.0 min, 95% B; and 3.2–5.5 min, 40% B. The mass spectrometer was operated in the negative electrospray ionization (ESI) mode and quantitation was performed by multiple reaction monitoring (MRM). The ion transitions for BSP and internal standard (IS) betamethasone were selected as m/z 815.0→771.0 and m/z 391.3→361.4, respectively. Structural Modeling of OATP2B1 and Molecular Docking of E3S to OATP2B1. Previously, we constructed a three-dimensional structure model of OATP1B3 based the crystal structure of EmrD (PDB entry 2GFP) by fold recognition method.18 In the present study, a structure model for OATP2B1 has been developed based on the structure model of OATP1B3 with the following procedures. First, the sequence alignment pattern between OATP2B1 and OATP1B3 was determined by a multiple sequence alignment of all 11 human OATPs. The sequence similarity between OATP2B1 and OATP1B3 is about 45%. Then, based on the alignment pattern and structure model of OATP1B3, the theoretical model of OATP2B1 was established by the Build Homology Models protocol incorporated in the Modeler program in Discovery Studio (DS) 2.5.23 The modeled structure was minimized with



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the CHARMM force field using the Minimization protocol.24 In minimizations the Generalized Born model was employed for a better approximation of the solvent effect, and the dielectric constant of solvent was set to be 80. To construct the complex structure of OATP2B1-E3S, molecular docking of E3S to the OATP2B1 model was carried out using AutoDock 4.2.25 The Lamarckian genetic algorithm (LGA) was used for docking with the following settings: a maximum number of 2500000 energy evaluations, an initial population of 150 randomly placed individuals, a maximum number of 27000 generations, a mutation rate of 0.02, a crossover rate of 0.8, and an elitism value of 1. For the adaptive local search method, the pseudo-Solis and Wets algorithm was applied with a maximum of 300 iterations per search. The conformer with the lowest binding free energy and reasonable conformation was selected as its binding conformation. The interaction between E3S and OATP2B1 was analyzed by Ligplot.26 Data Analysis. Uptake experiments were performed in triplicates and data with error bars represent mean ± standard deviation. Kinetic parameters were calculated using non-linear regression analysis incorporated in Prism 5 (GraphPad Software, La Jolla, CA). Unpaired t test was used to compare the difference between means of two groups. When comparing two or more groups with control, one-way ANOVA was performed followed by Dunnett’s test. The p value for statistical significance was set to be < 0.05 at 95% confidence interval.

RESULTS


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Surface Expression and Functional Characterization of OATP2B1 and its Six Tryptophan-to-Alanine Mutants. To investigate the functional role of six tryptophan residues in OATP2B1, first we mutated each of them to alanine and obtained six mutants, namely W272A, W276A, W277A, W523A, W611A, and W629A, and measured their transport activities for E3S which is a model substrate for OATP2B1. As shown in Figure 2A, the transport activities of mutants W272A, W276A, W277A, W611A, and W629A are significantly decreased as compared to that of wild-type OATP2B1, with mutant W611A showing most significant reduction of its activity which was only about 7% of that of OATP2B1. The reduction of activity could be due to lowered surface expression or reduced transport function. To rule out the factor of surface expression, we determined their surface expression levels by surface biotinylation and immunoblot analysis and normalized their activities to their surface expression levels. As shown in Figure 2B, mutants W272A, W276A, and W277A show most significant reduction of their surface expression as compared to OATP2B1. The surface expression of mutant W629A also decreases but to a lesser extent, and the surface expression of mutants W523A and W611A is comparable to that of OATP2B1. Therefore, it seems that W272, W276, and W277 have most significant effect on the surface expression of OATP2B1. If these three tryptophan residues are mutated to alanine simultaneously which results in mutant W272/276/277A, its surface expression level is decreased to be only about 10% of that of OATP2B1 (Figure 2B).



