(Received for publication, September 25, 1996, and in revised form, December 16, 1996)
From the Department of Biochemistry, University of
Minnesota Medical School, Minneapolis, Minnesota 55455 and the
§ Department of Chemistry, St. Cloud State University of
Minnesota, St. Cloud, Minnesota 56301
The crystal structure of the recombinant form of
rat liver fatty acid-binding protein was completed to 2.3 Å and
refined to an R factor of 19.0%. The structural solution
was obtained by molecular replacement using superimposed polyalanine
coordinates of six intracellular lipid-binding proteins as a search
probe. The entire amino acid sequence of rat liver fatty acid-binding protein along with an amino-terminal formyl-methionine was modeled in
the crystal structure. In addition, the crystal was obtained in the
presence of oleic acid, and the initial electron density clearly showed
two fatty acid molecules bound within a central cavity. The
carboxylate of one fatty acid molecule interacts with arginine 122 and is shielded from free solvent. It has an overall bent conformation.
The more solvent-exposed carboxylate of the other oleate is located
near the helix-turn-helix that caps one end of the -barrel, while
the acyl chain lies in the interior. The cavity contains both polar and
nonpolar residues but also shows extensive hydrophobic character around
the nonpolar atoms of the ligands. The primary and secondary oleate
binding sites appear to be totally interdependent, mainly because
favorable hydrophobic interactions form between both aliphatic
chains.
Liver fatty acid-binding protein
(LFABP),1 is a member of the intracellular
lipid-binding protein (iLBP) family (1,2). The functions of LFABP, like
other family members, are thought to include lipid uptake, lipid
transport, regulation of lipid metabolism, and cellular protection by
maintaining the concentrations of free cytosolic fatty acids below
toxic levels (3). The iLBP family is characterized by their small size
of approximately 130 amino acids, their affinity for hydrophobic
molecules, and their tertiary structure. Both crystal and solution
structures have been determined for different iLBP family members that
primarily bind fatty acids in adipocytes (ALBP), intestine (IFABP),
heart muscle (HFABP), locust muscle (L-MFABP), myelin (P2), and
hornworm midgut (MFB2); retinol in liver (CRBPI) and intestine
(CRBPII); or retinoic acid in testis (CRABPI) and skin (CRABPII).
References to these structural data for the iLBP family are cited in
Table III. Although the homology of the amino acid sequences vary, all
iLBPs are composed of a 10-stranded, antiparallel -barrel with two
short antiparallel
-helices positioned over one end of the barrel.
Within the confines of the
-strands is a cavity that forms the lipid
binding site.
|
The various genes for iLBPs have expression levels that are related to the cell or tissue type. Several of the different genotypes have been cloned and are available in expression vectors. In some instances, measurements have been made of their expression levels in different cell types. For example, LFABP has been detected in abundance in tissue from liver, adipose deposits, myocardium, kidney, and large and small intestinal epithelia of rats (4, 5). In the small intestine, it is expressed at highest concentrations in tips of the villi in the jejunum, where it can make up to 5% of the soluble protein (5, 6). In the liver, it also comprises up to 5% of the cytoplasmic protein (7).
The gene for rat LFABP contains three introns, and the initial study was followed by the insertion of the corresponding cDNA into an expression system (8, 9). The expression system has been used to provide protein for the structural studies described in this report. Previously, introductory structural data was described for LFABP from chicken (10). Although the crystal structure of chicken liver LFABP suggested that few conformational differences existed between it and other family members, no information was given for any bound fatty acid or on the full structure with side chains (10).
Beyond its ubiquity across a variety of cell types, LFABP is unique within the iLBP family in its broad ligand binding specificity. In addition to fatty acids, their CoA esters, and lysophosphatidic acid, LFABP also binds heme, squalene, certain eicosanoids, bilirubin, and a host of other hydrophobic compounds (3, 4, 11-14). Whether LFABP binds cholesterol is uncertain. Using radiolabeled cholesterol, a Kd of 1.53 µM and a stoichiometry of 0.83 mol cholesterol/mol of protein has been reported (15). However, no cholesterol binding was detected by other workers using a similar technique (11).
Another distinguishing feature of LFABP is its ability to bind two fatty acids per protein molecule; other iLBPs bind only one. For the dissociation of oleate from rat and bovine LFABP, one site has a Kd ranging from 0.009 to 0.2 µM, and the other has a Kd of 0.06-4.0 µM (11, 16, 60). The weaker binding was determined by titration calorimetry (11, 16), while the small Kd values were measured with a fluorescent reagent, itself a fatty acid-binding protein (60). In some studies, a third molecule has even been reported to bind to LFABP (17). Considering the unusual stoichiometry of fatty acid binding, the variety of lipids that can bind to LFABP, and the greater solvent accessibility of the backbone amides (18), it has been hypothesized that the cavity size of LFABP is larger than that of other members of this family.
The multiple binding sites found in LFABP have distinct chemical shifts from the single site in IFABP according to NMR studies using carboxyl-labeled 13C-fatty acids (17). From the differences it is postulated that the fatty acids are bound to LFABP with their carboxyl termini in a more solvent-accessible conformation than in IFABP. By studying the NMR data as a function of pH, it has also been suggested that the fatty acid carboxylates have relatively normal pKa values when bound to LFABP. In IFABP and probably other iLBPs, the carboxylates of bound fatty acids have abnormal pKa values because of ion pair formation with arginine residues within the cavity. In the case of LFABP, interpretation of the pH effects are somewhat more difficult due to the fact that there are at least two binding sites (17).
