Towards the elucidation of molecular determinants of cooperativity in the

liver bile acid binding protein

Massimo Pedo`,1 Mariapina D’Onofrio,1 Pasquale Ferranti,2Henriette Molinari,1

and Michael Assfalg1*

1 Dipartimento di Biotecnologie, Universita` di Verona, Strada le Grazie 15, Verona 37134, Italy

2 Dipartimento di Scienza degli Alimenti, Universita` di Napoli Federico II, Parco Gussone, Portici 80055, Italy

INTRODUCTION

In recent years, the discovery of a number of transporter proteins expressed in the liver and intestine, specifically involved in bile acid transport, has led to an improved understanding of bile acid homeostasis and the enterohepatic circulation. Bile acid transporters are now recognized to play central roles in driving bile flow, as well as in the adaptation to various patho- logical conditions, with complex regulation of activity and function in the nucleus, cytoplasm and membrane.1–4

In a continuous effort to understand the mechanism underlying bile acid binding and release within the cytoplasm,5–8 we describe here the study of protein-ligand interactions involving liver bile acid binding proteins (L-BABPs), which act in parallel with the ileal transporters (ileal bile acid binding proteins, I- BABPs) to ensure vectorial transport of bile salts within hepatocytes and enterocytes, respectively.5

L-BABPs are small molecular mass proteins (14–15 kDa), belonging to the fatty acid binding protein family (FABP), exhibiting the typical and well-conserved fold of the family in which 10 strands of antiparallel b-sheets surround the hydro- phobic ligand binding cavity and two short a-helices are located between the first and second strands.

I-BABP was shown to be able to bind bile salts with high cooperativity, although the structural basis of this feature was not clarified.9–11 Given the expected parallel function of L- BABP in hepatocytes, we reasoned that the identification of a similar cooperative binding mechanism would firmly set this parallelism, adding new insights into the mechanism of molecular recognition of this protein subfamily.

In a previous work, focussed on the structural and dynamic properties of chicken liver bile acid binding protein (cL-

Additional Supporting Information may be found in the online version of this article. Grant sponsor: FIRB 2003; Grant number: RBNE03PX83; Grant sponsors: Fondazione Cari- verona, Universita` degli Studi di Verona, CIRMMP.

*Correspondence to: Michael Assfalg, Dipartimento di Biotecnologie, Universita` degli Studi di Verona, Strada le Grazie, 15 Verona 37134, Italy. E-mail: michael.assfalg@univr.it

Received 9 April 2009; Revised 21 May 2009; Accepted 1 June 2009

Published online 9 June 2009 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/prot.22496

ABSTRACT

Bile acid binding proteins (BABPs) are cytosolic lipid chaperones contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Liver BABPs act in parallel with ileal transporters to ensure vectorial transport of bile salts in hepatocytes and enterocytes, respectively. We describe the investigation of ligand binding to liver BABP, an essential step in the understanding of intracellular bile salt transport. Binding site occupancies were monitored in NMR titration experiments using 15N- labelled ligand, while the relative populations of dif- ferently bound BABP forms were assessed by mass spectrometry. This site-specific information allowed the determination of intrinsic thermodynamic parameters and the identification of an extremely high cooperativity between two binding sites. Protein- observed NMR experiments revealed a global structural rearrangement which suggests an allosteric mechanism at the basis of the observed cooperativity. The view of a molecular tool capable of buffering against significant concentrations of free bile salts in a large range of solution conditions emerges from the observed pH-dependence of binding. We set to determine the molecular determinants of cooperativity by analysing the binding properties of a protein contain- ing a mutated internal histidine. Both mass spectrometry and NMR experiments are consistent with an overall decreased binding affinity of the mutant, while the measured diffusion coefficients of ligand species reveal that the affinity loss concerns essentially one of the two binding sites. We therefore iden- tified a mutation able to disrupt energetic communi- cation functional to efficient binding and conclude that the buried histidine establishes contacts that sta- bilize the ternary complex.

Proteins 2009; 77:718–731.

VC 2009 Wiley-Liss, Inc.

