A Single Amino Acid Mutation in Zebrafish (Danio rerio) Liver Bile Acid-binding Protein Can Change the Stoichiometry of Ligand Binding
2007; Elsevier BV; Volume: 282; Issue: 42 Linguagem: Inglês
10.1074/jbc.m705399200
ISSN1083-351X
AutoresStefano Capaldi, Mara Guariento, G. Saccomani, Dimitrios Fessas, Massimiliano Perduca, Hugo L. Monaco,
Tópico(s)Protein Interaction Studies and Fluorescence Analysis
ResumoIn all of the liver bile acid-binding proteins (L-BABPs) studied so far, it has been found that the stoichiometry of binding is of two cholate molecules per internal binding site. In this paper, we describe the expression, purification, crystallization, and three-dimensional structure determination of zebrafish (Danio rerio) L-BABP to 1.5Å resolution, which is currently the highest available for a protein of this family. Since we have found that in zebrafish, the stoichiometry of binding in the protein cavity is of only one cholate molecule per wild type L-BABP, we examined the role of two crucial amino acids present in the binding site. Using site-directed mutagenesis, we have prepared, crystallized, and determined the three-dimensional structure of co-crystals of two mutants. The mutant G55R has the same stoichiometry of binding as the wild type protein, whereas the C91T mutant changes the stoichiometry of binding from one to two ligand molecules in the cavity and therefore appears to be more similar to the other members of the L-BABP family. Based on the presence or absence of a single disulfide bridge, it can be postulated that fish should bind a single cholate molecule, whereas amphibians and higher vertebrates should bind two. Isothermal titration calorimetry has also revealed the presence in the wild type protein and the G55R mutant of an additional binding site, different from the first and probably located on the surface of the molecule. In all of the liver bile acid-binding proteins (L-BABPs) studied so far, it has been found that the stoichiometry of binding is of two cholate molecules per internal binding site. In this paper, we describe the expression, purification, crystallization, and three-dimensional structure determination of zebrafish (Danio rerio) L-BABP to 1.5Å resolution, which is currently the highest available for a protein of this family. Since we have found that in zebrafish, the stoichiometry of binding in the protein cavity is of only one cholate molecule per wild type L-BABP, we examined the role of two crucial amino acids present in the binding site. Using site-directed mutagenesis, we have prepared, crystallized, and determined the three-dimensional structure of co-crystals of two mutants. The mutant G55R has the same stoichiometry of binding as the wild type protein, whereas the C91T mutant changes the stoichiometry of binding from one to two ligand molecules in the cavity and therefore appears to be more similar to the other members of the L-BABP family. Based on the presence or absence of a single disulfide bridge, it can be postulated that fish should bind a single cholate molecule, whereas amphibians and higher vertebrates should bind two. Isothermal titration calorimetry has also revealed the presence in the wild type protein and the G55R mutant of an additional binding site, different from the first and probably located on the surface of the molecule. The liver bile acid-binding proteins, formerly called liver "basic" fatty acid-binding proteins (FABPs), 2The abbreviations used are: FABP, fatty acid-binding protein; BABP, bile acid-binding protein; L-BABP, liver BABP; z-L-BABP, zebrafish liver bile acid-binding protein; ITC, isothermal titration calorimetry; r.m.s., root mean square.2The abbreviations used are: FABP, fatty acid-binding protein; BABP, bile acid-binding protein; L-BABP, liver BABP; z-L-BABP, zebrafish liver bile acid-binding protein; ITC, isothermal titration calorimetry; r.m.s., root mean square. belong to the conserved multigene family of the fatty acid-binding proteins (1Ockner R.K. Manning J.A. Poppenhausen R.B. Ho W.K. Science. 