There are a number of other biochemical properties that distinguish LFABP from other iLBP family members. For example, the mechanism by which LFABP binds fatty acids also differs from ALBP and HFABP (19). According to fluorescent studies, the transfer of fatty acids from LFABP to liposomes is not dependent on the liposomal concentration. This is not the case for ALBP and HFABP (19). The transfer studies suggest that while ALBP and HFABP form a transient collisional complex with membrane-like acceptors, LFABP favors monomeric soluble fatty acids during the binding reaction (19).
Titration calorimetry demonstrates that a larger proportion of LFABP's affinity for fatty acids derives from entropic contributions when compared with binding by IFABP (16). This suggests that the hydrophobic effect plays a more important role in ligand binding to LFABP than it does in IFABP and possibly other iLBPs that bind fatty acids. In all iLBP members that bind negatively charged ligands, two arginines are usually present in the binding cavity. The interaction between the internalized arginine(s) and the carboxylate of the ligand is probably a major factor in the enthalpic contribution to the binding energy. Arg122 of LFABP is homologous with one of these arginines in several other fatty acid-binding proteins. It is also protected from chemical modification by phenylglyoxal when oleic acid is bound (20). Other evidence for Arg122 involvement in ligand binding comes from site mutations where a change to a glutamine weakened LFABP's affinity for fatty acids (12, 21, 64).
In summary, LFABP is something of an iLBP family outcast. It binds more than 1 mol of ligand. The chemical nature of the ligand can vary more widely than most members of the fatty acid-binding proteins. When fatty acids or their analogues are bound, LFABP appears to have different properties from other siblings in the iLBP family. Crystallographic data would help explain some of the similarities and differences. Crystallographic studies were undertaken in 1990, but over a period of 3 years very few crystals were obtained. The initial efforts to solve the structure using molecular replacement were unsuccessful until a composite probe was used as described below.
Unless indicated, the DAUDA and
all other chemicals were purchased from Sigma and were
reagent grade or better. LFABP fractions were identified by using a
DAUDA fluorescence binding (22). LFABP concentrations were determined
by absorption at 280 nm using a molar absorption coefficient of 6400 M1 cm
1.
Rat liver LFABP was obtained from Escherichia coli using the pJBL2 expression (8). Although the expression of recombinant LFABP in E. coli K12 H1 followed that of Winter et al. (23), the purification procedures differed. A suspension of roughly 110 g of wet cells was made in buffer A, which consisted of 0.05 M Tris, 10% sucrose, 0.05% sodium azide, 1 mM EDTA, 5 mM 2-mercaptoethanol, and 1.4 ml of a protease inhibitor mixture at a pH of 7.4. The protease inhibitor mixture contained 100 mM benzamidine, 143 µM pepstatin A, and 200 µM leupeptin in isopropyl alcohol.
Cell lysis resulted from sonication for 5 min at 4-15 °C. After removal of cellular debris by centrifugation at 5,000 × g, insoluble nucleic acid and some protein were extracted at 25,000 × g in two steps with 1% protamine sulfate and 65% ammonium sulfate in buffer A. The supernatant was dialyzed overnight into buffer B containing 20 mM potassium phosphate, 10 mM 2-mercaptoethanol, 0.05% sodium azide, 1 mM EDTA, and 1.4 ml of the protease inhibitor mixture at pH 7.4. It was then concentrated to 40 ml by ultrafiltration with an Amicon YM5 membrane. Pure LFABP was obtained after passage over a Sephadex G50 column (1 m × 5 cm), equilibrated in buffer B and run at 17 ml/h.
The purified LFABP was run through a hydroxyalkoxypropyl dextran column (Lipidex 1000, VI) at 37 °C to remove bound endogenous E. coli lipids. SDS-PAGE (24) of the sole eluant showed a single band at 14 kDa. The first 10 amino acids match that of LFABP but with an additional amino-terminal methionine present: MNFSGKYQVQ (Applied Biosystems gas phase sequenator). Since the x-ray results to be described herein indicate an N-formyl group at the amino-terminal, the amino acid sequence data suggest that some of the purified LFABP was deformylated by E. coli deformylase partially unblocking the amino terminus. However, for unknown reasons, a relatively large percentage of the crystalline protein still contains the N-formyl group on the terminal methionine as is evident in the electron density maps.
CrystallizationThe LFABP is equilibrated with a 10 × molar excess of oleic acid (Nu Check Prep, Elysian, MN) overnight at 5 °C in 100 ml of buffer C (20 mM potassium phosphate and 30 mM sodium chloride at pH 7.4). Unbound lipid is removed by ultrafiltration, and the LFABP-oleate complex is concentrated to about 10-15 mg/ml. The protein stock was stable at 4 °C for up to 5 months. A single 0.5 × 0.25-mm hexagonal rod crystal grew by hanging drop diffusion against a 1-ml well volume of 3 M ammonium sulfate, 200 mM LiSO4, 100 mM citrate, at a pH of 5.6. The 10-µl drop was a 1:1 mixture of the protein stock (13 mg/ml) and the well contents.
Data CollectionThis crystal was mounted 12 cm from a
Siemens X-1000 multiwire area detector operating at room temperature. A
Rigaku RU-200 generator operating at 45 kV and 200 mA and fitted with a
graphite monochromator produced a Cu K beam collimated to 0.5 mm.
Data frames were counted for 3 min before each crystal rotation of 0.25°. An attempt was made to measure a complete P1 data set to the
maximum extent of diffraction at 2.1 Å, although this was not possible
due to radiation decay and the availability of only one crystal. The
indexing, integrating, scaling, and merging of the diffraction data
were handled with the XENGEN package of programs (25). Data collection
statistics are given in Table I.
|
Reflections were present from a resolution range of 35-2.1 Å.