Key words: NMR spectroscopy; fatty acid binding protein; bile acids; cooperativity; molecular recognition.

unbound protein, which resulted capable of sampling a variety of conformations including the one stabilized by the ligands.12 Molecular dynamics simulations suggested that this ‘‘allosteric’’ binding behavior was related to the protonation state of a buried histidine (H98), involved (at least in the simulation) in triggering a conformational change, namely the opening/closure of the EF loop, at the protein open end.8 Specifically, the highly conserved H98 was indicated to play a central role in establishing a network of hydrogen bonds and salt bridges among buried residues, defining a sort of continuous polar ‘‘spine’’ connecting remote strands of the apo protein.6 In this light the comparison of the binding behavior of WT cL- BABP and its mutant devoid of H98 should provide the means to describe the elusive structural and dynamic basis of cooperativity in the ternary complex with two bile salt molecules. Indeed, understanding the molecular mechanism underlying cooperativity is an elaborate task involving detailed characterization of all ligated states. Precious insights would be gained by analyzing the structure and dynamics of singly bound states, in comparison with data obtained for the free and fully loaded forms. However, due to cooperativity, the half-saturated states are always too scarcely populated to allow direct experimental observation. One way to circumvent this problem is to reduce binding affinity of one of the sites by amino acid mutation. This strategy, here delineated and followed, has found extensive application for example, in NMR studies of EF-hand calcium binding proteins.13,14

To analyse ligand binding and gain insight into the structural basis of cooperativity we have applied to both the WT protein and its mutant H98Q a variety of NMR methods which provide site-specific and residue-specific information, both at the structural and dynamic level. The data are complemented with a mass spectrometry analysis of the bound proteins.

MATERIALS AND METHODS Sample preparation

The expression plasmid for H98Q cL-BABP was obtained from that of wild type (WT) cL-BABP using the Quickchange (Stratagene) mutagenesis kit. The proteins were expressed as previously reported.8 A number of different mutations at position 98 were essayed but they resulted in scarcely soluble proteins. All the protein preparations were checked by 1D 1H NMR prior and af- ter delipidation and reproducible spectra were always obtained for the apo and undelipidated forms. The molecular weight of the two proteins and the extent of label- ling was verified by MALDI mass analysis.15N Glychoche- nodeoxycholic acid was prepared as described.7

A Q-TOF Ultima mass spectrometer (Waters, UK) was used in positive ion mode in all the experiments. Ten microliter aliquots of reaction mixture were introduced through a Rheodyne external loop injector into the ion source at a flow rate set to 1 lL/min with a Phoenix 20 CU HPLC pump driving a gas-tight syringe connected to the instrument capillary. To obtain maximum signals for the ions of the putative PL complex, a solution of 5% acetonitrile in 10 mM ammonium hydrogen carbonate, pH 7.0, was used. Different conditions of orifice voltage (20–80 V) and temperature (40–1008C) were employed. The declustering potential of 40 V and the source temperature of 608C were the chosen parameters at which no fragmentation was observed and optimum sensibility for the adduct peaks was attained. Spraying was achieved, using nitrogen as the nebulizing gas, by charging the probe at 3.6 kV. Calibration of the mass scale was performed using the multiple charged ions of horse heart myoglobin from a separate sample introduction in positive ion mode. Full-scan mass spectra were generated in continuous data-acquisition mode. The spectra reported are an average of three scans from m/z 800 to m/z 2200 (scan rate 5 10 s/scan). Quantitative analysis of compo- nents was performed by integration of the multiple- charge ions relative to each species.

NMR spectroscopy

NMR experiments were performed on a Bruker Avance 500 spectrometer equipped with a triple-resonance TXI probe and incorporating x, y, z-axes gradient coils, and on a Bruker Avance III 600 spectrometer equipped with a pulsed field gradient triple resonance TCI cryoprobe. Standard sequence schemes employing pulsed field gradients were employed to achieve suppression of the sol- vent signal and spectral artefacts. Direct and indirect dimensions were normally apodized by use of 908-shifted squared sine-bell functions (for 13C- and 15N-edited dimensions) or Lorentzian-to-Gaussian functions (for the

H dimension), followed by zero filling and Fourier transform. The NMR data were processed with Topspin 2.1 (Bruker) and analyzed with the same software or with Sparky 3.11 (UCSF). Typical samples contained 0.5 mM protein dissolved in 30 mM phosphate (Na2HPO4/NaH2PO4), 90% H2O-10% D2O. Proton

chemical shifts were referenced to external 3-(trimethyl- silyl)-3,3,2,2-d4-propionic acid while nitrogen chemical

shifts were referenced indirectly as described.15 The mea- surement temperature was 298 K.