1972; 177: 56-58Crossref PubMed Scopus (513) Google Scholar, 2Banaszak L. Winter N. Xu Z. Bernlohr D.A. Cowan S. Jones T.A. Adv. Protein Chem. 1994; 45: 89-151Crossref PubMed Google Scholar, 3Zimmerman A.W. Veerkamp J.H. Cell. Mol. Life Sci. 2002; 59: 1096-1116Crossref PubMed Scopus (405) Google Scholar, 4Coe N.R. Bernlohr D.A. Biochim. Biophys. Acta. 1998; 1391: 287-306Crossref PubMed Scopus (271) Google Scholar, 5Schaap F.G. van der Vusse G.J. Glatz J.F. Mol. Cell. Biochem. 2002; 239: 69-77Crossref PubMed Scopus (116) Google Scholar, 6Haunerland N.H. Spener F. Prog. Lipid Res. 2004; 43: 328-349Crossref PubMed Scopus (329) Google Scholar). Originally, the different members of this family were named according to the tissue from which they were first isolated. More recently, an alternative nomenclature has been proposed, since it is well known that different FABP types can be present in the same tissue (7Hertzel A.V. Bernlohr D.A. Trends Endocrinol. Metab. 2000; 11: 175-180Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). In particular, in the liver of several vertebrates, two paralogous groups of FABPs have been described: the liver FABPs and another group that was initially called the liver "basic" FABPs and is currently referred to as the liver bile acid-binding proteins (L-BABPs) (8Ceciliani F. Monaco H.L. Ronchi S. Faotto L. Spadon P. Comp. Biochem. Physiol. Part B. 1994; 109: 261-271Crossref PubMed Scopus (46) Google Scholar, 9Santomé J.A. Di Pietro S.M. Cavagnari B.M. Córdoba O.L. Dell'Angelica E.C. Trends Comp. Biochem. Physiol. 1998; 4: 23-38Google Scholar). The reason for this unusual nomenclature was that the isoelectric point of the first protein of this group that was described, chicken liver BABP, is 9.0, and although it was evident that this protein was different from the canonical liver FABP, its specific ligand was unknown (10Scapin G. Spadon P. Pengo L. Mammi M. Zanotti G. Monaco H.L. FEBS Lett. 1988; 240: 196-200Crossref PubMed Scopus (37) Google Scholar). When it was shown that this protein could specifically bind cholic acid, a change in its designation was suggested to avoid confusion (11Nichesola D. Perduca M. Capaldi S. Carrizo M.E. Righetti P.G. Monaco H.L. Biochemistry. 2004; 43: 14072-14079Crossref PubMed Scopus (53) Google Scholar). The cytosolic bile acid-binding proteins mediate the intracellular movement of bile acids to the membranes across which they exit or enter the cells via membrane-bound transporters (12Alrefai, W. A., and Gill, R. K. (2007) Pharm. Res., in pressGoogle Scholar). The L-BABPs have been shown to be present in birds, amphibians, reptiles, and fish but not in mammals that express the very similar ileal BABPs, formerly called gastrotropins. It is suspected, although not proved, that the function that they perform in the liver of the species in which they have been found may be carried out in mammals by the other paralogous protein in the liver, L-FABP. X-ray crystallography and NMR spectroscopy have been used to experimentally determine the three-dimensional structure of many members of the FABP family both in their apo form and complexed with lipophilic ligands. All of the members of the family share the same fold, a 10-stranded β barrel in which two short helices are inserted between the first and the second strand of antiparallel β sheet. The first L-BABP three-dimensional structure determined by x-ray diffraction was that of chicken liver BABP (13Scapin G. Spadon P. Mammi M. Zanotti G. Monaco H.L. Mol. Cell. Biochem. 1990; 98: 95-99Crossref PubMed Scopus (54) Google Scholar), followed by toad (14Di Pietro S.M. Corsico B. Perduca M. Monaco H.L. Santomé J.A. Biochemistry. 2003; 42: 8192-8203Crossref PubMed Scopus (33) Google Scholar) and axolotl L-BABP (15Capaldi S. Guariento M. Perduca M. Di Pietro S.M. Santomé J.A. Monaco H.L. Proteins. 2006; 64: 79-88Crossref PubMed Scopus (14) Google Scholar). NMR and other biophysical techniques have also been used to study chicken L-BABP, which remains the best characterized member of the L-BABP family (16Schievano E. Quarzago D. Spadon P. Monaco H.L. Zanotti G. Peggion E. Biopolymers. 1994; 34: 879-887Crossref PubMed Scopus (24) Google Scholar, 17Beringhelli T. Goldoni L. Capaldi S. Bossi A. Perduca M. Monaco H.L. Biochemistry. 