However, for the resolution range of 2.3-2.1 Å, only about one-third of the x-ray data has been recorded. The DATAMAN program (26) (version
4.1.2) indicates an effective resolution of roughly 2.3 Å with
|F|/|F|
1 for
all reflections. The data from 20 to 2.3 Å are complete, well
measured, and uniformly distributed in reciprocal space. Only
reflections from 20 to 2.3 Å were used in the structural study.
The symmetry of the x-ray data indicated that the space group was either P3121 or P3221 with unit cell dimensions of a = b = 84.34 Å and c = 46.8 Å. The data were isomorphous (Rmerge = 0.12) to that described for an earlier LFABP x-ray data set (23). Calculation of the Matthews' coefficient (27), VM = 2.6 Å3/Da, suggested 1 LFABP molecule/asymmetric unit.
Structure DeterminationThe initial phases were determined by molecular replacement using X-PLOR (version 3.1) (28). Cross-rotation and translation function computations with various resolutions and integration radii were unsuccessful when a variety of complete, polyalanine, or fragmented iLBP coordinates were used as search probes. However, when a composite structure consisting of six superimposed iLBPs was as used as the search probe (29), the solution for the cross-rotation and translation searches was easily determined. The composite structure was composed of the complete polyalanine coordinates of ALBP, CRABPI, CRBPI, CRBPII, IFABP, and HFABP.
For the cross-rotation function, reflections between 10 and 6 Å were
used with a 2 cut-off on |Fo|. Patterson
search vectors between 5 and 20 Å sampled one asymmetric unit of
rotational space with a step size of 3.5°. The highest rotation
function peak, 3.2
above the mean, was correct. Patterson
correlation refinement (30) was used to refine this orientation using
10-4 Å data. The translation function was calculated over the same
resolution range with a 2
cutoff on |Fo|. The
highest peak was 5.7
over the mean value. At this point, the
crystallographic symmetry was confirmed as P3221. Rigid
body refinement in X-PLOR was used to optimize this molecular
replacement solution using 1115 |Fo| values
2
between 6 and 4 Å. The resulting R factor was
49.3%. A |2|Fo|
|Fc|| map (8-2.3 Å) showed appropriate
electron density for 87% of the omitted amino acid side chains.
Furthermore, unbiased electron density for two oleate molecules was
found within the binding cavity, confirming the completion of the
initial phase determination.
A 126-residue model was built using the main
chain fragments that best fit the electron density from the six protein
models in the search probe. The initial electron density for LFABP side chains was also modeled. Extensive use was made of the rotamer data
base provided in the O program (version 5.10) (31). Temperature factors
were preset to 20 Å2. A randomly selected set of 496 or
5.3% of the reflections were set aside for
Rfree calculations (32) to monitor the
refinement process and the appropriate weights for the x-ray
pseudoenergy terms in X-PLOR. The Engh and Huber force field was used
(33). No ionic charges were assigned to protein side chains.
Reflections from 8 to 2.3 Å with |Fo| values
1
were used. The first cycle of rigid body and energy
minimization reduced the R factor to 35.1%
(Rfree = 42.0%). Next, one round of slow
cooling-simulated annealing (34) was carried out, starting from 4000 K
with 25 K temperature decrements. Finally, the structure was subjected to several macrocycles of energy minimization, grouped B
factor refinement, and model rebuilding before completion. The atomic occupancies were not refined.
Data from 20 to 2.3 Å (>1 ), modified by a bulk solvent
correction, were used in refinement macrocycles run in parallel with the 8-2.3-Å refinements described above. The resultant coordinates and maps from both processes were then viewed together during remodeling. However, only the 8-2.3-Å refined models were actually manipulated and rebuilt. The bulk solvent correction was accomplished by a modified X-PLOR script. It utilized the available solvent mask
calculation to generate Fsolvent terms for the
addition to the |Fc| values. Required parameters
such as solvent density (0.36 e
/Å3), radii of mask probe (0.25 Å), and Bsolvent (50 Å2) were
methodically optimized by evaluation of the
Rfree. The plot of R factor and
Rfree in the resolution range of 20-2.3 Å closely matched theoretical Luzzati plots (35) in slope and value only
after the bulk solvent correction was applied.
At each stage, LFABP models were critically examined with help from the
programs X-PLOR, O, and PROCHECK (36). When structural adjustments were
contemplated, omit maps were calculated with and without the
solvent-corrected low resolution data for every five residues along the
protein's sequence to help verify the map interpretation. In the
initial refinement cycles, |2m|Fo| D|Fc|| and
m||Fo|
|Fc|| omit maps (37) were used to help
guarantee unbiased electron density with respect to the model being
fit; m is a figure of merit based on the similarity of
|Fo| to |Fc|.
D is estimated from the coordinate error. After the
R factor was reduced to 23.7% (29.8%
Rfree) and the model displayed acceptable
geometry,
|2|Fo|
|Fc|| and
||Fo|
|Fc|| omit
maps were used instead. Simulated annealing omit maps (38) calculated
for residues 21-25, 26-30, 71-75, and 76-80 were nearly equivalent
to these later omit maps.
