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Bile Acid Recognition by Mouse Ileal-Bile Acid Binding Protein Mohsen Badiee, and Gregory P. Tochtrop ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00865 • Publication Date (Web): 23 Oct 2017 Downloaded from http://pubs.acs.org on November 15, 2017

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ACS Chemical Biology

Bile Acid Recognition by Mouse Ileal-Bile Acid Binding Protein Mohsen Badiee, and Gregory P. Tochtrop* Department of Chemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, USA Corresponding Author * E-mail: [email protected] Phone: (216) 368-2351.

ACS Paragon Plus Environment


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ABSTRACT Ileal bile acid binding protein (I-BABP, gene name FABP6), is a component of the bile acid recycling system, expressed in the ileal enterocyte. The physiological role of I-BABP has been hypothesized to be either an intracellular buffering agent to protect against excess intracellular bile acids or separately as a modulator of bile acid controlled transcription. We investigated mouse I-BABP (mI-BABP) to understand the function of this protein family. Here, we studied energetics and site selectivity of binding with physiological bile acids using a combination of isothermal calorimetric analysis and NMR spectroscopy. We found that the most abundant bile acid in the mouse (β-muricholic acid) binds with weak affinity individually and in combination with other bile acids. Further analysis showed that mI-BABP like human I-BABP (hI-BABP) specifically recognizes the conjugated form of cholic acid: chenodeoxycholic acid (CA:CDCA) in a site-selective manner, displaying the highest affinity of any bile acid combination tested. These results indicate that I-BABP specifically recognizes the ligand combination of CDCA and CA, even in a species such as mouse where CDCA only represents a trace component of the physiological pool. Specific and conserved recognition of the CDCA and CA ligand combination suggests that I-BABP may play a critical role in the regulation of bile acid signaling in addition to its proposed role as a buffering agent.


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Bile acids (BAs) are steroidal detergents derived from cholesterol that facilitate absorption of dietary lipids and fat-soluble vitamins during digestion. Although all bile acids share a rigid steroidal scaffold and a short aliphatic side chain, they comprise a wide range of modifications that make up a structurally diverse family of small molecules. Differences primarily arise from variations in steroidal hydroxylation patterns and differences in the length, functionality, and conjugation state of the side-chain. Most bile acids in higher vertebrates are defined by a C24 skeleton (shown in Figure.1A) and are typically found conjugated to either taurine or glycine. The hydroxylation pattern of the steroidal core represents a key source of heterogeneity when examining bile acid pools across closely related species. (1-3) For example, humans produce two bile acids: cholic acid (CA; hydroxyl groups at 3α, 7α, and 12α positions) and chenodeoxycholic acid (CDCA; 3α, 7α) with a final 1:1 ratio in the bile. These bile acids are found to be conjugated to both glycine and taurine in a 3:1 ratio, affording a pool of nine possible molecules when including the unconjugated molecules. (4) This can be contrasted to the homogenous bile acid pool of the fox squirrel with TCDCA comprising the only major bile acid molecule. (3) Position, number, and stereochemistry of hydroxyl groups form almost forty structurally distinct isoforms of C24 bile acids across vertebrate species. (3) Several hypotheses have been put forth as to the origin of this biosynthetic diversity. The most cited are from Hofmann, who proposed that variations in hydroxylation patterns represent an evolutionary response to the oxidation, epimerization, and dehydroxylation processes carried out by colonic bacteria.(1) These bacterial processes have been hypothesized to represent an evolutionary response to abrogate the detergent functions of bile acids in the colon. These modified molecules are passively reabsorbed in the colon and recycled expanding the diversity of the bile acid pool. Common examples of these molecules include lithocholic acid (LCA) and



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deoxycholic acid (DCA) derived from bacterial 7α- dehydroxylation of CDCA and CA, respectively. (5, 6) Interestingly, gut microbiota composition is highly variable between individuals, which results in corresponding variability in bile acid pools. (7) While the source of heterogeneity is well understood, the relevance and functional consequences of bile acid pool heterogeneity are not clear. Physiologically, bile acids are secreted into the proximal small intestine via the gall bladder, and are efficiently recycled via a process termed the enterohepatic circulation. (2) It is generally accepted that ileal bile acid binding protein (I-BABP) plays an important role in bile acid transport through binding interactions with bile acids that occur in the enterocytes of the distal ileum. The unique binding properties of I-BABP have provided some clues as to the role of this protein. Specifically, human, rabbit (8), zebrafish (9), and chicken I-BABP (10) bind two ligands with a moderate affinity and variable cooperativity. (11) This stands in contrast to other members of the intracellular lipid binding protein (iLBP) family which bind ligands with relativity high affinity and 1:1 stoichiometry. (12) Recently, liver FABP which is closely related to I-BABP in the iLBP family, was also shown to bind BAs with moderate affinity along with other hydrophobic ligands.(13) Human I-BABP (hI-BABP) has been shown to bind the two most abundant BAs preferentially and with site specificity, GCDA at Site 1 and GCA at Site 2. (14) These observations supported two possible hypotheses about the physiologic role of I-BABP. First, it was postulated in human and rabbit that I-BABP acts as an intracellular buffering agent to protect cells against the harmful effects of bile acids such as cytolysis and apoptosis. Second, our group has speculated that IBABP directly interacts with and functions to ‘deliver’ specific bile acid ligands to FXR, a nuclear receptor that binds to and is activated by bile acids. Related to this hypothesis, FXR is