2001; 40: 12604-12611Crossref PubMed Scopus (17) Google Scholar, 18Ragona L. Catalano M. Luppi M. Cicero D. Eliseo T. Foote J. Fogolari F. Zetta L. Molinari H. J. Biol. Chem. 2006; 281: 9697-9709Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The most important result of these structural studies is that the stoichiometry of binding for cholate is of two ligands per protein molecule (11Nichesola D. Perduca M. Capaldi S. Carrizo M.E. Righetti P.G. Monaco H.L. Biochemistry. 2004; 43: 14072-14079Crossref PubMed Scopus (53) Google Scholar, 15Capaldi S. Guariento M. Perduca M. Di Pietro S.M. Santomé J.A. Monaco H.L. Proteins. 2006; 64: 79-88Crossref PubMed Scopus (14) Google Scholar), whereas that for oleate is of only one molecule per binding site (15Capaldi S. Guariento M. Perduca M. Di Pietro S.M. Santomé J.A. Monaco H.L. Proteins. 2006; 64: 79-88Crossref PubMed Scopus (14) Google Scholar, 17Beringhelli T. Goldoni L. Capaldi S. Bossi A. Perduca M. Monaco H.L. Biochemistry. 2001; 40: 12604-12611Crossref PubMed Scopus (17) Google Scholar). It is an unusual characteristic of the paralogous L-FABPs that they bind two molecules of oleate in their binding sites (19Thompson J. Winter N. Terwey D. Bratt J. Banaszak L. J. Biol. Chem. 1997; 272: 7140-7150Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 20Thompson J. Reese-Wagoner A. Banaszak L. Biochim. Biophys. Acta. 1999; 1441: 117-130Crossref PubMed Scopus (51) Google Scholar). Another important aspect described is the conformational changes that take place upon ligand binding that have been studied in chicken L-BABP by both x-ray diffraction (11Nichesola D. Perduca M. Capaldi S. Carrizo M.E. Righetti P.G. Monaco H.L. Biochemistry. 2004; 43: 14072-14079Crossref PubMed Scopus (53) Google Scholar) and NMR spectroscopy that has highlighted the important role of a buried histidine (18Ragona L. Catalano M. Luppi M. Cicero D. Eliseo T. Foote J. Fogolari F. Zetta L. Molinari H. J. Biol. Chem. 2006; 281: 9697-9709Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Although L-BABPs have been studied in catfish (Rhandia sapo) (21Di Pietro S.M. Dell'Angelica E.C. Veerkamp J.H. Sterin-Speziale N. Santomé J.A. Eur. J. Biochem. 1997; 249: 510-517Crossref PubMed Scopus (33) Google Scholar), lungfish (Lepidosiren paradoxus) (22Di Pietro S.M. Santomé J.A. Arch. Biochem. Biophys. 2001; 388: 81-90Crossref PubMed Scopus (13) Google Scholar), Japanese sea perch (Lateolabrax japonicus) (23Odani S. Baba K. Tsuchida Y. Aoyagi Y. Wakui S. Takahashi Y. J. Biochem. (Tokyo). 2001; 129: 69-76Crossref PubMed Scopus (7) Google Scholar), gilthead sea bream (Sparus aurata) (24Sarropoulou E. Power D.M. Magoulas A. Geisler R. Kotoulas G. Aquaculture. 2005; 243: 69-81Crossref Google Scholar), salmon (Salmo salar L.) (25Jordal A.E. Hordvik I. Pelsers M. Bernlohr D.A. Torstensen B.E. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2006; 145: 147-158Crossref PubMed Scopus (35) Google Scholar), shark (Halaelurus bivius) (26Cordoba O.L. Sanchez E.I. Santome J.A. Eur. J. Biochem. 1999; 265: 832-838Crossref PubMed Scopus (20) Google Scholar), and zebrafish (Danio rerio) (27Denovan-Wright E.M. Pierce M. Sharma M.K. Wright J.M. Biochim. Biophys. Acta. 2000; 1492: 227-232Crossref PubMed Scopus (47) Google Scholar), there is currently no three-dimensional structure available of an L-BABP belonging to this vertebrate group. The zebrafish (Danio rerio) is a powerful animal model that is widely used to study vertebrate development. The presence in this species of the gene coding for liver BABP has been reported, and the cDNA clone coding for the protein has been isolated (27Denovan-Wright E.M. Pierce M. Sharma M.K. Wright J.M. Biochim. Biophys. Acta. 2000; 1492: 227-232Crossref PubMed Scopus (47) Google Scholar). In this paper, we report the expression, purification, crystallization, and three-dimensional structure determination of zebrafish liver BABP. We have prepared and examined co-crystals with cholate of the wild type protein and two mutants. The wild type protein and one of the mutants bind one cholate molecule per binding site, whereas the other mutant has the stoichiometry found in chicken and axolotl L-BABP of two ligands per binding site. Isothermal titration calorimetry has yielded results that are consistent with the x-ray data. Protein Expression, Purification, Complex Formation, and Crystallization—The cDNA coding for zebrafish liver BABP (IMAGE ID 5410136), obtained from RZPD (Deutsches Ressourcenzentrum fuer