At this point, the fitting of ||Fo| |Fc|| electron density for oleic acid inside
the LFABP cavity was begun. An amino-terminal formyl-methionine and
modified cysteine, residue 69, were also placed in available electron
density. Nonsolvent ||Fo|
|Fc|| electron density over 5
and roughly
80 Å3 in volume was found for an unknown molecule near the
amino-terminal end of the first LFABP
-helix. This density was
ultimately modeled by one molecule of butanoic acid. Additional
||Fo|
|Fc||
electron density (3
) found adjoining that of one of the oleic acid
molecules near its
-terminal end was also definitely nonsolvent in
nature. This density proved difficult to interpret and was modeled by a
single carbon pseudoatom. The carbon atom is residue 131 in the
deposited list of atomic coordinates. During the ligand modeling and
refinement process, the B factors of individual atoms were
refined. Moreover, preliminary stereochemical restraints applied to the
fatty acid chains were relaxed in the last stages of refinement to
allow a better fit of the available electron density. Sixty-one ordered
solvent molecules were added, and waters were restrained in their
position by a constraint of 10 kcal Å mol
1. Solvent
molecules were removed after either 8-2.3-Å or solvent-corrected 20-2.3-Å refinement if the volume of the electron density became insufficient, their temperature factors surpassed 90 Å2,
or their location moved by more than 2.0 Å. The electron density clearly displayed discrete disorder for Asp34, and two
conformations were modeled. Multiple conformations for the
solvent-surface residues Thr94 and Thr114 were
weakly observable but not modeled.
The atomic coordinates for the 20-2.3-Å and 8-2.3-Å refined LFABP-oleate complexes have been deposited in the Protein Data Bank (39). The identification code is 1lfo. The residue-numbering scheme begins with the formyl-methionine as number 1 and is complete for 127 amino acids or the entire LFABP polypeptide chain. The two complete oleate molecules, the butanoic acid, a single carbon atom, and 61 waters are consecutively numbered 128-192 following the protein residues.
The results from refinement and the model statistics are contained in Table II. The final structure of LFABP in complex with oleate results in an R factor of 19.0% (20-2.3 Å) or 20.2% (8-2.3 Å), while the Rfree is 25.1% (20-2.3 Å) or 26.2% (8-2.3 Å). The differences between the R factor and Rfree are centered within the empirically observed range (40). Initially, the R factor and Rfree determined from bulk solvent-corrected 20-2.3-Å refinement were 3 and 5% less, respectively, than the corresponding 8-2.3-Å refinement. These differences decreased to 1.1%, shown here, commensurate with the significant improvements in the LFABP model.
|
The inclusion of the corrected low resolution data had a number of
effects. (a) The increase in electron density contrast facilitated the correct placement of amino acid side chains,
particularly the loop containing residues 73-76. (b)
Alternative side chain conformations were resolved and tested, which
removed problematic errors in placement. (c) There was a
generalized reduction of electron density for solvent-facing lysines,
glutamines, and side chains of the second helix. (d) As
expected, differences between the atomic coordinates were small in
comparison with r.m.s. deviations always less than 0.6 Å but generally
around 0.2 Å. A conservative estimate of the average coordinate error
is about 0.47 Å, obtained from a Luzzati plot with the
Rfree rather than the R factor as one
of the variables (41). (e) Finally, the use of the solvent correction systematically increased the average B factor of
protein (+6 Å2) and oleate (+7 Å2) atoms but
decreased that for bound solvent (5 Å2) compared with a
trial refinement using just the 5-2.3-Å data. The overall average
B factor for all atoms is 5 Å2 higher. The
systematic discrepancy between |Fo| and |Fc| is expected to be slight for those
reflections higher than 5 Å in resolution because the amplitudes are
not as dependent on the contrast between the protein and the solvent.
We currently have no explanation for these B factor results
but believe that the use of low resolution x-ray data aided
significantly in the early stages of interpreting the electron
density.
The stereochemistry of the final crystal coordinates was compared with a protein data base of 118 high resolution structures using PROCHECK. Assessments of the Ramachandran plot, bad contacts, bond geometry, and side chain geometry are quantified and summarized in the G factor. For the LFABP-oleate complex, the value is 0.29. This result is better than the expected value for equivalent resolution structures and is indicative of the strong geometric restraints applied during the refinement.
All of the non-glycine amino acids in LFABP have and
angles
that fall within the "most favored" (93.5%) or "additional allowed" (6.5%) regions. No residues have angles that lie in either the "generously allowed" or "disallowed" sections. Even the
glycine angles are within or near energetically stable conformations. Only Asn14 and Lys96 show clear left-handed
-helical character (
,
: 59.9°, 36.0° and 49.9°, 37.1°,
respectively). These two residues occur in turns between elements of
secondary structure connecting the first
-strand to the following
-helix and the seventh and eighth
-strands.
The electron density for the polypeptide main chain exhibits no breaks
at the level of 1 . However, |2|Fo|
|Fc|| density is incomplete (<1
) for at
least one side chain atom of the following solvent-exposed residues:
formyl-Met1, Glu26, Asp27,
Lys47, Lys57, Glu62,
Glu70, Lys80. The correlation between the model
electron density and the |2|Fo|
|Fc|| electron density was calculated for each
residue with the program O; 0.87 is the average value for protein. The equivalent computation for oleate 128 is 0.81, while for oleate 129 it
is 0.84. Low values are only found for Glu26 (0.68) and
Asp27 (0.50). These two residues are located on the
solvent-accessible side of the second
-helix. Although significant
enough to roughly place both side chains, the disordered density fit
poorly within the geometric limits of acceptable rotamer positions.
The difference Fourier map produced before refinement was adequate to determine the positioning of the two oleate carboxyls, but they were not included in the coordinates until the R factor was less than 0.25. In addition, to ensure that the fatty acids were placed in the correct orientation through the contiguous density, four possibilities were initially tested. Thus, all combinations for placing the carboxylates of the model fatty acids at both ends of the two "sausage-like" segments of electron density were tried. The current carboxyl locations result in the lowest Rfree and agree with the positions implied by the original difference Fourier.
