From the Structural Biochemistry Group, University of
Edinburgh, Swann Building, King's Buildings, Mayfield Road, Edinburgh
EH9 3JR, Scotland and § Tecnologia se los Alimentos,
Veterinary Faculty, University of Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain
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ABSTRACT |
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Bovine 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 (1).
The crystal structures so far determined reveal the typical lipocalin
to be an eight-stranded antiparallel Not only is The biological function of 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. (19) reported a possible binding site for retinol on the outer surface of the molecule in a groove formed between the helix and the The crystalline complex was prepared in two distinct ways. In
the first method, bovine -lactoglobulin (
-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,
-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
-strands that are typical of the lipocalins. We have now
cocrystallized
-Lg with palmitic acid, and the refined structure
(R = 0.204, Rfree = 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
-Lg.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
-barrel arranged to form a
conical central calyx or cavity in which the hydrophobic ligand is
located (2). There is an
-helix on the outer surface of the
-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 (1) 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
-sheet proteins, although with 10
-strands rather
than 8. However, in common with their distant lipocalin relatives,
their binding site is also within a central cavity (3).
-lactoglobulin
(
-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. 4-6) 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 (7). A further reason for studying
-Lg is its
involvement in the loss of functionality during the heat processing of
milk (8). 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 (9-12). The initial stage of the thermal denaturation process also
involves dimer dissociation (12, 13).
-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
-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 (1, 2, 14-16). 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
-Lg that has been
freshly isolated from milk, and Perez et al. (17) have
suggested that by removing free fatty acids as they are formed by
pregastric lipases,
-Lg could facilitate the digestion of milk fat.
If such a role is the true function of
-Lg, it is interesting that
ruminant, but not mare and sow,
-Lgs exhibit significant fatty acid
binding (18), whereas one might expect all
-Lgs to perform this
function. On the other hand,
-Lg from all species appears to bind
retinol, but this ligand is found associated not only with
-Lg but
also with other milk proteins such as serum albumin and
-lactalbumin
(18), indicating that this interaction seems to be rather unspecific.
Thus, the functional relevance of retinol binding to
-Lg is also
open to question.
-sheet. This result, which has been
questioned (16, 20, 21), 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 Cho
et al. (23). The existence of two independent ligand binding
sites as proposed by Narayan and Berliner (20) 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
-Lg
co-crystallized with palmitate from sodium citrate solution at pH
7.5 and observed the binding of the ligand in the central calyx.
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Fig. 1.
A general view of -lactoglobulin, a
typical lipocalin, prepared by the program MOLSCRIPT (37). The
binding site (filled atoms) is shown in the central calyx,
and the putative binding site (open atoms) is indicated on
the outer surface of the protein. The structurally conserved regions
are at the rear of the molecule on strand A, the FG
loop, and the loop before the
-helix.
EXPERIMENTAL PROCEDURES
-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 (24) using 1.34 M
sodium 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
-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
-Lg of mixed genetic variants A and B was
prepared according to Puyol et al. (25) and delipidated by
charcoal treatment at pH 3 as described by Chen (26). 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
-Lg was dissolved in 0.29 M NaCl, 2.5 mM KH2PO4, 16 mM
K2HPO4, 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 Table
I).
Data collection statistics
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-K 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 (27) and reduced with SCALEPACK (27). The statistics are given in Table I. The structure was solved by molecular replacement using AMORE (28) with the refined -Lg lattice X monomer (space group
P1: a = 37.8 Å, b = 49.6 Å,
c = 56.6 Å,
= 123.4°,
= 97.3°,
=103.7°) as the search model. Data within the resolution range of
10-4 Å and a Patterson radius of 18 Å were used to calculate the
rotation and translation functions. The maximum peak (4.86
) in the
rotation function and next highest peak (3.70
) were used to
calculate the translation function, which gave a distinct peak at a
height of 9
for the maximum rotation peak, whereas the next highest
peak was 4.5
. The second rotation peak did not give a distinct
solution in the translation function. The space group P3221
was also confirmed by the translation function. The highest peak in
P3221 was 9
(R-factor = 38.9%),
whereas the highest peak in P3121 was 5.2
(R-factor = 53.1%). Rigid body refinement of the
correct solution reduced the R-factor slightly from 38.9 to
38.0%. Positional, occupancy, and temperature factor refinement was
performed using SHELX97 (29). The refinement calculation was
interleaved with several rounds of model-building with the program O
(30). Water molecules were added using the program SHELXWAT (29). The
final refinement statistics are summarized in Table
II.