Resonance assignments

The complete resonances assignments for both apo- and holo WT cL-BABP were available.6,8 Amide resonances assignments for apo- and holo H98Q cL-

tration data (addition of ligand or change of pH) as well as by analysis of four pairs of TOCSY-[1H,15N]-HSQC and NOESY-[1H,15N]-HSQC spectra registered at 600 MHz. The NOESY mixing time was set to 120 ms, while a spin-lock time of 70 ms was used for TOCSY spectra.

Hydrogen exchange experiments

Hydrogen exchange (HX) experiments were performed at p2H 7.4 and 6.5 (p2H 5 pHread 10.4). The HX sample of 15N H98Q cL-BABP was prepared by dissolving 5 mg of delipidated and lyophilized protein in 600 lL of 30 mM deuterated Na2HPO4/NaH2PO4 buffer. The final

protein concentration was about 0.4 mM. The solution was centrifuged briefly at 48C to remove insoluble protein and transferred to a 5 mm NMR tube. SOFAST- HSQC experiments16 were collected at 600 MHz, with 700 points in F2 and 192 complex data points in F1,

using a relaxation delay of 0.5 s with 2 scans and a duty cycle of 7.6%. The band-selective 1H excitation (PC9) and refocusing (RSNOB) pulses were centered at 4.7 ppm with pulse lengths of 2.33 and 0.817 ms, respectively. 13C decoupling was achieved through an adiabatic pulse (smoothed chirp) employing a 500 ls pulse, where the carbon hard pulse corresponded to 11.8 ls. An adiabatic pulse of 2 ms was employed for 15N decoupling, where the nitrogen hard pulse was 45 ls. HX rate constants were determined by fitting cross-peak volumes to a first-order exponential decay:

IðtÞ ¼ Ið0Þ expðkexÞ ð1Þ

where I(t) represents the volume of the cross-peaks at the time t, I(0) the volume of the cross peak at t 5 0; kexis the observed rate of hydrogen exchange and t is the

time in minutes. Here, t 5 0 corresponded to 11 minutes and kex greater than 1021 min21 could not be deter-

mined.

The free energy of opening, DGop, was derived from

the equation:

DGop¼ RTInKop ð2Þ

where Kop 5 kex/kchand kch for model random peptides

was derived as previously reported.17 Data were fitted with the program Sigmaplot (Jandel Scientific).

Protein-ligand titration experiments

Protein stock solution concentrations were determined by UV while the ligand stock solutions were determined by measuring dry weights using a microbalance. Random samples were prepared three times and the error on the measured points reflects the standard deviation from the mean. Typical 1H-15N HSQC spectra were collected at 500MHz with 40 and 12 ppm spectral widths and 256

frequency dimensions, respectively. Thirty-two scans were collected for observation of 15N-labelled protein samples, while 256 scans were used for observation of15N-labelled ligand. For the latter experiments only 64 data points in F1(15N) were collected.

The NMR isotherms for the binding of 15N-GCDA to WT cL-BABP were analyzed simultaneously according to the site-specific model depicted in the inset of Figure 5. The volumes for the unbound, site 1, and site 2 resonances were estimated considering the following relationships: P¼ CP=½1 þ ðj1þ j2ÞL þ c12ðj1þ j2ÞL2; PL0¼ P j1 L;

PL00¼ P j2 L; PL2¼ P c12j1j2 L2 ð3Þ

where j1and j2are the intrinsic affinity constants and c12

the intrinsic cooperativity factor. P, L, PL0_{, PL@, PL}

2 are

the equilibrium concentrations of unbound protein, unbound ligand, protein singly bound at site 1, protein singly bound at site 2, and double bound protein, respectively. CPis the total protein concentration. L was obtained

by solving analytically the cubic equation: c12j1j2 L3þ ½ð2CP CLÞc12j1j2þ j1þ j2 L2

þ ½ðCP CLÞðj1þ j2Þ þ 1 L CL¼ 0 ð4Þ

where CL is the total ligand concentration. This analysis

was performed with an in-house Matlab routine.