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The normalized transport activities of OATP2B1 and six single mutants are shown in Figure 2C. As compared to that of OATP2B1, the activities of mutants W272A, W276A, W277A, and W611A for E3S transport are significantly decreased with the activity of mutant W611A decreasing most dramatically which is only about 9% of that of OATP2B1. Therefore, W611 is critical for the function of OATP2B1 for E3S transport. (Figure 2)

Kinetic Study of OATP2B1 and Mutants W272A, W276A, W277A, and W611A. To determine whether the reduced function of mutants W272A, W276A, W277A, and W611A is due to reduced binding affinity or translocation capacity, kinetic studies have been carried out on E3S transport mediated by OATP2B1 and mutants W272A, W276A, W277A, and W611A. The saturation curve and kinetic parameters for each protein are showed in Figure 3. The Vmax/Km values for OATP2B1, W272A, W276A, W277A, and W611A (the slope of linearly increased activity as an alternative of Vmax/Km) are 25.6, 36.4, 35.0, 30.6, and 0.3 µL/normalized mg/min, respectively. The uptake of E3S by wild-type OATP2B1 exhibits a monophasic saturation kinetics (Figure 3A, inset) with Km and Vmax values being of 7.1 ± 1.1 µM and 182 ± 7 pmol/normalized mg/min, respectively (Figure 3A), which are comparable to the values reported in the literature.27-30 The Km values for mutants W272A, W276A, and W277A are increased to 36.0 ± 6.2, 31.7 ± 5.8, and 39.6 ± 9.2 µM, respectively. However, their Vmax values are also increased (Figure 3B, C, D) and


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their Vmax/Km values are even higher than that of OATP2B1. The decreased activity of these three mutants shown in Figure 2 can be ascribed to the used substrate concentration (0.1 µM), which is low and may not include the factor of their higher Vmax values. Therefore, the reduced function at low substrate concentration of these mutants is due to the lowered binding affinity instead of translocation capacity. For mutant W611A, its kinetic curve changes to be a linear curve instead of a saturable curve with transport rate decreasing dramatically as compared to OATP2B1 and other mutants (Figure 3E). This result indicates that the Km value of mutant W611A is extremely higher than those of OATP2B1 and other three mutants. Therefore, the tryptophan residue of 611 is very important for substrate binding and thus the function of OATP2B1. (Figure 3)

Functional Comparison of Alanine and Phenylalanine Mutants of W272, W276, W277, and W611, and Kinetic Study of Mutant W611F. To investigate whether the structural characteristics of tryptophan residue is a key factor for the reduced function of mutants W272A, W276A, W277A, and W611A, we substituted these four tryptophan residues with phenylalanine which also contains an aromatic ring and obtained four phenylalanine mutants W272F, W276F, W277F, and W611F. The surface expression levels of these four phenylalanine mutants have been determined (Figure 4A) and used to normalize their transport activities. As shown in Figure 4B, the transport function of all phenylalanine mutants is significantly higher



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than their corresponding alanine mutants with the function of W611F increasing most dramatically which is 9.1-fold of that of W611A. As compared to OATP2B1, W272F fully recovers its function and W276F, W277F, and W611F recover most of their function (up to 70% of that of OATP2B1). These results indicate that the aromatic ring structure is crucial for these four residues, especially for W611 which shows the highest fold of increase between its alanine and phenylalanine mutants. To further characterize the effect of aromatic ring on the function of OATP2B1, we carried out the kinetic study of mutant W611F. As shown in Figure 4C, mutant W611F recovers to show a typical transporter kinetics with a saturable curve in contrast to mutant W611A which shows a linear kinetic curve (Figure 3E). Therefore, the aromatic structure of the side chain of residue 611 is very important for substrate binding and OATP2B1’s function. (Figure 4)

Functional Characterization of Additional Mutants for W611. For E3S and many other substrates of OATP2B1 are organic anions which contain polar and/or negatively charged group(s), we want to check whether hydrogen-bond forming and positively charged groups at position 611 will be beneficial for the function of OATP2B1. Therefore, additional three mutants, namely W611S, W611Y, and W611H, were constructed and their transport activities were measured. Serine and tyrosine have an additional hydroxyl group as compared to alanine and phenylalanine, respectively. Like phenylalanine, histidine is also aromatic but is positively charged


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(although only partially ionized at physiological pH). As shown in Figure 5, the activity of mutant W611S doesn’t show any significant increase as compared to that of mutant W611A, and mutant W611Y even shows a decreased activity as compared to mutant W611F. When residue 611 is changed from a phenylalanine to a histidine, its function is also decreased. These results indicate that hydrogen-bond forming and positively charged groups at position 611 are not beneficial for the function of OATP2B1. (Figure 5)