In the later stages of refinement, some ambiguity developed regarding
the placement of the side chain of Met74 and the
-terminal carbon atoms for oleate 129 as shown in Fig. 1. The SD atom belonging to Met74 is only
3.0 Å from the
terminus of the oleic acid in the final model. The
side chain of Met74 is far from a typical rotamer
conformation; the r.m.s. deviation from the closest rotamer is 4.2 Å.
Moreover, both the SD and CE atoms have very high temperature factors.
Nonetheless, during the refinement, the side chain position of residue
Met74 and the last few carbons of the bound oleic acid were
thoroughly investigated for possible alternate conformations and
discrete disorder. Although many alternative interpretations were
tried, the correlation of the model and map shown in Fig. 1 appeared to
be the best.
During the evaluation, particularly close attention was paid to the
bulge jutting from electron density around the oleate at the position
labeled X in Fig. 1. This density consistently reappeared in
omit maps and in difference maps calculated during refinement. It does
not fit any criteria normally attached to a bound water molecule. The
stereodiagram in Fig. 1 reveals the ease by which this density could be
alternatively modeled by rotation about the C13-C14 bond of the fatty
acid, but then the density would only accommodate a C16 fatty acid.
Furthermore, no other satisfactory conformation for this oleate could
be found without generating significant negative
||Fo| |Fc|| density features surrounding the
terminus atoms. No conformer of
Met74 was large enough to fully model the
|2|Fo|
|Fc|| density remaining in the region of the bad contact. The dilemma was not
solved by refining both configurations simultaneously assuming discrete
disorder, although the simultaneous refinement with discrete disorder
of both C18 and C16 fatty acids was not attempted. Finally, the
coordinates as shown in Fig. 1 result in the lowest
Rfree values. As we were unable to resolve the
difficulties in interpretation, we have included the crystallographic
coordinates of a pseudoatom with a residue name of C16 and an atom name
of CX. Its purpose is to remind anyone studying the crystallographic model that there was difficulty in interpreting the electron density map at this point.
Electron density appearing after refinement in omit maps showed clear evidence for an amino-terminal formyl-methionine, which was subsequently included. Also present in omit maps was additional density, which indicated that the side chain of the sole cysteine, residue 69, was chemically modified. The latter density could not be represented by solvent. Its shape and size was not adequate to support cysteinylation or glutathionylation as has been previously reported (42), nor was it sufficient for an oxidized, -SO2 or -SO3, moiety. Instead, a methyl group was added, which best represents the additional volume of electron density.
Clear non-solvent ||Fo| |Fc|| electron density (>5
) for an
unknown bound molecule was found near the amino-terminal end of the
first LFABP helix. Attempts to reinterpret this density with protein
atoms, especially with the side chain atoms of N14, were not
successful. All known chemical components from the purification and
crystallization were modeled into the density, but none fit
satisfactorily. Electron density in approximately the same position was
observed in the structural studies of ALBP when it was in a complex
with oleate, stearate, palmitate, or hexadecanesulfonic acid (43). In
the latter two cases, butanoic acid was modeled and refined. The
residue name in the coordinate list is C4. Since the shape and volume
of this mystery density near LFABP is comparable with that in the ALBP
studies, butanoic acid coordinates were again used. Nevertheless, a
third oleic acid molecule with a disordered hydrocarbon tail is a
second explanation. At very low contouring levels, < 0.5
, the
difference Fourier exhibits three nearly contiguous segments of
electron density, which in toto could encompass this third
bound oleate.
Within the iLBP
family, LFABP has a uniquely different ligand binding stoichiometry and
is more widespread in its tissue expression pattern but has essentially
a similar overall conformation. Comparison of the crystal coordinates
of other family members fitted to the LFABP model reveals the same
basic intracellular hydrophobic ligand binding skeleton. It includes
(a) an antiparallel -barrel with an internalized
cavity, (b) a combination of polar and nonpolar residues and
bound water within this cavity, (c) a hydrophobic ligand, in
this case one of two bound oleic acids, forming an ion pair with an
internalized arginine side chain, (d) a gap between the
D
and
E strands of the
-barrel, and (e) a lid or portal to the cavity made by the hairpin turns forming the connection between
C and
D,
D and
E,
G and
H, and a
helix-turn-helix.
For a more analytical comparison to the other family members, the
crystal coordinates of other known structures were fitted to those of
LFABP by least squares methods. The overall r.m.s. distance differences
between LFABP and representatives of the iLBP family are listed in
Table III. Based on these average differences, the
myelin fatty acid-binding protein P2 appears to be closest in
structure. However, the range of differences given in Table III is not
very broad (1.3-1.9 Å). The percentage amino acid identities between
LFABP and the listed iLBPs range from 22 to 26%. Analogous comparisons
of the level of amino acid sequence identities for ALBP, HFABP, and P2
are in the range of 58-67%. In terms of the LFABP linkage to other
family members, there appears to be little or no systematic
relationship between the level of amino acid sequence identities and
the r.m.s. deviation values of the C positions among the various
crystal structures.
A more detailed comparison with a sampling of the family members is
shown in Fig. 2. Overall, the largest conformational
differences appear to occur between LFABP and the two retinoid-binding
proteins, CRBPII and CRABPI. Starting from the amino terminus, the
first conformational difference occurs with residues between 23 and 28. The discrepancy in this region is due to the movement of the first
helix and the previous turn toward the cavity interior in the crystal
structure of LFABP. However, this inward movement of 1-3 Å fails to
fully close off access to the cavity. Many of the other differences
occur in the intervening turns between elements of secondary structure.
In general, these regions are also indicated by the shaded
bar at the bottom of Fig. 2 as the most
solvent-accessible. Exceptions to this trend occur at residues between
the indicated E, F, and F"
-strands.