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RESULTS AND DISCUSSION |
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A monomer of the crystal structure of -Lg refined at 1.8-Å
resolution in triclinic lattice X (16) was used as the search model in
the structure determination of the lattice Z crystal form containing
the palmitate. Although the 3.0-Å lattice Z structure (16) could have
been used, the higher resolution lattice X structure was preferred as
the better starting model. Baker and co-workers (31) have refined the
structure of the lattice Z form at three distinct pH values (pH 6.2, 7.1, and 8.2) showing that there is a distinct movement of a loop as
the pH value is raised. This movement uncovers a buried carboxyl group,
observed during titration by Tanford et al. (32), probably
identified as Glu-89 by Brownlow et al. (16) but confirmed
convincingly by Qin et al. (31). Glu-89 is part of the EF
loop whose movement allows access for the binding of palmitate in that
it is the movement of the loop as the pH is raised from pH 6 to 7.5 that opens up the entrance to the calyx (Fig.
2, A and B). The
occlusion of, or at least hindrance to, the binding site at low pH may
provide an explanation for the failure of our soaking experiments with
the lattice X crystal form at pH 6.5. We also noted that
cocrystallization appears to favor the lattice Z crystal form. In a
control experiment without palmitate added, only crystals of the
orthorhombic lattice Y grew at pH 7.5, whereas in drops with palmitate
added also at pH 7.5, we never obtained this form but only obtained
lattice Z.
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Fig. 2C shows the electron density map with the final
refined structure superimposed. The final R-factor was
0.204, and Rfree was 0.240 for the 6,888 unique
reflections to 2.5-Å resolution. The geometry is acceptable, and the
Ramachandran plot shows that essentially all residues are in the
allowed regions, with Tyr-99 the notable exception, adopting a classic
-turn conformation common to nearly all of the lipocalin structures.
The density for the palmitate is clear and shows a kink at C-6
associated with the movement of the side chain of Met-107. To allow
access to the calyx, the EF loop, associated with the Tanford
transition (16, 31), is also repositioned. The local environment of the palmitate within the pocket is shown in Fig.
3, which indicates the distances between
side chains and the fatty acid. The binding site in
-Lg is rather
fully extended, but there is space for longer fatty acid molecules such
as stearate and oleate to be accommodated within the calyx, with the
carboxyl group making the same interactions with Lys-60 and Lys-69. The
association constants for both acids are similar (33, 34). In the fatty acid-binding protein family, palmitate is also observed in an extended
form, although alternative conformations of bound fatty acids have been
observed (35, 36).
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As perturbation of the Trp fluorescence can be used to monitor binding
of hydrophobic ligands to -Lg, the distances to the two Trp residues
were determined directly. The closest approach of C-2 is 10.36 Å from
Trp-61, and C-15 is 6.98 Å from Trp-19. It is possible, therefore,
that there is perturbation of signals from both Trp residues, although
the Trp at position 19 is in a significantly more restricted environment.
Careful comparison of the B-factors of the palmitate carbons well within the calyx (C-8 to C-16) shows that they are similar in magnitude to those of the adjacent side chains. Those nearer the opening of the calyx increase in magnitude, presumably reflecting the larger volume of the cavity and hence less specific nature of the binding in the region of the highly flexible EF and GH loops. The electron density is not well defined for either of these loops.