Role of Tryptophan Residues in OATP2B1-mediated Transport for Other Substrates. To further explore the role of the six tryptophan residues in the function of OATP2B1, transport activities of the six tryptophan-to-alanine mutants for other OATP2B1 substrates including BSP, atorvastatin, fluvastatin, and rosuvastatin were measured. As shown in Figure 6, only mutant W611A exhibits decreased transport activities for all of these four substrates, demonstrating that the activity reduction of mutant W611A is not substrate-specific and residue W611 is critical for the function of OATP2B1. (Figure 6)

Structural

Model

of

OATP2B1-E3S

Complex

and

Analysis

of

OATP2B1-E3S Interactions. To elucidate the important role of W611 from a structural perspective, we constructed a three-dimensional structure model for



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OATP2B1 by molecular modeling and developed a complex model for OATP2B1-E3S by molecular docking. Molecular dynamics simulations indicate that the obtained complex model is stable (Figure S1). As shown in Figure 7, the putative substrate translocation pathway is composed of amino acid residues from TMs 1, 2, 4, 5, 7, 8, 10, and 11. W611 is located at the middle of TM 11 and its side chain is facing towards the translocation pathway, where it could interact with E3S directly. The substrate translocation pathway is relatively large and E3S only occupies part of the substrate binding site. The rest of space could afford extra room for larger OATP2B1 substrates such as BSP and statins. (Figure 7) Interaction analysis by Ligplot indicates that E3S might interact with W611 and several other non-polar residues by aromatic and hydrophobic interactions (Figure 8). In addition, the negatively charged sulfate group of E3S might form a salt bridge with arginine 607 of OATP2B1. This residue is highly conserved in human OATPs (Figure 1B). Studies show that R580 (corresponding to R607 in OATP2B1) is important for the function of OATP1B1 and 1B3.31,32 To verify our model, we mutated arginine 607 to an alanine and measured its function and surface expression. As shown in Figure 9A, mutation of arginine 607 to an alanine leads to the complete loss of function of OATP2B1. However, mutant R607A has almost normal surface expression with expression level slightly lower than wild-type OATP2B1 (Figure 9B), indicating that the loss of function is not due to the change of protein surface expression. These


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results demonstrate that R607 is indeed very important for the function of OATP2B1 and indicate that our model is reliable. (Figure 8) (Figure 9)

DISCUSSION OATP2B1 which is highly expressed in enterocytes and hepatocytes could be a key determinant for the intestinal absorption and hepatic uptake of its substrate. OATP2B1

transports

BSP,

E3S,

dehydroepiandroserone

sulfate

(DHEAS),

taurocholate, thyroxine, and some drugs such as aliskiren, fexofenadine, glibenclamide and statins,2,4 most of which are amphipathic organic anions. Tryptophan residues may possess a multitude of functions for a transport protein through aromatic interactions, e.g. maintaining the proper protein structure, guiding the depth of membrane insertion, or interacting directly with substrates.33,34 There are totally six tryptophan residues in human OATP2B1, namely W272, W276, W277, W523, W611 and W629. Among them, W272, W276, W277, and W629 are conserved among different OATP family members, but W523 is totally unconserved. W611 is conserved in OATP2 family, but it is not conserved within other OATP families (Figure 1B). While five of these six tryptophan residues are located at the extracellular membrane-aqueous border or extracellular loops of OATP2B1, W611 is located at the middle of the α-helical segment of TM 11 (Figure 1A). In this study, the



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role of these six tryptophan residues in the expression and function of OATP2B1 has been investigated. Surface expression and functional studies of mutants W272A, W276A, W277A, W523A, W611A, and W629A show that W272, W276, and W277 have the greatest effect on the surface expression of OATP2B1 and the reduction of activity of mutants W272A, W276A, and W277A is mainly due to their decreased surface expression levels. W276 and W277 have moderate effects on the function of OATP2B1 (Figure 2C), while their counterparts W258 and W259 are critical for the function of OATP1B1.20 However, the reduction of activity of mutant W611A is exclusively due to its decreased transport function (Figure 2). W523 and W629 did not show any significant effect on both the surface expression and function of OATP2B1. Kinetic studies of OATP2B1-mediated E3S transport confirmed the critical role of W611 in the function of OATP2B1. While mutants W272A, W276A, and W277A exhibit typical transporter-mediated kinetics with a saturable curve like wild-type OATP2B1, mutant W611A shows a linear curve with dramatically decreased transport rate as compared to OATP2B1 (Figure 3), demonstrating that W611 is very crucial for the substrate binding and function of OATP2B1. For tryptophan residues potentiate a range of aromatic interactions such as pi stacking interactions which might contribute to the stable binding of aromatic OATP2B1 substrates, we investigated the role of aromatic ring of W272, W276, W277, and W611 in the function of OATP2B1 by mutating these four tryptophan residues to phenylalanine and comparing their function with their alanine counterparts.