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strongly activated by CDCA which binds with high affinity to hI-BABP. This hypothesis is strengthened with data from Sato et al., (15) showing that I-BABP functionally interacts with FXR in the nucleus and data from Noy et al., (16) showing nuclear colocalization with receptors is also observed in other members of the iLBP family. To further define the physiological role of I-BABP we began investigating the ligand-binding properties of this protein across species. The rationale behind these experiments was to differentiate between the two stated hypotheses. For example, if the primary role was as a buffering agent, we would expect to see the ligand specificity of I-BABP reflect the composition of the bile acid pool in another organism. On the other hand, if the primary role of I-BABP is modulating ligand availability for FXR, we would expect that I-BABP would recognize ligands specific for FXR specifically CDCA. (17) We focused our studies on mouse I-BABP. Although mouse is closely related evolutionarily to human, it has a drastically different bile acid pool specifically deficient in CDCA as illustrated in Table 1. While the primary BAs in human are CDCA and CA, the dominant molecules in the mouse are CA and the family of muricholic acids (MCA). Among muricholic acids, β-MCA is more prevalent than ω-MCA and α-MCA in mouse bile. Additionally, mouse bile acids are primarily taurine conjugated while human is predominately conjugated with glycine. (3) We began investigating the binding properties of four bile acids (CA, CDCA, β-MCA, α-MCA) and their taurine conjugates to mI-BABP using a strategy that first utilized isothermal titration calorimetry (ITC) to explore the macroscopic binding properties, followed by the use of NMR to explore the site specificity of binding.



Figure 1. (A) Bile acid chemical structure and nomenclature. (B) Hydroxylation pattern on ring B and C of bile acids used in this study. Table 1: Human and mouse primary bile acids (derived from cholesterol in the liver). Secondary bile acids (derived from primary bile acids by intestinal bacteria) Bile acid

Primary

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Chenodeoxycholic acid Glycochenodeoxycholic acid Taurochenodeoxycholic acid Cholic acid Glycocholic acid Taurocholic acid β-muricholic acid d Glyco β-muricholic acid d Tauro β-muricholic acid d

Abbreviation

CDCA GCDCA TCDCA CA GCA TCA β-MCA Gβ-MCA Tβ-MCA

Hydroxylation R1, R2, R3 7α, 7α, 7α, 7α,12α 7α,12α 7α,12α 6β,7β 6β,7β 6β,7β

Conjugation

Human(18, 19)

none glycine taurine none glycine taurine none glycine taurine


abundancea ND 19.6-46.3 % 1.6-23.7 % 0-0.9 % 17.0-43.5 % 6.8-18.8 % ND c ND ND

Mouse (20-22) abundanceb 0.0-1.7 % ND 0.7-2.7 % 0.1-1.9 % 0.1-0.2 % 27.0-46.0 % 0.1-14.4 % ND 14.1-32.1 %

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Secondary

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α-muricholic acid Glyco α-muricholic acid Tauro α-muricholic acid Deoxycholic acid Glycodeoxycholic acid Taurodeoxycholic acid Lithocholic acid Glycolithocholic acid Taurolithocholic acid

α-MCA Gα-MCA Tα-MCA DCA GDCA TDCA LCA GLCA TLCA

6β,7α 6β,7α 6β,7α 12α 12α 12α

none glycine taurine none glycine taurine none glycine taurine

ND ND ND 0.0-0.1 % 0.1-32.9 % 0.0-10.7 % 0.0-1.0 % 0.0-2.2 % 0.0-0.9 %

a

percent of total bile acid in biliary track or gall bladder, healthy human.

b

percent of total bile acid in biliary track or gall bladder, conventional mouse.

c

below the detection limit.

d

primary bile acid only in mouse.