Genomforschung GmbH), was amplified by PCR using primers designed to introduce restriction sites for BamHI and HindIII endonucleases and a sequence coding for a digestion site for thrombin in the C-terminal end in the amplified fragment. After purification, the fragment and the expression vector pQE50 (Qiagen) were digested with the restriction enzymes mentioned above and incubated with ligase to insert the cDNA in the vector respecting the reading frame. SG13009 E. coli cells were transformed with the resulting vector and grown at 37 °C, and protein synthesis was induced overnight at 20 °C with 0.5 mm isopropyl β-d-thiogalactopyranoside. Under these conditions of subcloning in pQE50, the expressed intracellular domain is fused to a histidine tag through its C terminus. The presence of the tag allowed the affinity purification of the fused protein by passing the bacterial extracts through a nickel-Sepharose column. The column was equilibrated with 20 mm Tris-HCl, pH 7.5, 0.5 m NaCl, 10 mm imidazole, and 0.02% NaN3, and the bound protein was eluted with a linear gradient of imidazole from 10 to 500 mm. After the affinity column, the tag was removed by thrombin digestion, and the protein was further purified by gel filtration in a Super-dex G-75 column equilibrated with 20 mm Tris-HCl, pH 7.5, 0.15 m NaCl, and 0.02% NaN3. Complete removal of the tag was assessed by Western blot analysis using an anti-His-horseradish peroxidase-conjugated antibody (Sigma). The purified protein showed one band in SDS-PAGE. Five times the molar protein concentration of sodium cholate was added to the apoprotein at a concentration of about 30 mg/ml in 20 mm Tris-HCl buffer, pH 7.5, in order to prepare the complex with the ligand. The solution was stirred overnight at 20 °C and used at this concentration for the initial screen of crystallization conditions. Hampton Research Screens were used at 20 °C with the hanging drop method, mixing 1 μl of the protein solution with the same volume of the precipitating solution and equilibrating versus a volume of 0.3 ml of the latter in the reservoir. The conditions yielding very small crystals were later refined, and the sitting drop method with larger volumes was also tested until crystals that were large enough for data collection were obtained. The best crystals of the wild type z-L-BABP cholate complex grow by mixing equal volumes of the protein solution and 0.1 m sodium citrate, pH 5.6, 20% polyethylene glycol 4000, and 20% isopropyl alcohol. They are thin rods of about 2.5 mm × 30 μm × 30 μm and grow to their full size in very few days at 20 °C. The mutants were generated using the commercial QuikChange kit (Stratagene) and primers designed to introduce the proper mutations. The presence of the desired mutations was confirmed by plasmid sequencing. Protein expression and purification followed the protocol used for the wild type protein. The co-crystals of both mutants were grown using a protocol identical to that of the wild type protein. In the case of the C91T mutant, the precipitant was a solution of 0.1 m sodium acetate, pH 4.6, 0.2 m ammonium acetate, and 30% polyethylene glycol 4000. Data Collection, Structure Solution, and Refinement—The co-crystals of wild type z-L-BABP and cholate are trigonal, space group P32, with a = b = 43.4 Å and c = 67.5 Å and contain one molecule in the asymmetric unit (see Table 1). The co-crystals of the G55R mutant with cholate are isomorphous with this form, whereas those of the C91T mutant are monoclinic, space group P21, with a = 28.0 Å, b = 63.3 Å, c = 35.7 Å, and β = 105.5° (Table 1).TABLE 1Data collection and refinement statisticsData setL-BABP wild type + cholateL-BABP G55R + cholateL-BABP C91T + cholateSpace groupP32P32P21a (Å)43.3643.4828.01b (Å)43.3643.4863.28c (Å)67.5367.3435.67α90.090.090.0β90.090.0105.51γ120.0120.090.0Resolution range (Å)33.8-1.5050.0-1.8721.1-1.50Observed reflections59,90639,46569,200Independent reflections21,47111,01619,136MultiplicityaThe values in parentheses refer to the highest resolution shells. For the wild type liver BABP complexed with cholate, the highest resolution interval is 1.58-1.50 Å, for the G55R mutant it is 1.94-1.87 Å, and for the C91T mutant it is 1.58-1.50 Å. The highest resolution shells used in the