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All phenylalanine mutants exhibit significantly increased activity as compared to their alanine counterparts with mutant W611F showing the highest fold of increase (Figure 4B). Kinetic study of mutant W611F shows that a phenylalanine substitution for alanine at position 611 rescues OATP2B1 to act as a normal transporter with a saturable kinetics (Figure 4C), indicating that W611F might have significantly higher affinity to E3S than W611A. These results suggest that the aromatic structure is very important for W611. However, hydrogen-bond forming and positively charged groups at position 611 are not beneficial for the function of OATP2B1 (Figure 5), although most of OATP2B1 substrates such as E3S, BSP, and statins are negatively charged organic anions. Molecular modeling and interaction analysis of OATP2B1-E3S complex indicate that W611 might interact with E3S by aromatic interactions and the negatively charged sulfate group of E3S might form a salt bridge with the positively charged R607 of OATP2B1 (Figure 8). This arginine residue is highly conserved in human OATPs (Figure 1B) and it is important for the function of OATP1B1 and 1B3.31,32 During the preparation of this manuscript, a paper reported that replacing Arg607 of OATP2B1 with Ala caused a significant decrease in E3S uptake.35 Our result also shows that R607 is very important for the function of OATP2B1 (Figure 9). These may explain why polar and positively charged groups at position 611 are not favorable for the function of OATP2B1. The important role of W611 for the function of OATP2B1 has been further demonstrated with the functional consequence of tryptophan-to-alanine mutation of 611 for other OATP2B1 substrates. Mutant W611A shows significant reduction of



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transport function for all tested substrates including BSP, atorvastatin, fluvastatin, and rosuvastatin (Figure 6), demonstrating that the reduction of transport activity of mutant W611A is not substrate-dependent and residue W611 is critical for the function of OATP2B1. In conclusion, in the present study the role of six tryptophan residues for the expression and function of OATP2B1 has been investigated. Our results show that while W272, W276, and W277 have a moderate effect on the surface expression of OATP2B1, W611 located at TM 11 is critical for the function of OATP2B1, which is not due to the change of OATP2B1’s surface expression but due to a bona fide reduction of its function. W611 might directly interact with substrates through aromatic interactions and thus the aromatic structure of side chain is important for its function. However, polar and positively charged groups at position 611 are not favorable for OATP2B1’s function. The importance of W611 for the function of OATP2B1 is not substrate-dependent. Taken together, this study demonstrates that W611 is critical for the function of OATP2B1.

ACKNOWLEDGEMENTS The authors gratefully acknowledge the technical assistance from Dr. Tingjun Hou’s laboratory on molecular modeling. This work is financially supported by grant from National Natural Science Foundation of China (31200623).


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(13) Shirasaka, Y.; Shichiri, M.; Mori, T.; Nakanishi, T.; Tamai, I. Major active components

in

grapefruit,

orange,

and

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juices

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for

OATP2B1-mediated drug interactions. J. Pharm. Sci. 2013, 102, 280-288. (14) Shirasaka, Y.; Shichiri, M.; Murata, Y.; Mori, T.; Nakanishi, T.; Tamai, I. Long-lasting inhibitory effect of apple and orange juices, but not grapefruit juice, on OATP2B1-mediated drug absorption. Drug Metab. Dispos. 2013, 41, 615-621. (15) Wen, F.; Shi, M.; Bian, J.; Zhang, H.; Gui, C. Identification of natural products as modulators of OATP2B1 using LC-MS/MS to quantify OATP-mediated uptake. Pharm. Biol. 2016, 54, 293-302. (16) Jacquemin, E.; Hagenbuch, B.; Stieger, B.; Wolkoff, A. W.; Meier, P. J. Expression Cloning of a Rat Liver Na+-Independent Organic Anion Transporter. Proc. Natl. Acad. Sci. USA 1994, 91, 133-137. (17) Wang, P.; Hata, S.; Xiao, Y.; Murray, J. W.; Wolkoff, A. W. Topological assessment of oatp1a1: a 12-transmembrane domain integral membrane protein with three N-linked carbohydrate chains. Am. J. Physiol. Gastrointest. Liver Physiol. 2008, 294, G1052-1059. (18) Gui, C.; Hagenbuch, B. Amino acid residues in transmembrane domain 10 of organic anion transporting polypeptide 1B3 are critical for cholecystokinin octapeptide transport. Biochemistry 2008, 47, 9090-9097. (19) Gui, C.; Hagenbuch, B. Role of transmembrane domain 10 for the function of organic anion transporting polypeptide 1B1. Protein Sci. 2009, 18, 2298-2306.



