β-Lactoglobulin Binds Palmitate within Its Central Cavity

Abstract

Bovine β-lactoglobulin (β-Lg) has been studied extensively in both the isolated and the naturally occurring states. It is a commercially important whey protein of obvious nutritional value but, so far, one that has no clearly identified biological function. In common with many of the other members of the lipocalin family to which it belongs, β-Lg binds hydrophobic ligands, and it appears possible that there are at least two distinct binding sites per monomer for a variety of ligands. By comparison with other members of the family, there is a probable binding site in the central cavity of the molecule that is formed by the eight antiparallel β-strands that are typical of the lipocalins. We have now cocrystallized β-Lg with palmitic acid, and the refined structure (R = 0.204, R free = 0.240 for 6,888 reflections to 2.5-Å resolution) reveals that the ligand binds in the central cavity in a manner similar to the binding of retinol to the related lipocalin, serum retinol-binding protein. The carboxyl group binds to both Lys-60 and Lys-69 at the entrance to the cavity. The hydrophobic tail stretches in an almost fully extended conformation into the center of the protein. This is the first direct observation of a ligand binding to β-Lg. Bovine β-lactoglobulin (β-Lg) has been studied extensively in both the isolated and the naturally occurring states. It is a commercially important whey protein of obvious nutritional value but, so far, one that has no clearly identified biological function. In common with many of the other members of the lipocalin family to which it belongs, β-Lg binds hydrophobic ligands, and it appears possible that there are at least two distinct binding sites per monomer for a variety of ligands. By comparison with other members of the family, there is a probable binding site in the central cavity of the molecule that is formed by the eight antiparallel β-strands that are typical of the lipocalins. We have now cocrystallized β-Lg with palmitic acid, and the refined structure (R = 0.204, R free = 0.240 for 6,888 reflections to 2.5-Å resolution) reveals that the ligand binds in the central cavity in a manner similar to the binding of retinol to the related lipocalin, serum retinol-binding protein. The carboxyl group binds to both Lys-60 and Lys-69 at the entrance to the cavity. The hydrophobic tail stretches in an almost fully extended conformation into the center of the protein. This is the first direct observation of a ligand binding to β-Lg. β-lactoglobulin. The lipocalin family is a large and diverse family of proteins with functions varying from insect camouflage to small hydrophobic molecule transport typified by the serum retinol-binding protein (1Flower D.R. Biochem. J. 1996; 318: 1-14Crossref PubMed Scopus (1389) Google Scholar). The crystal structures so far determined reveal the typical lipocalin to be an eight-stranded antiparallel β-barrel arranged to form a conical central calyx or cavity in which the hydrophobic ligand is located (2Newcomer M.E. Jones T.A. Aqvist J. Sundelin J. Erikkson U. Rask L. Peterson P.A. EMBO J. 1984; 3: 1451-1454Crossref PubMed Scopus (336) Google Scholar). There is an α-helix on the outer surface of the β-barrel, and the amino acid sequence contains three structurally conserved regions (sequence motifs) together with one or more disulfide bridges (see Fig. 1). The calycin superfamily (1Flower D.R. Biochem. J. 1996; 318: 1-14Crossref PubMed Scopus (1389) Google Scholar) extends the family to include proteins such as the fatty acid-binding proteins, which do not contain the second of these three sequence patterns but which are also antiparallel β-sheet proteins, although with 10 β-strands rather than 8. However, in common with their distant lipocalin relatives, their binding site is also within a central cavity (3Banaszak L. Winter N. Xu Z.H. Bernlohr D.A. Cowan S. Jones T.A. Adv. Protein Chem. 1994; 45: 89-151Crossref PubMed Google Scholar). Not only is β-lactoglobulin (β-Lg),1 from the milk of the domestic cow, a typical lipocalin, but it has also been studied extensively over the past 60 years (see Refs. 4Tilley J.M.A. Dairy Sci. Abstr. 