0.2-2.4 % ND 2.8-9.8 % 0.0-0.2 % ND 0.9-4.4 % 0.0-0.2 % ND 0.0-0.2 %

RESULTS To determine macroscopic binding constants, isothermal titration calorimetry (ITC) was utilized. This technique is advantageous as a complete thermodynamic analysis can be completed in a single experiment. When examining the data in Figure 2, it is important to note that the discontinuity in the raw data is due to differential injection volumes as described in Methods. This approach allowed us to sample the early portion of the binding curve, before the inflection, with higher resolution. The heat of injections was corrected for heats of dilution and normalized to a ligand protein molar ratio for analysis. The data were fit to a two-site stepwise binding model illustrated in Equation 1 and described previously where P is protein, L is ligand, PL, and PL2 are protein –ligand complexes, K1obs and K2obs are the apparent dissociation constants for the first and second molar equivalent of ligand respectively.(11, 14, 23)

(Equation 1) The data were fit utilizing the nonlinear least square analysis program SCIENTIST. As we have thoroughly documented earlier, this method of data fitting cannot definitively identify a unique set of stepwise binding constants, especially in the context of ligand systems that display



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observed positive cooperativity. (24) However, we only required identification of preferred ligand combinations within the heterogeneous bile acid pool which can be determined using the product of Kd1obs and Kd2obs as a measure of the overall binding affinity of the ligand system for a given experiment. For example, the overall binding affinity (Kd1obs × Kd2obs) for a homotypic ligand system composed of either TCA or T-βMCA is 1.7 × 10-9 M2 and 2.1 × 10-7 M2, respectively. This means that mI-BABP has a higher overall binding affinity for TCA compared to T-βMCA when considering the doubly ligated state (Table.2). Bile acids and mouse I-BABP homotypic binding. Given that macroscopic data is available for hI-BABP(11, 14, 23), we initially determined macroscopic binding constants for the complement of bile acids present in the mouse. We measured overall affinities for mI-BABP to each bile acid individually in what we will refer to as a homotypic complex (see Table 1). All of the homotypic ligand combinations produced high-quality isotherms. An example of this raw data can be seen in Figure 2 top panel followed by fit analyses of the tauro-conjugates of the majority of these bile acids in Figure 2 middle panel, and S1. A similar analysis can be seen for unconjugated bile acids in Figure S1. It is noteworthy that at the macroscopic level, many significant differences exist between human and mouse I-BABP binding profiles. For example although hI-BABP displayed similar affinities and positive cooperativity for all bile acids examined, the affinity of mI-BABP varied greatly and not all bile acids displayed positive cooperativity. In fact, CDCA displayed negative cooperativity. However, the most striking observation of the mI-BABP binding data is that the major mouse-specific constituents of bile, βMCA and its conjugates, displayed weak observed binding. Further investigation into the muricholic acid family, specifically α-MCA and its conjugates, again revealed weak observed binding. These results were not predicted as these two bile acids (β-MCA and α-MCA) and their


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taurine conjugates comprise ~40 % of the bile acid pool in the mouse. Another remarkable observation was that α-MCA and CDCA affinity improved 10 and 100 times, respectively as a result of conjugation to taurine. Taurine conjugation did not significantly improve binding of CA and β-MCA.

Figure 2. Isothermal titration calorimetry data for binding of mI-BABP and bile acids at 25oC. Top panel is the heat profile (raw data after baseline correction) for successive injections of 12 mM taurocholic sodium acid into 0.16 (± 0.02) mM mI-BABP in 20 mM phosphate buffer pH=7.2. Homotypic and heterotypic combinations are shown in the middle and bottom panels,



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respectively. Data points are representative integrated heat of injections for indicated bile acids. The solid black line represents a least-squares fit of the data using a step-wise binding model.


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Table 2. Macroscopic binding data for homo and heterotypic mixtures of bile acids with mIBABP calculated from the product of two step-wise binding constants from ITC analysis. Kd1obs (M) 8.1 (±0.7) × 10-5 CA 1.0 (±1.0) × 10-4 CDCA 1.1 (±0.2) × 10-3 β-MCA 8.9 (±7.0) × 10-3 α-MCA 2.7 (±0.6) × 10-4 TCA 2.1 (±0.8) × 10-4 TCDCA 5.9 (±2.0) × 10-4 Tβ-MCA 1.3 (±0.4) × 10-2 Tα-MCA 1.1 (±1.0) × 10-2 CA + CDCA 2.5 (±1.0) × 10-4 CA + β-MCA 4.5 (±3.0) × 10-5 CA + α-MCA 7.8 (±5.0) × 10-4 CDCA + β-MCA 1.5 (±na) × 10-5 CDCA + α-MCA 1.2 (±na) × 10-3 α-MCA + β-MCA 1.1 (±0.5) × 10-4 TCA + TCDCA 4.3 (±3.0) × 10-3 TCA + Tβ-MCA 2.2 (±0.2) × 10-4 TCA + Tα-MCA TCDCA + Tβ-MCA 1.5 (±0.9) × 10-2 TCDCA + Tα-MCA 3.1 (±3.0) × 10-2 Tα-MCA + Tβ-MCA 1.5 (±1.0) × 10-2 Bile Acid