The Hill coefficient determined at half saturation was used as an indicator of macroscopic cooperativity and calculated as:

nH¼ 2=½1 þ ðKd2=Kd1Þ0:5 ð5Þ

where Kd1 and Kd2 are the stepwise dissociation con-

stants, related to the intrinsic affinity constants as fol- lows: Kd15 2/(j11j2), Kd25 (c12Kd1j1j2)21.

pH titration experiments

A sample of 0.5 mM 15N,13C-labelled WT cL-BABP was subjected to pH change and an echo-antiecho

H,13C-HSQC spectrum was acquired at 600 MHz at each of the following pH values: 4.1, 4.7, 5.0, 5.1, 5.5, 5.9, 6.2, 6.7, 7.1, 7.5, and 7.9. Thirty-two scans were acquired over a spectral window of 12 (F2,1H) 3 40 (F1, 13

C) ppm. The nitrogen carrier was placed at 125 ppm, and the INEPT delay was set to 1,725 ms. The pH change was obtained by dialysis of the protein sample against the already equilibrated buffer. At selected titration points, a long-range 1H,15N-HSQC experiment was acquired, optimized for observation of histidine resonances with the following parameters: spectral window of 17 (F2, 1H) 3 110 (F1, 15N) ppm, 512 scans, nitrogen car-

rier centered at 205 ppm, INEPT delay 17 ms. Carbon and nitrogen decoupling was introduced. The long-range

labelled H98Q cL-BABP sample at different pH values. The pH-dependent proton and carbon chemical shift values obtained from the above 1H,13C-HSQC spectra of the wild type protein were used to estimate the pKaval-

ues of the corresponding histidine residues according to the following equation:

dobs¼ ddþ ðdp ddÞ=½1 þ 10ðpHpK aÞ ð6Þ

where dpand ddare the chemical shifts of the protonated

and the deprotonated state, respectively.

Two series of standard 1D and 1H,15N-HSQC (only first increment) titrations were also performed on the following holo protein samples: (i) WT cL-BABP 1 15N- GCDA (P:L 5 1:2) at pH 7.3, 5.9, 4.5, 4.3, 4.1, 3.4, 3.0, 7.1; (ii) H98Q cL-BABP 115N-GCDA (P:L 5 1:2) at pH 6.9, 6.1, 5.1, 4.4, 4.2, 4.1, 3.3. The pH was changed by step-wise addition of small aliquots of hydrochloric acid to the samples in starting neutral conditions.

Relaxation rate analysis

N relaxation experiments were acquired at 600 MHz on 15N-labelled 0.4 mM protein samples, both at pH 7.0 and 5.6. The pulse program for longitudinal relaxation measurements, run in interleaved fashion and including a water flip-back scheme, was provided by Daniel Cicero (University of Rome, Tor Vergata), while for transverse relaxation measurements a standard Bruker two-dimensional pulse sequence was used. Nine delays (0.01, 0.18, 0.36, 0.54, 0.72, 0.90, 1.08, 1.26, 1.44 s) were used for R1

measurements, and nine delays (16.96, 33.92, 50.88, 67.84, 101.76, 135.68, 169.6, 220.48, and 237.44 ms) were used for R2measurements. The delay in the Carr-Purcell-

Meiboom-Gill pulse train was set to 0.45 ms. The number of scans was set to 8 for R1 and to 16 for R2, the

relaxation delay between each scan was 3 s, and a matrix of 2048 3 128 data points was acquired. Cross-peaks were integrated with Topspin 2.1, and the volume sets were fitted to a monoexponential decaying function using two adjustable parameters within the program RELAXFIT.18 The errors were obtained using a standard Monte Carlo approach.

Diffusion experiments

N-edited diffusion experiments were performed on samples of WT cL-BABP:15N-GCDA and H98Q cL- BABP:15N-GCDA with protein:ligand ratios of 1:3, to determine the diffusion coefficients of protein-bound ligands compared to that of the free molecules. The pulse program was obtained by combining the standard HSQC pulse scheme with a PFG-STE (pulsed field gradient stimulated echo) module employing bipolar gradients19 (the pulse program was kindly provided by Daniel Cic- ero, University of Rome ‘‘Tor Vergata’’). PFG-STE-HSQC

tion (d) and 80 ms between the pulsed field gradients (D). The spectral widths and other acquisition parameters were the same as those used for standard HSQC experiments for ligand observation. However, 512 or 768 scans per t1-point and a recycling delay of 3 s were used.