1960; 22: 111-125Google Scholar, 5Lyster R.L.J. J. Dairy Res. 1972; 39: 279-318Crossref PubMed Scopus (74) Google Scholar, 6Hambling S.G. McAlpine A.S. Sawyer L. Fox P.F. Advanced Dairy Chemistry, I: Protein Chemistry. Elsevier, Amsterdam1992: 141-190Google Scholar) because of its size, convenience (∼3 g/liter of milk), and stability. The ruminant protein is normally a dimer at room temperature and physiological pH, although at pH values below 3 and above 8, it dissociates into monomers, for which the structure at low pH is similar to that at pH 7 (7Ragona L. Pusteria F. Zetta L. Monaco H.L. Molinari H. Fold. Des. 1997; 2: 281-290Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). A further reason for studying β-Lg is its involvement in the loss of functionality during the heat processing of milk (8Fox P.F. Heat Induced Changes in Milk. 2nd Ed. International Dairy Federation, Brussels, Belgium1995Google Scholar). Thus, many studies on the pure protein are being directed toward unraveling the molecular mechanisms that are responsible for its thermal denaturation because this denaturation is thought to initiate the wider aggregation of milk proteins during thermal processing (9Roefs S.P.F.M. De Kruif K.G. Eur. J. Biochem. 1994; 226: 883-889Crossref PubMed Scopus (390) Google Scholar, 10Noh B. Richardson T. Creamer L.K. J. Food Res. 1989; 54: 889-893Crossref Scopus (45) Google Scholar, 11Iametti S. De Gregori B. Vecchio G. Bonomi F. Eur. J. Biochem. 1996; 237: 106-112Crossref PubMed Scopus (238) Google Scholar, 12Gezimati J. Creamer L.K. Singh H. J. Agric. Food Chem. 1997; 45: 1130-1136Crossref Scopus (114) Google Scholar). The initial stage of the thermal denaturation process also involves dimer dissociation (12Gezimati J. Creamer L.K. Singh H. J. Agric. Food Chem. 1997; 45: 1130-1136Crossref Scopus (114) Google Scholar, 13Qi X.L. Holt C. McNulty D. Clarke D.T. Brownlow S. Jones G.R. Biochem. J. 1997; 324: 341-346Crossref PubMed Scopus (298) Google Scholar). The biological function of β-Lg is unknown. The amino acid composition and the quantities present in ruminant species support a nutritional role. However, the ligand binding properties that have emerged, coupled with the structural similarity of β-Lg to retinol-binding protein and possibly even to fatty acid-binding protein, have led to the suggestion that it has a transport role for ligands such as retinol or fatty acids (1Flower D.R. Biochem. J. 1996; 318: 1-14Crossref PubMed Scopus (1389) Google Scholar, 2Newcomer M.E. Jones T.A. Aqvist J. Sundelin J. Erikkson U. Rask L. Peterson P.A. EMBO J. 1984; 3: 1451-1454Crossref PubMed Scopus (336) Google Scholar, 14Perez M.D. Calvo M. J. Dairy Sci. 1995; 78: 978-988Abstract Full Text PDF PubMed Scopus (220) Google Scholar, 15Rocha T.L. Brownlow S. Saddler K.N. Fothergill-Gilmore L.A. Sawyer L. J. Dairy Res. 1996; 63: 575-584Crossref Google Scholar, 16Brownlow S. Morais Cabral J.H. Cooper R. Flower D.R. Yewdall S.J. Polikarpov I. North A.C.T. Sawyer L. Structure. 1997; 5: 481-495Abstract Full Text Full Text PDF PubMed Scopus (652) Google Scholar). The majority of the ligands that have been examined are hydrophobic, so that a transport role, in keeping with the other lipocalins, is certainly possible, although no real proof of such a role has been published. Fatty acids have been found bound to ruminant β-Lg that has been freshly isolated from milk, and Perez et al. (17Perez M.D. Sanchez L. Aranda P. Ena J.M Oria R. Calvo M. Biochim. Biophys. Acta. 1991; 1123: 151-155Crossref Scopus (79) Google Scholar) have suggested that by removing free fatty acids as they are formed by pregastric lipases, β-Lg could facilitate the digestion of milk fat. If such a role is the true function of β-Lg, it is interesting that ruminant, but not mare and sow, β-Lgs exhibit significant fatty acid binding (18Puyol P. Perez M.D. Ena J.M. Calvo M. Agric. Biol. Chem. 1991; 55: 2515-2520Crossref Scopus (2) Google Scholar), whereas one might expect all β-Lgs to perform this function. On the other hand, β-Lg from all species appears to bind retinol, but this ligand is found associated not only with β-Lg but also with other milk proteins such as serum albumin and α-lactalbumin (18Puyol P. Perez M.D. Ena J.M. Calvo M. Agric. Biol. Chem. 1991; 55: 2515-2520Crossref Scopus (2) Google Scholar), indicating that this interaction seems to be rather