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(20) Huang, J.; Li, N.; Hong, W.; Zhan, K.; Yu, X.; Huang, H.; Hong, M. Conserved tryptophan residues within putative transmembrane domain 6 affect transport function of organic anion transporting polypeptide 1B1. Mol. Pharmacol. 2013, 84, 521-527. (21) Nozawa, T.; Nakajima, M.; Tamai, I.; Noda, K.; Nezu, J.; Sai, Y.; Tsuji, A.; Yokoi, T. Genetic polymorphisms of human organic anion transporters OATP-C (SLC21A6) and OATP-B (SLC21A9): Allele frequencies in the Japanese population and functional analysis. J. Pharmacol. Exp. Ther. 2002, 302, 804-813. (22) Gui, C.; Miao, Y.; Thompson, L.; Wahlgren, B.; Mock, M.; Stieger, B.; Hagenbuch, B. Effect of pregnane X receptor ligands on transport mediated by human OATP1B1 and OATP1B3. Eur. J. Pharmacol. 2008, 584, 57-65. (23) Sali, A.; Potterton, L.; Yuan, F.; van Vlijmen, H.; Karplus, M. Evaluation of comparative protein modeling by MODELLER. Proteins 1995, 23, 318-326. (24) Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. J.; Swaminathan, S.; Karplus, M. CHARMM: A program for macromolecular energy, minimization, and dynamics calculations. J. Comput. Chem. 1983, 4, 187-217. (25) Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S.; Olson, A. J. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J. Comput. Chem. 2009, 30, 2785-2791. (26) Wallace, A. C.; Laskowski, R. A.; Thornton, J. M. LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein Eng. 1995, 8, 127-134.


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(27) Nozawa, T.; Imai, K.; Nezu, J.; Tsuji, A.; Tamai, I. Functional characterization of pH-sensitive organic anion transporting polypeptide OATP-B in human. J. Pharmacol. Exp. Ther. 2004, 308, 438-445. (28) Ogura, J.; Koizumi, T.; Segawa, M.; Yabe, K.; Kuwayama, K.; Sasaki, S.; Kaneko,

C.;

Tsujimoto,

T.;

Kobayashi,

M.;

Yamaguchi,

H.;

Iseki,

K.

Quercetin-3-rhamnoglucoside (rutin) stimulates transport of organic anion compounds mediated by organic anion transporting polypeptide 2B1. Biopharm. Drug Dispos. 2014, 35, 173-182. (29) Noe, J.; Portmann, R.; Brun, M. E.; Funk, C. Substrate-dependent drug-drug interactions between gemfibrozil, fluvastatin and other organic anion-transporting peptide (OATP) substrates on OATP1B1, OATP2B1, and OATP1B3. Drug. Metab. Dispos. 2007, 35, 1308-1314. (30) Grube, M.; Kock, K.; Karner, S.; Reuther, S.; Ritter, C. A.; Jedlitschky, G.; Kroemer, H. K. Modification of OATP2B1-mediated transport by steroid hormones. Mol. Pharmacol. 2006, 70, 1735-1741. (31) Weaver, Y. M.; Hagenbuch, B. Several conserved positively charged amino acids in OATP1B1 are involved in binding or translocation of different substrates. J. Membr. Biol. 2010, 236, 279-290. (32) Glaeser, H.; Mandery, K.; Sticht, H.; Fromm, M. F.; Konig, J. Relevance of conserved lysine and arginine residues in transmembrane helices for the transport activity of organic anion transporting polypeptide 1B3. Br. J. Pharmacol. 2010, 159, 698-708.