A comparison of hydrogen bonds shows that 10 -strands form the
-barrel in all existing iLBP crystal structures, except in LFABP.
Analysis of the crystal structure of LFABP using DSSP (44) suggests 11
-strands with the additional strand labeled
F" in Fig. 2.
F
involves residues 78-79, while
F" comprises residues 84-86.
Definitions of
-structure are based on strict rules of hydrogen
bonding ladders (44). The positioning of both
F
and
F" (and the
residues between) does not deviate significantly from the homologous
F strand in other iLBPs, but the hydrogen bonding pattern differs.
Thus, the low solvent accessibility for some residues between the E,
F
, and F"
-strands is explainable. We conclude that the definition
of the two strands
F
and
F" has little conformational meaning
and is certainly not a departure from the basic 10-stranded barrel
typical of the iLBPs. All further reference to the
F strand includes
F
,
F", and the residues between them.
A neighboring region visible in the schematic representation at the
top of Fig. 2 near the C position labeled 96 is conformationally different in LFABP. Here, both
G and
H are
two residues shorter than the average for the iLBP family. These
conformational differences are not fully portrayed in the distance
graph, since the additional amino acids present in other
superpositioned iLBPs do not pair with any LFABP residues. The
shortening of the
G and
H strand length has relatively dramatic
consequences as shown in detail in Fig. 3. In addition
to the normally observed gap or missing interstrand hydrogen bonds
between
D and
E, the
-barrel of LFABP has a second gap in the
hydrogen bonding scheme located between
F
and
G. At least four
interstrand hydrogen bonds are missing that generate the assignment of
two
-strands,
F
and
F" instead of one. Meanwhile, only four
hydrogen bonds are present between
F" and
G,
Lys84-Val92 and
Glu86-Lys90, and no bonds are found between
F" and
E because the
D
E loop is immediately adjacent.
This second gap is unique to LFABP. Near the vicinity of the largest
separation between main chain atoms, about 7.5 Å, a salt bridge is
formed between the residues Glu77 and Lys96.
Furthermore, solvent access to the fatty acid binding cavity through
this opening appears to be blocked by the side chains of
Met22, Met74, Val79, and
Phe95. The function of this second gap in the intrastrand
hydrogen bonding pattern is presently unknown. Its existence does not
add any substantial volume to the fatty acid binding cavity. However, the nearby associated residues of the E
F and
G
H hairpin
turns do define part of the hypothetical portal region for alleged
ligand entry and exit. A decrease in the number of intra-main chain
hydrogen bonds girding the portal suggests a greater degree of possible conformational motion in LFABP compared with the other iLBPs.
Data supporting extraordinary conformational motion in the iLBP
secondary elements surrounding the portal, the C
D,
E
F, and
G
H turns, and the two helices come from a variety of experiments. First, the cavities of both the apo- and the holocrystal structures of
IFABP (61, 63) and CRBPII (58) are sealed by protein atoms, and the
atomic coordinates vary little with or without ligand. This means that
in these two family members, conformational fluctuations must accompany
the binding reaction. Conformation motion in this region is further
supported by comparisons of apo- and holo- forms of ALBP where only a
slightly uncovered cavity opening is present. In crystallographic
studies of other family members, other changes are evident. In the
crystal structure of apo-CRABPI, the cavity is completely exposed,
while in the holo- form, the cavity is only partially accessible (45).
In CRABPI, the differences are due to variations in the
C and
D
strand conformation (45). Finally, the B factors for
residues in the reverse turns near the helix-turn-helix lid in LFABP
are higher than the average. A similar phenomenon is noted in almost
all iLBP crystal structures.
Kinetic experiments also support localized conformational flexibility. A rate-limiting step found in the association of oleate with IFABP was interpreted as a requirement for some conformational change allowing the ligand access into the binding cavity (46). After the helical region in IFABP was deleted, the dissociation rate for oleate increased, and although the association rate was mainly unaffected, it was no longer rate-limiting (46). Furthermore, the apo- forms of LFABP (47), CRABPI (48), CRBPI (49), and CRBPII (49) are significantly more susceptible to limited proteolysis in these components than the holo- forms, suggestive of greater accessibility at least transiently in the apo- form.
Finally, NMR studies of several family members point to a degree of
conformational flexibility in narrowly defined portions of the
-barrel structure. In NMR structural studies of CRABPI (48), the
portal elements of apo- forms generally had fewer nuclear Overhauser
effect constraints than that for the holo- form. Similar results were
obtained during NMR studies of HFABP (50). The largest conformational
differences between the crystal and solution structures of HFABP
occurred at the
-turns between strands
E and
F, between
strands
G and
H, and in the region of helix
II (50).
The stoichiometry of fatty acid binding
to LFABP is uniquely different from other iLBP family members in that
more than one fatty acid is bound. Two molecules of bound oleate are
observed in the holocrystal structure described here. We have tried to account for this difference in stoichiometry by carefully comparing LFABP with the crystal structures of other family members, focusing mainly on the internalized binding cavity. The size and shape of the
binding cavity in crystalline LFABP is shown in stereo in Fig.
4. This is a solvent-accessible surface calculated with the GRASP program (51) using a probe radius of 1.4 Å. The cavity in
LFABP is essentially a flattened rectangular box with rough dimensions
of 13 × 9 × 4 Å. A corner of this interior box is linked to a surface opening by a narrow but contiguous channel of about 10 Å in length and 3-4 Å in width. Approximately 48% of this surface is
identified with hydrophobic side chains, while 52% is associated with
the side chains of charged or polar residues.