unspecific. Thus, the functional relevance of retinol binding to β-Lg is also open to question. As part of our continuing study of the relationship between the structure of the protein and the thermally induced aggregation, we sought convincing evidence of the binding of hydrophobic ligands to the molecule. Monaco et al. (19Monaco H.L. Zanotti G. Spadon P. Bolognesi M. Sawyer L. Eliopoulos E.E. J. Mol. Biol. 1987; 197: 695-706Crossref PubMed Scopus (333) Google Scholar) reported a possible binding site for retinol on the outer surface of the molecule in a groove formed between the helix and the β-sheet. This result, which has been questioned (16Brownlow S. Morais Cabral J.H. Cooper R. Flower D.R. Yewdall S.J. Polikarpov I. North A.C.T. Sawyer L. Structure. 1997; 5: 481-495Abstract Full Text Full Text PDF PubMed Scopus (652) Google Scholar, 20Narayan M. Berliner L.J. Biochemistry. 1997; 37: 1906-1911Crossref Scopus (107) Google Scholar, 21Sawyer L. Brownlow S. Polikarpov I. Wu S.-Y. Int. Dairy J. 1998; 8: 65-72Crossref Scopus (122) Google Scholar), is based upon an unrefined difference electron density map. Apart from the location by analogy and modeling ((21, 22), some experimental evidence for an internal binding site comes from the fluorescence and site-directed mutagenesis work of Choet al. (23Cho Y. Batt C.A. Sawyer L. J. Biol. Chem. 1994; 269: 11102-11107Abstract Full Text PDF PubMed Google Scholar). The existence of two independent ligand binding sites as proposed by Narayan and Berliner (20Narayan M. Berliner L.J. Biochemistry. 1997; 37: 1906-1911Crossref Scopus (107) Google Scholar) adds further support to the possible existence of a central site (see Fig.1). However, to provide unequivocal evidence, we have now refined the crystal structure of β-Lg co-crystallized with palmitate from sodium citrate solution at pH 7.5 and observed the binding of the ligand in the central calyx. The crystalline complex was prepared in two distinct ways. In the first method, bovine β-Lg (B variant, Sigma) was dissolved in H2O to a concentration of 40 mg/ml and crystallized at 20 °C by the sitting drop method (24Ducruix A. Giegé R. Crystallisation of Nucleic Acids and Proteins. IRL Press, Oxford1992: 73-98Google Scholar) using 1.34 msodium citrate, 0.1 m HEPES, pH 7.5, as the precipitant. Typically, a microbridge was placed in 1.0 ml of precipitant solution (the reservoir) in a 24-well Linbro tissue culture plate. For a sitting drop, 4 μl of β-Lg solution was added to 12 μl of reservoir solution. Then, 0.4 μl of 100 mm palmitic acid in ethanol was added to the drop (a molar ratio of 10/protein dimer) and mixed by pipette before the well was sealed with a glass coverslip. Because the palmitic acid was supersaturated with respect to the aqueous phase, a white precipitate appeared in the drop. After ∼4–5 days, the white precipitate had disappeared, and lattice Z crystals (space group P3221) grew from the clear drops. The pH of the crystals was assumed to be 7.5, although it was not directly measured. In the second method, bovine β-Lg of mixed genetic variants A and B was prepared according to Puyol et al. (25Puyol P. Perez M.D. Calvo M. J. Dairy Sci. 1994; 77: 1494-1502Abstract Full Text PDF Scopus (59) Google Scholar) and delipidated by charcoal treatment at pH 3 as described by Chen (26Chen R.F. J. Biol. Chem. 1967; 242: 173-181Abstract Full Text PDF PubMed Google Scholar). Palmitic acid at a molar concentration ratio of 2:1 with respect to the protein dimer was dissolved in chloroform and dispensed in a glass tube. After the organic solvent was evaporated under nitrogen, a solution of delipidated β-Lg was dissolved in 0.29 m NaCl, 2.5 mm KH2PO4, 16 mmK2HPO4, pH 7.4, and the mixture was incubated overnight at 37 °C. The saturated protein solution was then dialyzed against distilled water and freeze-dried. Analysis by gas chromatography showed that the complex had about 1 mol of palmitate bound to 1 mol of dimeric protein. The freeze-dried material was dissolved in H2O to a concentration of 40 mg/ml and crystallized as a sitting drop (4 μl of protein solution + 12 μl of well solution) over a well solution of 1.4 m sodium citrate, 0.1 m HEPES, pH 7.5. Crystals, also of the trigonal lattice Z form, grew in a few days and appeared identical to the crystals from the