a

Kd2obs (M) 6.4 (±1.4) × 10-5 1.9 (±0.8) × 10-3 3.5 (±2.0) × 10-4 5.6 (±5.0) × 10-4 6.7 (±1.2) × 10-6 1.0 (±0.1) × 10-5 1.2 (±0.4) × 10-4 1.9 (±0.4) × 10-5 5.0 (±5.8) × 10-5 1.1 (±0.5) × 10-4 4.6 (±2.1) × 10-5 8.4 (±5.1) × 10-4 3.6 (±na) × 10-4 1.7 (±na) × 10-4 1.1 (±0.3) × 10-5 1.9 (±2.0) × 10-5 1.9 (±0.3) × 10-5 2.7 (±1.4) × 10-6 1.2 (±1.0) × 10-6 2.3 (±0.4) × 10-5

Kd1obs × Kd2obs (M2) 5.2 (±1.0) × 10-9 1.8 (±2.0) × 10-7 3.5 (±1.0) × 10-7 1.4 (±0.3) × 10-6 1.7 (±0.5) × 10-9 2.0 (±0.7) × 10-9 0.8 (±0.5) × 10-7 2.4 (±0.2) × 10-7 1.6 (±0.3) × 10-8 2.3 (±0.2) × 10-8 2.7 (±2.0) × 10-9 3.9 (±0.6) × 10-7 2.0 (±na) × 10-8 2.1 (±na) × 10-7 1.1 (±0.4) × 10-9 2.8 (±0.4) × 10-8 4.3 (±0.4) × 10-9 2.9 (±0.3) × 10-8 1.9 (±1.0) × 10-8 3.0 (±2.0) × 10-7

∆Ho overall (kcal/mol) - 7.7 (±0.2) -41.2 (±17.7) -21.2 (±8.2) -1.7 (±4.3) -7.8 (±0.8) -7.6 (±0.9) -9.2 (±0.3) -14.7 (±1.0) -18.4 (±15.8) -8.9 (±1.5) -5.9 (±0.9) -30.3 (±12.2) -7.5 (±0.0) -10.6 (±0.0) -8.7 (±0.1) -7.1 (±3.8) -10.1 (±0.7) -30.6 (±3.1) -11.8 (±10.9) -35.4 (±21.7)

T∆So overall (kcal/mol) 3.6 (±0.3) -31.4 (±17.6) -12.4 (±8.3) 6.3 (±4.4) 4.2 (±0.6) 4.3 (±0.7) 0.6 (±0.7) -5.7 (±1.0) -7.8 (±15.9) 1.6 (±1.6) 6.3 (±1.8) -21.5 (±12.1) 3.7 (±0.0) -1.5 (±0.0) 3.5 (±0.2) 3.2 (±3.8) 1.3 (±0.7) -20.3 (±3.2) -1.1 (±11.1) -26.0 (±22.7)

not performed in triplicate.

Bile acids and mouse I-BABP heterotypic binding. In the ileum, mI-BABP is exposed to a mixture of bile acids, and likely will bind to bile acids heterotypically. Previous studies revealed that hI-BABP preferentially binds the heterotypic combination of the conjugated forms of CA: CDCA, the most abundant bile acids in human. Given the observed differences in the binding profile of homotypic bile acids between mouse and human, we next investigated mI-BABP binding in mixtures of physiologically relevant mouse bile acids. We measured macroscopic binding constants utilizing all possible heterotypic combinations of two different bile acids in a 1:1 ratio with mI-BABP (see Figure 2, Figure S1, Table 2 and S1).