The gradient strength was calibrated from a diffusion experiment on 99.8% D2O using the HDO apparent dif-

fusion coefficient of 1.902 3 1029 m2s21 at 258C. The length of all delays and pulses was held constant while the gradient strength was varied from 2 to 95% of its maximum value (53.5 G/cm). The measured signal volumes as a function of the applied gradient were fitted to the following equation using a nonlinear least squares minimization:

I ¼ Ið0Þ exp½Dg2G2d2ðD d=3 s=2Þ ð7Þ

where D is the translational diffusion coefficient, g is

H gyromagnetic ratio, G is the gradient strength, D and d are defined as above and s is the gradient pulse separation.

RESULTS

Structure and stability assessment of WT and H98Q cL-BABP

The assignments for free and bound WT cL-BABP were determined previously at pH 7.2 and 5.6, and the solution structures for apo and holo forms at pH 7.2 were reported.6,8,20 The choice of amino acid for mutation of the highly conserved H98 was based on the observation of the presence of glutamine in few sequences of proteins belonging to the BABP family. The H98Q mutant exhibited well-dispersed 1H,15N-HSQC spectra [Fig. 1(d)] and the assignment of the amide resonances was obtained at pH 7.2 and 5.6 by use of 1H,15N-NOESY- HSQC and 1H,15N-TOCSY-HSQC spectra. The comparison of peak positions of the wild type and mutant proteins is reported in Figure 1 for both apo and holo proteins. It appears that while the N-terminal half of the apo protein is completely unaffected by the mutation, non-negligible perturbations are evidenced in the C-terminal half, with combined (proton and nitrogen) chemical shift changes up to 0.4 ppm. While the largest effects are, as expected, close to the mutation (residues in the range 89–111, belonging to strands G, H, I), the changes involve also distant residues located mainly in loop DE and strands E, F [Fig. 1(a,e)]. The distances of these residues from position 98 are larger than 6.5 A˚, implying that these effects cannot be merely ascribed to ring cur- rent shifts21 but are indicative of an altered stereo-elec- tronic environment. The NOESY patterns of H98Q cL- BABP residues which showed chemical shift variations compared to the wild type protein were analyzed. Practi-

Figure 1

Chemical shift differences between H98Q cL-BABP and WT cL-BABP. (a) Combined average chemical shift differences (Dd 5 {[(DdH)21(DdN/5)2]/

2}1/2_{) between the apo proteins; the protein regions displaying the largest differences are highlighted with a dotted circle (residues close to the}

mutation) and dotted rectangles (residues far from the mutation). (b) Chemical shift differences between holo and apo H98Q cL-BABP (grey bars) and holo and apo WT cL-BABP (black bars). (c) Chemical shift differences between holo H98Q cL-BABP and holo WT cL-BABP; the largest differences experienced by residues (56–59) distant from the mutation site are highlighted by a circle. (d) Superposition of1H,15N-HSQC spectra of apo WT (black) and H98Q cL-BABP (grey). (e) Mapping of the chemical shift differences of plot (a) on the three-dimensional structure of cL-BABP, the residues experiencing the largest perturbations are represented by spheres, histidine98 is represented in sticks. (f) Mapping of the chemical shift differences of plot (c) on the three-dimensional structure of cL-BABP, the residues experiencing the largest perturbations are represented by spheres.

observed in the wild type protein were retrieved in the spectrum of the mutant, thereby assessing the substantial invariance of the protein scaffold following the mutation. As already noticed previously7, saturation of the binding sites in the WT protein by two bile salt molecules produces chemical shift perturbations that are spread over the whole protein and are as large as 1.4 ppm. The same comparison was here performed on the H98Q mutant by recording an HSQC spectrum on a P:L 5 1:4 sample. The assignment of amide HN signals was made by use of 1H,15N-NOESY-HSQC and 1H,15N-TOCSY- HSQC spectra at pH 7.2 and 5.6. By plotting the chemical shift differences observed upon binding for both the WT protein and the mutant [Fig. 1(b)], it appears that the corresponding residues are affected by essentially the same amount of shift change. However, the direct chemical shift comparison [Fig. 1(c)] indicates that the shift

Nel documento Liver bile acid intracellular transporter: NMR studies of unfolding, cooperative binding and interaction with membrane mimetic. (pagine 90-107)