first preparation (see TableI).Table IData collection statisticsData collection statisticsFirst preparationSecond preparationSpace group, P3221a =b = 53.48 Åa = b = 53.52 Åc = 111.64 Åc = 111.37 Åα = β = 90°, γ = 120°α = β = 90°, γ = 120°Resolution range20–2.5 Å25–2.3 ÅNumber of reflections measured97,14248,843Number of unique reflections6,8888,763Completeness99.9%97.8%Multiplicity14.15.57R merge (outer shell)aR merge = Σh‖ I − <I> ‖/Σh‖ I‖, where <I> is the mean intensity of all observation of reflection. h =hkl.7.3% (36.6%)6.3% (37.6%)I/ς (I) (outer shell)bς (I) is the S.D. of the measured intensity.21.2 (3.95)17.38 (2.57)a R merge = Σh‖ I − <I> ‖/Σh‖ I‖, where <I> is the mean intensity of all observation of reflection. h =hkl.b ς (I) is the S.D. of the measured intensity. Open table in a new tab A crystal of about 0.3–0.4 mm in length was collected in a 0.5-mm Cryoloop (Hampton Research, Inc.), dipped briefly in immersion oil (Type B, Cargille), and frozen by plunging into liquid N2. The frozen crystal was then transferred to a magnetic goniometer head in a stream of N2 at 100 K (Cryostream; Oxford Cryosystems). Diffraction data were collected on a 300 mm MarResearch imaging plate system mounted upon an ENRAF-Nonius FR571 rotating anode generator operating at 40 kV and 80 mA and producing Cu-Kα radiation from a graphite crystal monochromator. At least 90° of data were collected in 1.5° oscillations (i.e.>60 images), each of a 20-min duration. The data were processed by DENZO (27Otwinowski Z. Minor 1997; PubMed Scopus Google Scholar) and with (27Otwinowski Z. Minor 1997; PubMed Scopus Google Scholar). The are in I. The structure was by molecular using J. P. 1997; PubMed Scopus Google Scholar) with the refined β-Lg lattice monomer (space group a = b = = = β = γ as the within the of and a of were to the and The in the function and were to the which a distinct at a of ς for the whereas the was The second not a distinct solution in the function. The group was also by the function. The in was ς = whereas the in was ς = of the solution the from to and temperature was using 1997; PubMed Scopus Google Scholar). The was with of with the T.A. Cowan M. A. 1991; PubMed Scopus Google Scholar). were added using the 1997; PubMed Scopus Google Scholar). The are in of of statisticsFirst preparationSecond free mean from in and regions of Open table in a new tab A monomer of the crystal structure of β-Lg refined at in lattice (16Brownlow S. Morais Cabral J.H. Cooper R. Flower D.R. Yewdall S.J. Polikarpov I. North A.C.T. Sawyer L. Structure. 1997; 5: 481-495Abstract Full Text Full Text PDF PubMed Scopus (652) Google Scholar) was as the in the structure of the lattice Z crystal form the the lattice Z structure (16Brownlow S. Morais Cabral J.H. Cooper R. Flower D.R. Yewdall S.J. Polikarpov I. North A.C.T. Sawyer L. Structure. 1997; 5: 481-495Abstract Full Text Full Text PDF PubMed Scopus (652) Google Scholar) could have been the lattice structure was as the and Creamer L.K. Biochemistry. 1998; 37: PubMed Scopus Google Scholar) have refined the structure of the lattice Z form at three distinct pH values and that there is a distinct of a as the pH value is This a carboxyl group, observed during by et al. C. Y. J. Chem. Scopus Google Scholar), identified as by Brownlow et al. (16Brownlow S. Morais Cabral J.H. Cooper R. Flower D.R. Yewdall S.J. Polikarpov I. North A.C.T. Sawyer L. Structure. 1997; 5: 481-495Abstract Full Text Full Text PDF PubMed Scopus (652) Google Scholar) but by et al. Creamer L.K. Biochemistry. 1998; 37: PubMed Scopus Google Scholar). is part of the for the binding of palmitate in that it is the of the as the pH is from pH to 7.5 that the entrance to the calyx A and The or at least the binding site at low pH provide an for the of our with the lattice crystal form at pH We also that appears to the lattice Z crystal In a palmitate only crystals of the lattice grew at pH 7.5, whereas in with palmitate added also at pH 7.5, we this form but only lattice Z. Fig. the electron density with the refined structure The was 0.204, and R free was 0.240 for the 6,888 unique reflections to 2.5-Å The is and the that all are in the with the a conformation common to all of the lipocalin The density for the palmitate is clear and a at associated with the of the of to the the associated with the (16Brownlow S. Morais Cabral J.H. Cooper R. Flower D.R. Yewdall S.J. Polikarpov I. North A.C.T. Sawyer L. Structure. 