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In the search for the preferred combinations, we initially compared the observed overall affinity (Kd1obs × Kd2obs) for all combinations. Surprisingly, our data showed that the heterotypic combinations of β-MCA displayed a low observed overall binding similar to the homotypic combination of β-MCA. Furthermore, the heterotypic combination Tβ-MCA:TCA, the two most abundant mouse bile acids, was not the ligation state of lowest energy. Two unexpected and interesting results were observed in subsequent experiments. First, α-MCA, β-MCA, and their taurine conjugates displayed differing binding profiles in heterotypic combinations with CA. αMCA heterotypic combinations exhibited a substantially higher overall affinity to mI-BABP than heterotypic combinations of β-MCA. This difference was specific to CA and did not occur with CDCA. This extends findings of Tochtrop et al., (23) in which they suggested that cooperativity in bile salt-I-BABP recognition is governed by the pattern of steroid B- and C-ring hydroxylation (Figure 1B). Finally and importantly, mI-BABP displays the highest overall affinity for the conjugated forms of CA:CDCA. This finding is significant for two reasons. First, the conjugated form of CDCA is not a major bile acid in mouse. Second, the heterotypic combination of CA: CDCA in conjugated form is also the preferred ligation state in human. Our ITC results revealed that the same preferential energetic profile exists between two species despite a vastly different biliary pool composition and differences in homotypic bile acid binding preferences. Specifically, I-BABP binds to the conjugated form of CA:CDCA with the highest affinity in both species. As discussed in the introduction, site selectivity is another unique binding property of hI-BABP bound to the heterotypic combination of conjugated CA: CDCA. We next wanted to determine if this was conserved in mI-BABP; however site selectivity cannot be determined by ITC. (25)


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NMR spectroscopy. In order to determine site selectivity of bile acid binding, we utilized the resolving power of NMR, more specifically standard 1H-15N HSQC in conjunction with isotopically labeled bile acids. This method was successfully developed and applied to observe the site selectivity displayed by hI-BABP. (11, 23) The key advantage of this approach is the ability to filter all resonances except for those arising from labeled bile acids in bound or unbound states. Using this method in a binding experiments provides valuable information about the number of binding sites and occupancy of each site within mI-BABP. This information can then be applied to competition experiments which monitor bile acid replacement in each binding site. NMR-active nuclei, nitrogen-15, was incorporated via

15

N-taurine to each bile acid via an

amide coupling. Each HSQC experiment provided a two-dimensional spectrum and each crosspeak in the spectrum corresponds to the correlation of 1H and the absence of mI-BABP at 10 °C, all

15

15

N nuclei in the amide bond. In

N-labeled tri-hydroxy-bile acids displayed one major

resonance at 7.87 ± .01 ppm (1H nuclei) and 120.98 ± .12 ppm (15N nuclei) and

15

N-TCDCA

displayed one major resonance at 7.83 ppm (1H nuclei) and 120.18 ppm (15N nuclei), as illustrated in Figure S2. This signal was assigned as unbound bile acid labeled as “U”. In some cases, either one or two minor resonances (U1 and U2), with intensity less than 1-2% were detected in the 2D spectrum. The critical micelle concentration (CMC) for bile acid solutions is often taken as the midpoint of a small concentration range over which micelles form. CMC values at room temperature for TCA and TCDCA are 6 mM, and 3 mM, respectively. (26-28) Based on these values, these two minor resonances most likely represent a low order of bile acid aggregation due to gradual micellization of bile acid anions. (29-31)



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Before collecting data to investigate site selectivity for the heterotypic combinations of bile acids, resonances of homotypic combinations with mI-BABP were assigned. Initial assessment revealed significant temperature dependency of the 1H-15N signal. All 2D-spectrum were collected over a range of 10-35 °C to find optimal temperatures to avoid broadening or disappearance of the 1H-15N signal. 2D-spectra of TCA and TCDCA are shown in Figure 3. Other bile acid combinations are illustrated in Figure S2. 15N-TCA in the presence of mI-BABP displays two resonances showing two distinct environments noted as site “1” and “2” in addition to unbound bile acid peak. This result was expected due to two binding sites within mI-BABP. 15

N-Tα-MCA and 15N-Tβ-MCA also showed two binding signals with different affinity for each

site. Interestingly, the observed signal for both

15

N-Tα-MCA and

15

N-Tβ-MCA at site “2”

showed a different chemical shift compared to 15N-TCA thereby providing name site “2*”. 15NTα-MCA binding signal was strong at site “1” while 15N-Tβ-MCA only showed a weak binding signal at high temperature. U1 and U2 unbound bile acid peaks were strongly observable in both 15

N-Tα-MCA and

15

N-Tβ-MCA spectrum. The primary “U” signal was observed in

15

N-Tβ-

MCA but not 15N-Tα-MCA consistent with differences in binding affinity measured by ITC. By contrast in the 2D spectrum of 15N-TCDCA with mI-BABP, only one strong signal for 15NTCDCA in addition to the weak U1 and U2 resonances was observed (Figure 3B). This strong signal marked as “1/U” indicates unbound and bound

15

N-TCDCA are in the fast chemical

exchange. The attempt to resolve them was not successful. No binding signal was detected at site “2”. This behavior is consistent with an intermediate chemical exchange rate. We next investigated mI-BABP site selectivity for heterotypic bile acid combinations by competition experiments in which unlabeled bile acid was added to a solution of mI-BABP/15N – taurine labeled bile acid (Figure 3 and Figure S2). We first investigated the effect of adding