refinements are: 1.54-1.50 Å for the wild type protein, 1.95-1.90 Å for the G55R mutant, and 1.54-1.50 Å for the C91T mutant. The three values listed in the first two columns as ligand atoms correspond to cholate, isopropyl alcohol, and glycerol, respectively. The average B factors are listed in the same order.2.8 (1.9)3.5 (3.3)3.6 (3.6)Rmerge (%)bRmerge = ∑h∑i|Iih - 〈Ih〉|∑h∑i 〈Ih〉, where 〈Ih〉 is the mean intensity of the i observations of reflection h.7.2 (8.3)7.2 (32.7)6.5 (13.3)I/∑13.8 (6.3)6.6 (1.7)16.8 (8.2)Completeness (%)94.3 (72.6)98.3 (92.1)99.4 (100.0)Reflections in refinement20,3389,96418,134Rcryst (%)cRcryst = ∑||Fo| - |Fc||/∑|Fo|, where |Fo| and |Fc| are the observed and calculated structure factor amplitudes, respectively. Summation includes all reflections used in the refinement.18.8 (18.3)20.7 (30.1)22.0 (24.0)Rfree (%) (test set 5%)dRfree = ∑||Fo| - |Fc||/∑|Fo|, evaluated for a randomly chosen subset of 5% of the diffraction data not included in the refinement.20.9 (25.3)24.5 (39.1)24.9 (33.4)Protein atoms9729791,001Ligand atoms29 + 12 + 629 + 12 + 658Water molecules135100128r.m.s. deviation on bond lengths (Å)eRoot mean square deviation from ideal values.0.0060.0090.008r.m.s. deviation on bond angles (degrees)1.1831.2801.217Planar groups (Å)0.0030.0040.003Chiral volume dev. (Å3)0.0730.0800.076Average B factor (Å2)10.217.612.1Protein atoms8.417.011.2Ligand atoms6.1 − 9.1 − 12.113.4 − 15.9 − 19.86.4Solvent atoms20.624.119.4a The values in parentheses refer to the highest resolution shells. For the wild type liver BABP complexed with cholate, the highest resolution interval is 1.58-1.50 Å, for the G55R mutant it is 1.94-1.87 Å, and for the C91T mutant it is 1.58-1.50 Å. The highest resolution shells used in the refinements are: 1.54-1.50 Å for the wild type protein, 1.95-1.90 Å for the G55R mutant, and 1.54-1.50 Å for the C91T mutant. The three values listed in the first two columns as ligand atoms correspond to cholate, isopropyl alcohol, and glycerol, respectively. The average B factors are listed in the same order.b Rmerge = ∑h∑i|Iih - 〈Ih〉|∑h∑i 〈Ih〉, where 〈Ih〉 is the mean intensity of the i observations of reflection h.c Rcryst = ∑||Fo| - |Fc||/∑|Fo|, where |Fo| and |Fc| are the observed and calculated structure factor amplitudes, respectively. Summation includes all reflections used in the refinement.d Rfree = ∑||Fo| - |Fc||/∑|Fo|, evaluated for a randomly chosen subset of 5% of the diffraction data not included in the refinement.e Root mean square deviation from ideal values. Open table in a new tab The data for the co-crystals with cholate of the wild type and C91T mutant of the protein were collected at the ID29 beam line of the European Synchrotron Radiation Facility in Grenoble (λ = 0.98 Å) at 100 K after a brief soaking in a mixture of 80% mother liquor and 20% glycerol. The data were indexed, integrated, and reduced using the programs MOSFLM and Scala (28Leslie A.G.W. Jnt. CCP4/ESF-EACMB Newslett. Protein Crystallogr. 1992; 26: 27-33Google Scholar, 29Collaborative Computational Project Number 4Acta Crystallogr. Sect. D. 1994; 50: 760-767Crossref PubMed Scopus (19668) Google Scholar). The data for the co-crystals of the G55R mutant were collected on the home source at 100 K after a brief soaking in a mixture of 80% mother liquor and 20% glycerol. The detector was a MarResearch imaging plate mounted on a Rigaku RU-300 rotating anode x-ray generator. The source was operated at 50 kV and 100 mA, and monochromatic copper Kα radiation was obtained using Xenocs mirrors. The data were indexed, integrated, and reduced using the program AUTOMAR. The diffraction data statistics are summarized in Table 1. The structure of the complex wild type zebrafish L-BABP-cholate was solved using the CCP4 suite of programs for crystallographic computing. The initial phases were calculated by the molecular replacement method as implemented in the program AMoRe (30Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5026) Google Scholar), with the coordinates of chicken L-BABP (Protein data bank accession code 1TW4) as the search probe. When the rotation function was calculated with the data in the 8.0-3.0 Å resolution range, the two highest peaks had correlation coefficients of 20.2 and 11.5. The translation