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(33) Ruddat, V. C.; Mogul, R.; Chorny, I.; Chen, C.; Perrin, N.; Whitman, S.; Kenyon, V.; Jacobson, M. P.; Bernasconi, C. F.; Holman, T. R. Tryptophan 500 and arginine 707 define product and substrate active site binding in soybean lipoxygenase-1. Biochemistry 2004, 43, 13063-13071. (34) Hassan, K. A.; Souhani, T.; Skurray, R. A.; Brown, M. H. Analysis of tryptophan residues in the staphylococcal multidrug transporter QacA reveals long-distance functional associations of residues on opposite sides of the membrane. J. Bacteriol. 2008, 190, 2441-2449. (35) Hoshino, Y.; Fujita, D.; Nakanishi, T.; Tamai, I. Molecular localization and characterization of multiple binding sites of organic anion transporting polypeptide 2B1 (OATP2B1) as the mechanism for substrate and modulator dependent drug-drug interaction. Med. Chem. Commun. 2016, DOI: 10.1039/C1036MD00235H. (36) Thompson, J. D.; Higgins, D. G.; Gibson, T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994, 22, 4673-4680. (37) Robert, X.; Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 2014, 42, W320-W324.


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FIGURE CAPTIONS Figure 1. (A) Predicted topological structure of human OATP2B1 with twelve transmembrane domains. The starting and ending residues for each transmembrane domain are determined and labeled. The locations of the six tryptophan residues are depicted in the model. The conserved and non-conserved tryptophan residues among most human OATPs are colored in black and in grey, respectively. (B) Multiple sequence alignment of all 11 human OATPs. Multiple sequence alignment was carried out by Clustal W36 and the result was plotted with ESPript.37 Regions containing the six tryptophan residues are shown. Amino acids are numbered according to the sequence of OATP2B1. Identical and similar residues among 11 OATPs are boxed in red and in yellow, respectively. The six tryptophan residues are marked with red arrows.

Figure 2. Surface expression and functional characterization of OATP2B1 and its six tryptophan-to-alanine mutants by uptake assay, cell surface biotinylation, and immunoblot analysis. (A) Uptake of 0.1 µM E3S by OATP2B1 and mutants W272A, W276A, W277A, W523A, W611A, and W629A was measured at 37 °C for 1 min with empty vector and OATP-transfected cells. The net uptake was obtained by subtracting the uptake of empty vector-transfected cells from the uptake of OATP-transfected cells. (B) Surface expression of OATP2B1 and its six tryptophan-to-alanine mutants. OATP2B1 and mutants expressed on cell surface were isolated by surface biotinylation and detected by immunoblot analysis with an



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anti-His antibody. The plasma membrane marker Na+/K+-ATPase α subunit was used as protein loading control. Band intensity for each protein was quantitated, normalized to that of OATP2B1, and given in numbers. (C) Normalized net uptake of OATP2B1 and its tryptophan mutants to their respective surface expression levels. Asterisks indicate a p < 0.05 level of significant difference from OATP2B1.

Figure 3. Kinetic study of E3S uptake mediated by OATP2B1 and mutants W272A, W276A, W277A, and W611A. An Eadie–Hofstee plot of OATP2B1-mediated E3S uptake is included as a figure inset. Uptake of increasing concentrations of E3S was measured at 37 °C for 1 min with empty vector and OATP2B1-transfected HEK293 cells. Net uptake was obtained by subtracting the uptake of empty vector-transfected cells from the uptake of OATP-transfected cells and was normalized to their respective surface expression levels. Normalized net uptake was used to fit the Michaelis–Menten equation to determine Km and Vmax values.

Figure 4. (A) Surface expression of mutants W272F, W276F, W277F, and W611F. Surface biotinylated proteins were detected with an anti-His antibody. The plasma membrane marker Na+/K+-ATPase α subunit was used as protein loading control. Band intensity for each protein was quantitated, normalized to that of OATP2B1, and given in numbers. (B) Functional comparison of alanine and phenylalanine mutants of W272, W276, W277, and W611. Uptake of 0.1 µM E3S by OATP2B1 and the alanine and phenylalanine mutants of W272, W276, W277, and W611 was measured