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unlabeled TCA to a solution containing mI-BABP/15N-TCDCA in a molar ratio of 1: 4. Interestingly, a strong binding signal for

15

N-TCDCA appeared in the presence of TCA at

binding site 1 (7.85, 119.95) and the U1 and U2 signals also disappeared. We performed the experiment again switching the NMR-observable bile acid to confirm this result. As illustrated in Figure 3C, the addition of unlabeled-TCDCA to a solution containing a mI-BABP⁄15N-TCA caused the intensity of

15

N-TCA binding signal at site “1” to decrease dramatically and the

unbound signal (“U”) to increase. There was no observed change in the intensity of binding signal at site “2”. Together these results suggest that TCDCA competes with TCA for site “1”, however, binding of TCA in binding site 2 promotes binding of TCDCA. This binding profile is very similar to the site-selective binding of GCA: GCDCA by hI-BABP. Site-selectivity and affinity to binding sites were investigated in the other heterotypic combinations of taurine-conjugate bile acids. Our data revealed that TCDCA and Tβ-MCA replaced TCA at site “1” while Tα-MCA replaced TCA at site “2”. Interestingly, TCDCA does it in site-selective manner and Tα-MCA and Tβ-TMCA replace TCA in non-site-selective fashion. Additionally, TCDCA showed highest affinity to site “1” and affinity for this site could be ranked in the following order TCDCA>Tα-MCA>Tβ-MCA>TCA. Taken together, ITC and NMR data indicate a significant difference between mI-BABP and hI-BABP for binding of homotypic combinations of bile acids, but a strikingly similar preference and binding mode for the heterotypic combination of conjugated CA: CDCA.



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Figure 3. A select region of TCDCA:mI-BABP, (C)

15

15

N-HSQC spectrum of (A)

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15

N-TCA:mI-BABP, (B)

N-TCA:TCDCA:mI-BABP, and (D) TCA:

15

N-

15

N-TCDCA: mI-BABP.

“U” indicates unbound, “U1” and “U1” show low ordered aggregation of unbound bile acid and “number” shows bound 15N-labeled bile acid to mI-BABP. The molar ratio of mI-BABP to total bile acid is 1:4. Color shade and arrows show temperature dependence. DISCUSSION In this study we have characterized the binding of the largest variety of homo- and heterotypic bile acid combinations to an I-BABP family member. This enabled us to gain critical insights into the binding properties of mI-BABP and the two proposed functional roles of I-BABP family


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members in bile acid metabolism: buffering and signaling. We found the striking result that while binding properties of homotypic complexes between bile acids and I-BABP differ widely between mouse and human, the preferred heterotypic combination of conjugated CA: CDCA is conserved. From the extensive ITC and NMR studies performed here, we are able to describe unique species variation in I-BABP as well as refine some general principles for binding of bile acids to the unique member of the iLBP family. In human, the Kd1obs × Kd2obs for homotypic combinations of nine studied bile acids (CA, CDCA, DCA and their conjugates) is in similar range, nanomolar. In contrast, mouse displays a wide range in the Kd1obs × Kd2obs in identical condition, from micromolar to nanomolar. Another difference is that in human, amino acid conjugation had minor effect on binding affinity (23) whereas in mouse it showed a substantial effect. For example in human, the effect of conjugation on CDCA resulted in a 6.5-fold increase in overall affinity while in mice we observed a 77-fold increase. The binding affinity for heterotypic combinations is generally higher than homotypic combinations in both species. Comparison of overall affinity (Kd1obs × Kd2obs) for the best homoand heterotypic combinations of bile acids were strikingly similar between human and mouse. GCA and GCDCA in human, and TCA and TCDCA in mouse are the best homotypic combination with measured affinities of 2.1 × 10-9 M2, 0.9 × 10-9 M2, 1.7 × 10-9 M2, and 2.0 × 109

M2 respectively. Likewise, the GCA:GCDCA heterotypic combination in human and

TCA:TCDCA combination in mouse have the highest affinity, 0.7 × 10-9 M2 and 1.1 × 10-9 M2, respectively. Our study also revealed the effects of hydroxylation pattern in ring B and C on the binding. The presence of a hydroxyl group at C-6 appears to weaken the binding interactions for homotypic combinations of bile acids and mI-BABP. For example, homotypic combinations of