function, calculated for the first peak, gave an unambiguous and convincing answer with a correlation coefficient of 26.3 and an R factor of 54.0% and, after rigid body refinement, with a correlation coefficient of 46.2 and an R factor of 49.4%. Using data up to 2.0 Å resolution, the model was rigid body-refined moving initially the entire molecule and, in a second stage, the elements of secondary structure using the program REFMAC (31Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13712) Google Scholar). After the proper side chains had been introduced, the model was subjected to a series of rounds of positional refinement alternated with manual model revisions with the program XtalView (32McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2016) Google Scholar) and the refinement program REFMAC. During the process of refinement and model building, the quality of the model was controlled with the program PROCHECK (33Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Solvent molecules were added to the model in the final stages of refinement according to hydrogen bond criteria and only if their B factors refined to reasonable values and if they improved the R free. A similar procedure was followed to solve the structure of the C91T mutant. In this case, the two highest peaks of the rotation function had correlation coefficients of 26.6 and 17.5, whereas the translation function of the first peak had a correlation coefficient of 34.1 and an R factor of 50.0%, which, after rigid body refinement, became 48.0 and 46.6%. Refinement of this structure was also carried out with the program REFMAC following essentially the same procedure described above. The crystals of the G55R mutant are isomorphous to the wild type protein. The model was refined using the program REFMAC. The final refinement statistics for the models of the three crystal forms are summarized in Table 1. Isothermal Titration Calorimetry (ITC)—The proteins and sodium cholate were dissolved in the following buffer: 20 mm Tris-HCl, pH 7.5, 0.15 m NaCl, and 0.02% sodium azide. The sodium cholate concentrations in the titrating solution and the initial protein concentration in the measurement cell were 0.62 and 0.026 mm, respectively, for the wild type protein, 0.90 and 0.028 mm, respectively, for the G55R mutant, and 1.0 and 0.0136 mm, respectively, for the C91T mutant. The titrations were performed at 25 °C using a CSC 4200 isothermal titration calorimeter. A total of 25 injections of 10-μl aliquots of titrating solution were added to the 1.3-ml protein solution cell. The heat of the injections was corrected for the heat of dilution of the ligand into the buffer. Several binding models were tested to interpret the calorimetric data, and the fitting functions are described in the literature (34Castronuovo G. Elia V. Fessas D. Velleca F. Viscardi G. Carbohydr. Res. 1996; 287: 127-138Crossref Scopus (29) Google Scholar, 35Wyman, J., and Gill, S. J. (1990) Binding and Linkage, pp. 49-59, University Science Books, Mill Valley, CAGoogle Scholar, 36Gill, S. J., Robert, C. H., and Wyman, J. (1988) in Biochemical Thermodynamics (Jones, M. N., ed) Elsevier Science Publishers B.V., AmsterdamGoogle Scholar). Briefly, the observable enthalpy is given by Equation 1, ΔH(T,p,μL)=−R[δlnQδ(1/T)]p,μL(Eq. 1) and the degree of association (i.e. the concentration ratio x¯=[bound ligand]/[total protein] is given by Equation 2, x¯=RT[δlnQδμL]T,P=RT[δlnQδln[L]]T,P(Eq. 2) where R is the universal gas constant, μL is the chemical potential of the free ligand, [L] is the concentration of the free ligand, and Q is the partition function of the system referred to the free protein state (35Wyman, J., and Gill, S. J. (1990) Binding and Linkage, pp. 49-59, University Science Books, Mill Valley, CAGoogle Scholar). Since we can approximate these systems as diluted solutions, the thermodynamic activities of the solutes may be replaced with their molar concentrations. Under this assumption, the partition function (Equation 3), Q=∑j=0n[Pj]/[P0](Eq. 3) is the sum of the concentrations of all protein species, Pj, referred to the free protein, P0. Q depends on the assumptions made on the association (binding) mechanism and is the key function used to simulate the enthalpy so as to check the