Fig. 2D shows the electron density for the second data set,
where the stoichiometry of the binding of palmitate was measured and
shown to be one palmitate per dimer. The final R-factor for the structure in this case was 0.233, and Rfree
was 0.285 for the 8,763 unique reflections to 2.3-Å resolution. The
geometry is again acceptable, with Tyr-99 again the exception in the
Ramachandran plot. It is clear that there is a bulge in the density
associated with the position of Met-107. The B-factors for the same
palmitate carbon atoms after refinement with the second data set were
significantly larger than those of the adjacent side chains, but when a
single occupancy term for all ligand atoms was used in refinement, the B-value became similar to those of the surrounding side chains. In the
lattice Z crystal form, the two binding sites in the dimer are
crystallographically identical. If the stoichiometry was one ligand per
dimer, the expected occupancy would be 0.5 because, on average, each
site contains half a palmitate molecule. The refined occupancy was
0.69. This is higher than 0.5, but because of the imprecise nature of
the occupancy determination and the high correlation that exists
between occupancy and B-factor, the result indicates a reasonable
agreement with the solution study. For comparison, an identical
calculation of the occupancy of palmitate in the first data set gave a
value of 0.96. The bulge in the electron density at Met-107 is thus
explained as the addition of two components for the Met-107 side chain,
that of the native structure (Fig. 2A) and that with bound
ligand (Fig. 2B). It is not clear why the preparation
protocols used should produce different results and why the second one
should have such a reproducibly precise palmitate to -Lg dimer
ratio. There is no obvious cooperativity between the monomers in which
the binding sites, some 35 Å apart, are approached from a direction
away from the dimer interface.
At this stage, little can be said about the existence of a second
binding site. We were surprised to find the palmitate in the calyx,
because the report of Narayan and Berliner (20) establishes fatty acid
binding at a site that is not perturbed by retinol, and the report of
Cho et al. (23) connects Lys-69 with retinol binding.
Further, contrary to the findings of Narayan and Berliner (20), Puyol
et al. (18) find that palmitate and retinol compete on
binding to -Lg, the former displacing the latter. Thus, the groove
identified by Monaco et al. (19) on the outer surface of the
protein (see Fig. 1) has yet to be
confirmed as a binding site for any ligand, despite several strands of
circumstantial evidence that point to its existence (see Ref. 21). Many
of the reported ligand binding studies have been made at pH values at
or above 7, the pH at which the inner binding site is known to be more
accessible. It is tempting to speculate that the inner site becomes
accessible at high pH, whereas at lower pH only the putative outer site
is available. This gating of the inner binding site is reminiscent of
the dynamic portal hypothesis in fatty acid-binding protein, where
ligand access and binding are mediated by flexible regions of the
protein backbone (Ref. 36 and references therein). Whereas in the
trigonal lattice Z form
-Lg, discrete arrangements of the
"portal" loop EF exist, in the triclinic lattice X and the
orthorhombic lattice Y apo forms, these loops have weak electron
density. To clarify this point, binding studies need to be performed at
lower pH values, both in the crystal structure and in solution.
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ACKNOWLEDGEMENTS |
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We thank Ted Baker, Carl Batt, Larry Berliner, Maria Bewley, Lawrie Creamer, Linda Gilmore, Tomasz Haertlé, Carl Holt, Geoff Jameson, Bin Qin, and Thales Rocha for stimulating discussions.
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Note Added in Proof |
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Since this paper was accepted, an
independent study on 12-bromododecanoic acid binding to the lattice Z
form of bovine -Lg has been published by Qin et al. (Qin,
B. Y., Creamer, L. K., Baker, E. N. and Jameson, G. B. (1998)
FEBS Let. 438, 272-278). Their findings are in
agreement with those presented here: the carboxylate associates with
Lys-60 and Lys-69, and the 12-bromo group is at the inner end of the
calyx. The coordinates for the co-crystallized complex have been
deposited in the Protein Data Bank with the accession number
1b0o.
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FOOTNOTES |
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* This work was supported by the U. K. Biotechnology and Biological Science Research Council, NATO, and the European Union.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Structural Biochemistry Group, University of Edinburgh, Swann Bldg., King's Bldgs., Mayfield Rd., Edinburgh EH9 3JR, Scotland. Tel: 44-131-650-7062; Fax: 44-131-650-7055; E-mail: L.Sawyer{at}ed.ac.uk.
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ABBREVIATIONS |
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The abbreviation used is:
-Lg,
-lactoglobulin..
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REFERENCES |
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