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TCDCA binds 84-fold better than Tα-MCA which differs only in the presence of the C-6 hydroxyl group. This observation explains why overall affinity of TCA and TCDCA in mIBABP is higher than both Tβ-MCA and Tα-MCA in homotypic settings. Another interesting finding was the different binding profiles of Tα-MCA:TCA compared to Tα-MCA:TCDCA where only TCA promoted strong binding of Tα-MCA. This observation indicates a critical role of TCA in positive cooperativity and high overal binding in heterotypic combinations of bile acids. Site selectivity is another significant property of I-BABP which has been studied in chicken and human via 2D-NMR spectroscopy. Both human and chicken appear site selective for the heterotypic combination of GCDCA:GCA; however, chicken shows less selectivity at one of the binding sites.(10) Here in mouse, site selectivity is similar to human wherein TCDCA is preferentially bound at site 1 and TCA at site 2. We also find that bile acids lacking a 12-OH preferentially bind to site 1 as was shown previously (14, 32). We further find that when two bile acids lacking the 12-OH (TCDCA:Tα-MCA or Tβ-MCA) are combined, we still observed some site selectivity, and TCDCA dominated binding at site 1 compared to Tα-MCA or Tβ-MCA suggesting that the presence of the C-6 hydroxyl group decreases binding affinity to site 1. Although mouse and human have distinct biliary pools, I-BABP amino acid sequence (80% identity Figure S3) and unique recognition of the CA:CDCA combination are highly conserved in the two species. One of the suggested roles of I-BABP is as bile acid buffering agent during reabsorption of bile acid from the intestine in order to protect enterocytes from detergent activity and other adverse effects. This role has been suggested based on studies which found that IBABP selectively buffers CDCA, an abundant human bile acids. Here, we found that one of the most abundant bile acids in mouse, β-MCA and its conjugates, does not bind well to mI-BABP


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homotypically nor heterotypically. Although mI-BABP may act as a bile acid buffering agent for alpha 7-OH bile acids like CDCA in mouse bile which are more toxic than other bile acids, our results suggest that buffering is not the primary function of I-BABP. Instead, our findings suggest that mI-BABP is tuned to recognize CDCA in combinations with other bile acids, specifically CA, for a role outside of buffering in human and mouse physiology. It is well known that CDCA is a natural ligand for FXR, a nuclear receptor in bile acid hemostasis. (17)Activation of FXR by CDCA increases transcription of the I-BABP gene and decreases transcription of cholesterol 7α-hydroxylase, the rate-limiting step in bile acid synthesis. (33) Recently, it has been reported that tauro-conjugated α- and β-muricholic acids can antagonize FXR.(34) Due to the unique binding properties of I-BABP to recognize CA:CDCA in a mixture of bile acids, it makes I-BABP an ideal candidate to modulate signal transduction by delivering CDCA to FXR. There are still questions remain to be addressed at a molecular level to better understand bile acid signaling. First, although I-BABP and FXR have been shown to functionally interact (15), it is not clear whether I-BABP and FXR physically interact to exchange the ligand. Second, it is also possible that they compete for the same ligands. Bile acid signaling through FXR has been implicated in several metabolic and digestive disorders (35), and understanding the role of I-BABP in this pathway may play an important role in developing therapeutics.

MATERIALS AND METHODS Materials. CA (Cat No: 21947) from chem-IMPEX international (Wood Dale, IL), CDCA (Cat No:101383) from MP Bio medicals LLC,

15

N-taurine powder 98% (Cat No:491330) from

and taurine powder from Sigma-Aldrich were purchased and used without further purification. α-



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MCA and β-MCA were synthesized utilizing the methodology of Iida, et al.(36). Conjugation of all bile acids studied here to 15N-labeled and unlabeled taurine was prepared according to Tserng, et al.(37) FABP6 cloning and Plasmid Construction. total RNA was extracted from the distal ileum of a C57BL/6 mouse via an RNA purification kit (Invitrogen, Life technology). cDNA of mouse fatty acid binding protein 6 was amplified by rtPCR with a gene specific primer carrying NdeI and XhoI sites in their 5`end (integrated DNA technologies™). mFABP6 cDNA ligated to pET22 b(+) and transfected to 5-alpha competent E. coli (New England Biolabs). Confirmation of the gene was determined by DNA sequencing which agreed to NCBI sequence for Mus musculus fatty acid binding protein 6, (GenBank: BC119289.1). The plasmid containing mFABP6 was transfected to T7 Express Competent E. coli from New England Biolab (C2566). Protein biosynthesis and purification. the bacteria harboring recombinant mFABP6 plasmid were grown in terrific broth (tryptone 12 g/L, yeast extract 24 g/L, glycerol 4 mL, KH2PO4 2.3 g/L and K2HPO4 12.5 g/L) containing 50-100 µg/L ampicillin. The cultures were incubated in a shaker incubator (Innova 4300, New Brunswick Scientific) at 37oC and 220 rpm. At OD600 of 0.6–0.9, IPTG was added to final concentration 1 mM to induce protein expression in the culture. After 4 hours at 37oC, cells were harvested by centrifugation 7000g for 20 min. The pellet was partially lysed by five cycles of freeze-thaw in dry ice/ethanol(38).Then cells were suspended in 20 mM Tris-HCl buffer containing a broad spectrum protease inhibitor cocktail (Roche). The suspension was centrifuged (7000 g) for 20 min at 4oC. The supernatant was chromatographed on three columns. First, on a 25 x 5 cm column of Q-Sepharose fast flow at 5 ml/min. then, the protein buffer was switched to 20 mM potassium phosphate, 135 mM potassium chloride, 10 mM sodium chloride by Amico Stirred Cells under nitrogen gas. Then, the protein was separated