The volume enclosed by the solvent-accessible surface over all of the
modeled atoms in the crystal structure is 28,600 Å3, while
the area is 7,070 Å2 (with a 1.4-Å probe). If only the
two bound oleate molecules are stripped away, the volume is reduced by
430 Å3, while the surface area increases by 560 Å2. As shown in Fig. 4, the two oleic acid molecules take
advantage of nearly all of the cavity space formed by the -barrel.
Table IV contains a surface and volume comparison with
other iLBP family members. For LFABP, calculation of the cavity area
and volume results in values of 610 Å2 and 440 Å3. In Fig. 4, the oleate molecule (oleate 129) is that
described above as interacting with an internalized arginine residue.
It will referred to as the primary binding site. The fatty acid is in a
bent or U-shaped conformation similar to bound fatty acids in other
family members, although it is in a notably different cavity location.
The second molecule of fatty acid (oleate 128) is bound with its
hydrocarbon tail in the space occurring between the limbs of the
"U" and has its carboxylate head group on the surface of the LFABP
molecule in what appears to be a fully solvent-accessible location. The
semiperpendicular orientation of the two oleic acids is consistent with
a lack of fluorescence self-quenching observed when cis- or
trans-parinaric acids were bound to LFABP (52, 65). The
conformation of the ligands will be described in a separate section
below.
|
An assessment of the cavity volume and area of holo-LFABP compared with that of other family members is contained in Table IV. Both GRASP and the program VOIDOO (53) were used to delineate the cavity and make measurements. Measurements by VOIDOO were made using all of the provided defaults and atomic radii. Because the binding cavity is not completely enclosed, the results are dependent on where an artificial cut-off is drawn separating the interior from the exterior. While automated in VOIDOO, in GRASP an arbitrary cut-off was chosen, and hypothetical atoms were placed to close off the cavity entrance for all of the iLBPs in a similar manner. This cutoff is indicated by the end of the accessible surface contour at the top of Fig. 4. The LFABP binding cavity is clearly the largest in the iLBP family, exceeding that of the next relative by an additional 26% in volume and 20% in area. A comparison of all cavity surfaces with that of LFABP shows that the majority of this increase is located around the binding site of oleate 129 (not shown).
The required increase in cavity volume for this binding site is
primarily due to reductions in side chain size or position at seven key
residue locations. The LFABP residues at these points are
Ser39, Asn61, Thr93,
Ser100, Thr102, Asn111, and
Ser124. A compilation of larger, homologous residues at
each spot is found in Table V for several iLBPs. If any
one of these residues were introduced into LFABP, it would sterically
interfere with binding at the primary site. Except perhaps for the
amino acids homologous to Ser100 and Thr93 on
the conformationally divergent G
H loop, the main chain atoms at
these locations vary little in position (see Fig. 2). Other nearby
residues among the various iLBPs differ only slightly in their
dimensions or placement.
|
The two oleic acids do not completely fill the binding cavity of LFABP.
This leaves room for six bound solvent molecules. The residue numbers
for these waters are 144, 179-182, and 185. The waters are involved in
hydrogen bond networks with the interior hydrophilic residues and the
carboxyl oxygen atoms of the oleate molecules (see below). The water
molecules presumably affect ligand affinity and probably play an
additional structural role. There are also two additional interior
buried waters in LFABP that are not located within the ligand binding
cavity. These solvent molecules, 167 and 168, reside between the
D
E loop and the
F strand near a conserved water position found
in most iLBP members. Both of these waters definitely play a structural
role. Even with all of the interior contents included, LFABP has three
smaller subcavities, a total of about 100 Å2, not modeled
by any atoms. These subcavities might presumably be occupied by three
to five thermodynamically disordered solvent molecules undetectable by
crystallography.
In the crystal structure, the more
internalized fatty acid, oleate 129, is completely surrounded by
protein atoms, structural water, and the nearby atoms of the second
bound fatty acid. Because of the location of the ligand within the
protein, the binding site for oleate 129 is likely to be the first
occupied. Since a portion of the fatty acid atoms in the second site
interact with the ligand at the primary site, their binding energies
must be linked in some presently unknown manner. As can be seen in Fig.
4, the terminus of oleate 128 is packed against oleate 129. Furthermore, in the absence of the primary fatty acid, much of the
hydrophobic character of the second binding site would not exist. In
fact, a 185-Å2 area is buried by the acyl chain
interactions between the two oleate conformations using a surface
calculated with a 1.4-Å probe. The rest of the second binding site
occupies the channel leading to the external milieu. Despite the fact
that the carboxyl of oleate 129 is exposed to the solvent, it is still
involved in a network of hydrogen bonds. From this single crystal
structure, it is unknown whether any conformational changes accompany
binding at the primary or secondary sites.
The U-shaped conformation of the more internalized fatty acid, oleate 129, appears to be facilitated by the C9=C10 double and C13-C14 single bonds, which both have synclinal dihedral angles. The remaining torsional angles along the aliphatic chain are roughly antiplanar, although the C7-C8 and C8-C9 angles are closer to anticlinal. It is important to remember that the ligand positions are time-averaged over the data collection period and that the resolution of this experiment will not differentiate small positional differences on the order of 0.5 Å.
Fig. 5 shows the omit ||Fo| |Fc|| electron density at a 3
level along
with all protein atoms, ligand atoms, and solvent within 4 Å from any
carbon atom of oleate 129. Beginning with the aliphatic end, the final
C17-C18 atoms are in close contact with the side chain of
Met74 located on the
E
F turn. As a result, the side
chain of Met74 deviates greatly from the normal rotamer
conformations and has a high B factor. Based on the
8-2.3-Å electron density, the least ordered region of the oleic acid
is that for the C14-C16 atoms. The possible existence of an
alternative conformation was investigated, but supporting electron
density was found to be incomplete. As noted under "Results," the
single atom sphere numbered 131 occupies the
|2|Fo|
|Fc||
electron density that has proven difficult to interpret. The sphere is
a darker shade than the water molecules in Fig. 5.