1997; 5: 481-495Abstract Full Text Full Text PDF PubMed Scopus (652) Google Scholar, Creamer L.K. Biochemistry. 1998; 37: PubMed Scopus Google Scholar), is also The of the palmitate within the is in which the between and the fatty The binding site in β-Lg is rather fully but there is for fatty acid such as and to be within the with the carboxyl group the with Lys-60 and The for both acids are similar M.D. Sanchez L. Aranda P. Ena J.M. Calvo M. J. Biochem. 1989; PubMed Scopus Google Scholar, D. T. J. Protein Chem. PubMed Scopus Google Scholar). In the fatty acid-binding protein family, palmitate is also observed in an extended form, although of bound fatty acids have been observed G. D. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M.E. Biochemistry. 1997; PubMed Scopus Google Scholar). As of the fluorescence be to binding of hydrophobic ligands to β-Lg, the to the two were determined The of is from and is from It is possible, that there is of from both although the at is in a more comparison of the of the palmitate well within the calyx to that they are similar in to of the the of the calyx in the of the cavity and of the binding in the of the and The electron density is not well for of these Fig. the electron density for the second data where the of the binding of palmitate was measured and to be one palmitate per The for the structure in this was and R for the unique reflections to The is with the in the It is clear that there is a in the density associated with the of The for the palmitate with the second data were than of the but a for all ligand was in the similar to of the In the lattice Z crystal form, the two binding sites in the dimer are If the was one ligand per the be on each site contains a palmitate molecule. The refined was This is than but because of the of the and the that between and the a with the solution For an identical of the of palmitate in the first data a value of The in the electron density at is as the of two for the that of the structure and that with bound ligand It is not clear the preparation and the second one have such a palmitate to β-Lg dimer There is no obvious between the in which the binding some are from a from the dimer At this be about the existence of a second binding We were to the palmitate in the because the of Narayan and Berliner (20Narayan M. Berliner L.J. Biochemistry. 1997; 37: 1906-1911Crossref Scopus (107) Google Scholar) fatty acid binding at a site that is not by retinol, and the of et al. (23Cho Y. Batt C.A. Sawyer L. J. Biol. Chem. 1994; 269: 11102-11107Abstract Full Text PDF PubMed Google Scholar) Lys-69 with retinol to the of Narayan and Berliner (20Narayan M. Berliner L.J. Biochemistry. 1997; 37: 1906-1911Crossref Scopus (107) Google Scholar), al. (18Puyol P. Perez M.D. Ena J.M. Calvo M. Agric. Biol. Chem. 1991; 55: 2515-2520Crossref Scopus (2) Google Scholar) that palmitate and retinol on binding to β-Lg, the the Thus, the groove identified by Monaco et al. (19Monaco H.L. Zanotti G. Spadon P. Bolognesi M. Sawyer L. Eliopoulos E.E. J. Mol. Biol. 1987; 197: 695-706Crossref PubMed Scopus (333) Google Scholar) on the outer surface of the protein (see Fig. has to be as a binding site for of evidence that to its existence (see 21Sawyer L. Brownlow S. Polikarpov I. Wu S.-Y. Int. Dairy J. 1998; 8: 65-72Crossref Scopus (122) Google Scholar). of the reported ligand binding studies have been at pH values at or above the pH at which the binding site is to be more It is to that the site at pH, whereas at pH only the outer site is This of the binding site is of the in fatty acid-binding protein, where ligand and binding are by regions of the protein M.E. Biochemistry. 1997; PubMed Scopus Google and in the trigonal lattice Z form β-Lg, of the in the lattice and the lattice these have electron this binding studies to be at pH both in the crystal structure and in solution. We and for in this was an independent study on acid binding to the lattice Z form of bovine β-Lg has been by et B. L. N. and G. B. are in with the with Lys-60 and and the group is at the of the calyx. The for the co-crystallized complex have been in the Protein with the