model with the experimental data and to obtain the association (or binding) constant, Kb, and the binding enthalpy ΔHb. The binding constant is a dimensionless quantity by definition. However, in order to stress the approximation of the thermodynamic activities with the molar concentrations, the use of m-1 units for this parameter is widely diffused and will be adopted in this paper. The fit based on the binding models was accomplished using the nonlinear Levendberg-Marquardt method (37Press, W. H., Flannery, B. P., Teukolsky, S. A., and Vetterling, W. T. (1989) Numerical Recipes: The Art of Scientific Computing, pp. 521-538, Cambridge University Press, Cambridge, UKGoogle Scholar). The errors of each fitting parameter were calculated with a 95.4% confidence limit by the Monte Carlo simulation method. Structure and Ligand-binding Stoichiometry of the Wild Type Protein—The final model of the wild type co-crystals of z-L-BABP corresponds to the full-length 125-amino acid chain, 972 protein atoms, one cholate, 3 isopropyl alcohol, one glycerol, and 135 water molecules. The conventional R factor is 18.8%, and the free R factor is 20.9% (Table 1). The R factors and r.m.s. of Table 1 were calculated with the program REFMAC (31Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13712) Google Scholar). The stereochemical quality of the protein model was assessed with the program PROCHECK (33Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). 94.5% of the residues are in the most favorable region of the Ramachandran plot, and the remaining 5.5% are in the additionally allowed region. The overall fold consists of the canonical β barrel with 10 strands of anti-parallel β chain and the two α helices inserted between the first and the second strand. The final secondary structure assignments are, for the β strands, the following: strand A, residues 4-12; B, residues 37-43; C, residues 46-53; D, residues 56-63; E, residues 67-71; F, residues 77-85; G, residues 88-92; H, residues 97-103; I, residues 106-113; and J, residues 116-124. The two α helices span residues 14-20 and 25-30. Fig. 1a is a schematic diagram of the zebrafish L-BABP molecule with the experimental electron density for the single cholate ligand present in the binding site. Resolution and quality of the maps are such that this stoichiometry of binding is completely unambiguous. This result is totally unexpected, since in the other two L-BABP species of this family studied by x-ray diffraction of co-crystals, chicken (11Nichesola D. Perduca M. Capaldi S. Carrizo M.E. Righetti P.G. Monaco H.L. Biochemistry. 2004; 43: 14072-14079Crossref PubMed Scopus (53) Google Scholar) and axolotl (15Capaldi S. Guariento M. Perduca M. Di Pietro S.M. Santomé J.A. Monaco H.L. Proteins. 2006; 64: 79-88Crossref PubMed Scopus (14) Google Scholar), the stoichiometry found was unequivocally of two ligand molecules per binding site. Furthermore, although there is currently no x-ray structure available for the closely related mammalian ileal BABPs, the same stoichiometry of two ligands is supported by NMR experiments (38Tochtrop G.P. Richter K. Tang C. Toner J.J. Covey D.F. Cistola D.P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1847-1852Crossref PubMed Scopus (82) Google Scholar, 39Tochtrop G.P. Bruns J.L. Tang C. Covey D.F. Cistola D.P. Biochemistry. 2003; 42: 11561-11567Crossref PubMed Scopus (37) Google Scholar). Fig. 1b represents schematically hydrogen bonds and hydrophobic interactions between the protein and the ligand. Using the program LSQKAB (40Kabsch W. Acta Crystallogr. Sect. A. 1978; 32: 922-923Crossref Scopus (2274) Google Scholar), the model of zebrafish L-BABP was superimposed to that of chicken L-BABP (11Nichesola D. Perduca M. Capaldi S. Carrizo M.E. Righetti P.G. Monaco H.L. Biochemistry. 2004; 43: 14072-14079Crossref PubMed Scopus (53) Google Scholar) (Protein Data Bank entry 1TW4). A stereo diagram of the two models with the ligands is represented in Fig. 1c. Interestingly, the single cholate molecule present in the zebrafish L-BABP binding site superimposes very well with one of the two molecules bound in the chicken liver protein, the one whose carboxylate is closer to the end of the second of the t
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