Of the two binding sites, oleate 129 is involved in much more extensive hydrogen bonding interactions at the carboxyl group. The residues shown in Fig. 5 that are involved in this network are Ser39, Arg122, and Ser124. Of the six bound waters found inside the cavity, three are members of this network. The waters appear as gray spheres and are numbered 179-181. Additional residues with atoms in contact with oleate 129 are Ile41, Phe63, Glu72, Thr73, Thr93, and Thr102. Last, a fourth solvent molecule, number 185, is within van der Waals distance of the hydrophobic acyl chain. Note that these residues and waters represent only a small portion of the cavity confines. The solvent-accessible surface of LFABP lost due to the interaction of this oleic acid is 400 Å2.
Earlier site-directed mutagenesis (21, 64) and chemical modification (20) data established the importance of Arg122 in the binding of at least one ligand. The mutants at this position show a reduced but not a total loss in binding of oleate. This may be explained by the fact that in the crystal structure, Arg122 is only one of the polar groups interacting with the carboxylate at the primary binding site. Also, the second binding site may remain fully functional. Furthermore, the remaining hydrophobic interactions may still favor binding. A recently expressed double mutant, T102Q/R122Q, was shown to lack lipid binding ability (21, 64). In the crystal structure, the T102Q mutation would clearly sterically block the primary binding site in LFABP.
Omit ||Fo| |Fc||
electron density for residues within 4 Å of the second bound oleate
appears in Fig. 6. The fatty acid at this site is nearly
fully extended in contrast to oleate 129. The C9=C10 double bond and
C1-C2, C7-C8, and C17-C18 single bonds show approximately synclinal
dihedral angles. The remaining torsional angles are all roughly
antiplanar. The electron density for this oleate molecule is weaker at
the solvent-exposed end, and the B factors rise as one
follows the hydrophobic tail toward the carboxylate. In fact, the
|2|Fo|
|Fc|| electron density was initially suggestive of two conformations for the
first five carbons of the oleic acid. However, strong negative
difference electron density arose for one configuration. The current
conformation resides in contiguous electron density and results in
lower B factors and in the formation of the largest possible
number of hydrogen bonds.
The carboxylate group is found near residues forming the portal into the cavity in a solvent-exposed position. Nonetheless, this group is still involved in a hydrogen bonding network as implied in Fig. 6. The side chain of Asp88 from another LFABP monomer is of less importance, since it interacts with the ligand because of crystal lattice packing. This location for the carboxylate agrees with results from NMR experimentation (17) and studies combining mutagenesis with fluorescence quenching (54). The total accessible surface buried by interactions with oleate 128 is 420 Å2. From C1 to C14 there are no interactions with atoms of the primary fatty acid. On this basis, the prior presence of fatty acid at the primary site may be required if anything longer than a C14 fatty acid were to bind at the secondary site.
As mentioned under "Results," the crystals also contained a small
amount of electron density of unknown origin. This density is located
in a position similar to electron density in ALBP and is shown in
stereo in Fig. 7 modeled as a 4-carbon carboxylic acid.
The good correlation in the LFABP map between butanoic acid and the
electron density is apparent. The unknown substance has the properties
of an anionic compound, since the two atoms at the carboxyl end are
within hydrogen bonding distance to main chain nitrogen atoms of
residues Asn14, Phe15, and Glu16.
The formation of four hydrogen bonds is possible. The amount of
solvent-accessible area buried by this interaction is 30 Å2. Phe15 and Glu16 are positioned
at the N-terminal end of helix 1 (Fig. 7, upper right).
Hence, the electron density is nearly at the precise position for
anionic interactions with the positive dipole of the N terminus of this
-helix. Although highly speculative, the appearance of the density
might suggest an exterior binding site for fatty acids. Only the first
few carbon atoms would be ordered; the remaining part of the ligand
would be assumed to be largely disordered.
Conclusions
The structure of holo-LFABP has been determined from crystals prepared with oleic acid. LFABP has the same overall conformation as other members of the intracellular lipid-binding protein family. However, a few differences in the conformation of the protein lead to dramatically different ligand binding properties. Instead of a single hydrophobic ligand as found with all of the other iLBPs, LFABP binds two molecules of oleic acid. The two binding sites interact in an unusual fashion. We have labeled a location characterized by an internalized carboxylate and a U-shaped hydrocarbon chain as the primary site. This bound fatty acid interacts with Arg122, which is homologous to arginines in other family members. The secondary site has the carboxylate near the surface with the hydrocarbon tail inserted toward the center of the molecule and the primary binding site. C18 fatty acids in the secondary site have the last few carbon atoms of the hydrocarbon tail interacting with the nonpolar portion of the fatty acid at the primary site. The carboxylate at the second site is solvent-accessible. Since the two ligands are in physical contact, one would predict an ordered binding mechanism and interactions in their relative affinities. In fact, from the structural data, it appears that the second site may not exist until the primary site is filled.
The atomic coordinates and structure factors (codes 1LFO and R1LFOSF) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
We thank Jeramia Ory for compiling several programs used to generate the figures. Ed Hoeffner has aided the study by maintaining the laboratory computing and x-ray diffraction hardware and software. We acknowledge several discussions with Dick Kostrewa of Hoffmann-La Roche Basel, on results from the use of solvent correction with low resolution data.