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at 37 °C for 1 min with empty vector and OATP-transfected cells. The net uptake was obtained by subtracting the uptake of empty vector-transfected cells from the uptake of OATP-transfected cells. Final results were obtained by normalizing net uptakes to their respective surface expression levels. Asterisks indicate a p < 0.05 level of significant difference between each pair of alanine and phenylalanine mutants, and the ratio of transport activities for each mutant pair is given. (C) Kinetic study of E3S uptake mediated by mutant W611F. Uptake of increasing concentrations of E3S was measured at 37 °C for 1 min with empty vector and W611F-transfected HEK293 cells. Net uptake was obtained by subtracting the uptake of empty vector-transfected cells from the uptake of OATP-transfected cells and was normalized to its surface expression level. Normalized net uptake was used to fit the Michaelis–Menten equation to determine Km and Vmax values.

Figure 5. (A) Uptake of E3S by OATP2B1 and mutants W611A, W611F, W611S, W611Y, and W611H. Uptake of 0.1 µM E3S was measured at 37 °C for 1 min with empty vector and OATP-transfected cells. The net uptake was obtained by subtracting the uptake of empty vector-transfected cells from the uptake of OATP-transfected cells. (B) Surface expression of OATP2B1 and mutants W611A, W611F, W611S, W611Y, and W611H. Surface biotinylated proteins were detected with an anti-His antibody. The plasma membrane marker Na+/K+-ATPase α subunit was used as protein loading control. Band intensity for each protein was quantitated, normalized to that of OATP2B1, and given in numbers. (C) Normalized net uptake of OATP2B1



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and mutants W611A, W611F, W611S, W611Y, and W611H. Uptake was normalized to their respective surface expression levels. Asterisks indicate a p < 0.05 level of significant difference from OATP2B1.

Figure 6. Uptake of (A) BSP, (B) atorvastatin, (C) fluvastatin, and (D) rosuvastatin mediated by OATP2B1 and its six tryptophan-to-alanine mutants. Uptake of 1 µM BSP, atorvastatin, fluvastatin, and rosuvastatin was measured at 37 °C for 2 min with empty vector and OATP-transfected HEK293 cells. The net uptake was obtained by subtracting the uptake of empty vector-transfected cells from the uptake of OATP-transfected cells. Final results were obtained by normalizing net uptakes to their respective surface expression levels. Asterisks indicate a p < 0.05 level of significant difference from OATP2B1.

Figure 7. Three-dimensional structure model of OATP2B1-E3S complex. (A) Side view and (B) intracellular view are presented. Protein is represented in solid ribbon and the twelve transmembrane domains of the protein are labeled. The tryptophan residue of 611 in TM 11 is shown in ball-and-stick and colored in purple. E3S is shown in CPK mode and colored in green.

Figure 8. Schematic diagram of interactions between OATP2B1 and E3S. Dashed line denotes a salt bridge between OATP2B1 and E3S, and spikes represent residues from OATP2B1 involved in aromatic and hydrophobic interactions with E3S.


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Figure 9. Function and surface expression of OATP2B1 and mutant R607A. (A) Uptake of 0.1 µM E3S by OATP2B1 and mutant R607A was measured at 37 °C for 1 min with empty vector and OATP-transfected cells. The net uptake was obtained by subtracting the uptake of empty vector-transfected cells from the uptake of OATP-transfected cells. (B) Surface expression of OATP2B1 and mutant R607A. OATP2B1 and mutant R607A expressed on cell surface were isolated by surface biotinylation and detected by immunoblot analysis with an anti-His antibody. The plasma membrane marker Na+/K+-ATPase α subunit was used as protein loading control.



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Figure 1A 381x184mm (96 x 96 DPI)


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Figure 1B 109x89mm (300 x 300 DPI)



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Figure 3C 123x83mm (300 x 300 DPI)


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Figure 3D 123x83mm (300 x 300 DPI)



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Figure 6D 103x90mm (300 x 300 DPI)



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Figure 8 289x289mm (72 x 72 DPI)


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For Table of Contents Use Only A Tryptophan Residue Located at the Middle of Putative Transmembrane Domain 11 is Critical for the Function of Organic Anion Transporting Polypeptide 2B1 Jialin Bian, Meng Jin, Mei Yue, Meiyu Wang, Hongjian Zhang, and Chunshan Gui 88x34mm (300 x 300 DPI)


Tryptophan Residue Located at the Middle of ... - ACS Publications

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