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by size 1ml/min on the second column, (90 x 2.5 cm column of Sephacryl S100). The third column, 15 cm x 5 cm lipophilic Sephadex G-50 type VI at 37oC with 0.5ml/min was used to delipidated mI-BABP. Protein purity was assessed by overloaded Coomassie-stained SDS-PAGE gel. Protein concentration was determined by Bradford assay. Isothermal Titration Calorimetry sample preparation. Bile acid titrant solutions were prepared from accurately determined stock solutions in methanol. Briefly, an aliquot of the stock solution was treated with 1.1 eq. of 2M KOH. An appropriate amount of this solution was added to ddH2O to allow for freezing and subsequent lyophilization. The resultant powder was then dissolved in an identical buffer to the protein solution and filtered through a 0.22 µm PTFE membrane. All experiments were carried on MicroCAl VP-ITC micro calorimeter (Northampton, MA). Titrations were performed in 20 mM potassium phosphate, 135 mM potassium chloride, 10 mM sodium chloride and 1 mM 2-mercaptoethanol adjusted to a pH of 7.2 at 25oC. For these titration, fifteen injections of 3 µL aliquots were followed by forty-two injections of 6 µL of 12 mM bile acid into a reaction cell containing 1.4626 ml of 0.16 (±0.02) mM mI-BABP. The concentration of mI-BABP was quantitated by Bradford assay. Briefly, The ITC data sets of all bile acid were fit to the stepwise binding model using the nonlinear least square analysis program SCIENTIST (Scientific Instrument Services, Ringoes, NJ). Then fitted values were used to construct the fitting line by the Origin software package (version 5.0) provided with the instrument. The analysis of the fitted curves yielded the stepwise dissociation constants (Kd1obs, Kd2obs) and the stepwise binding enthalpies (∆H1obs, ∆H2obs). NMR sample preparation and NMR data collection. Stock solutions were prepared for each 15

N-taruine bile acids as described in ITC section. Each sample for 1H-15N heteronuclear

correlation spectra (1H-15N HSQC) experiment was prepared as follows. An appropriate amount



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of bile acid from stock solution was taken. Followed by solvent removal under reduced pressure, added 60 µl of the identical buffer as protein to resultant oil and lyophilized overnight. Then addition a 540 µl of mI-BABP (1.1 mM), 60 µl of D2O and 2 µl of 0.3 M dithiothreitol (DTT) to lyophilized bile acid produce a final 600 µl NMR sample containing 1 mM mI-BABP, 3 mM bile acid, 1mM DTT, 20 mM potassium phosphate, 135 mM potassium chloride, 10 mM sodium chloride, pH=7.2, in H2O/D2O (9:1). Samples were equilibrated for few hour prior NMR data collection. All NMR spectra were performed at 10, 15, 29, 25, and 35oC on a Bruker 800 Avance II spectrometer equipped with TXI cryoprobe operating at 800.16 MHz. The gradient- and sensitivity – enhanced 1H-15N HSQC spectra were collected with the standard Bruker 1H-15N HSQC pulse sequence. All the HSQC spectra were acquired with a 1H spectra width of 9615 Hz and 1024 data points, no zero-filled, and 8 number of scan. In the

15

N dimension, spectra were

acquired with a 15N spectral width of 1297 HZ and 80 increments were collected and zero-filled to a total of 1024 points. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. ACKNOWLEDGMENT This work was supported by grants from National Science Foundation (NSF-MCB-0844801) and National Institutes of Health (NIH CA 157735). We would also like to thank X. Mao (Case Western Reserve University) for his technical assistance in the collection of HSQC data at Cleveland center for membrane and structural biology 800 MHz NMR facility. ASSOCIATED CONTENT


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35x17mm (300 x 300 DPI)


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Bile Acid Recognition by Mouse Ileal-Bile Acid ... - ACS Publications

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