(Received for publication, August 13, 1996, and in revised form, January 23, 1997)
From the Department of Biochemistry, ¶ College of Biological
Sciences, University of Minnesota, St. Paul, Minnesota 55108 and
the Institute of Human Genetics and the Department of
Biochemistry,
Medical School, University of
Minnesota, Minneapolis, Minnesota 55455
A number of crystallographic studies of the
adipocyte lipid-binding protein have established that the fatty
acid-binding site is within an internalized water-filled cavity. The
same studies have also suggested the existence of a region physically
distinct from the fatty acid-binding site which connects the cavity of the protein with the external solvent, hereafter referred to as the
portal. In an effort to examine the portal region, we have used
site-directed mutagenesis to introduce the mutations V32D/F57H into the
murine ALBP cDNA. Mutant protein has been isolated, crystallized, and its stability and binding properties studied by biochemical methods. As assessed by guanidine-HCl denaturation, the mutant form
exhibited a slight overall destabilization relative to the wild-type
protein under both acid and alkaline conditions. Accessibility to the
cavity in both the mutant and wild-type proteins was observed by
stopped-flow analysis of the modification of a cavity residue, Cys117, by the sulfhydryl reactive agent
5,5-dithiobis(2-nitrobenzoic acid) at pH 8.5. Cys117 of
V32D/F57H ALBP was modified 7-fold faster than the wild-type protein.
The ligand binding properties of both the V32D/F57H mutant and
wild-type proteins were analyzed using a fluorescent probe at pH 6.0 and 8.0. The apparent dissociation constants for
1-anilinonaphthalene-8-sulfonic acid were approximately 9-10-fold
greater than the wild-type protein, independent of pH. In addition,
there is a 6-fold increase in the Kd for oleic acid
for the portal mutant relative to the wild-type at pH 8.0. To study the
effect of pH on the double mutant, it was crystallized and analyzed in
two distinct space groups at pH 4.5 and 6.4. While in general the
differences in the overall main chain conformations are negligible,
changes were observed in the crystallographic structures near the site
of the mutations. At both pH values, the mutant side chains are
positioned somewhat differently than in wild-type protein. To ensure
that the mutations had not altered ionic conditions near the binding site, the crystallographic coordinates were used to monitor the electrostatic potentials from the head group site to the positions near
the portal region. The differences in the electrostatic potentials were
small in all regions, and did not explain the differences in ligand
affinity. We present these results within the context of fatty acid
binding and suggest lipid association is more complex than that
described within a single equilibrium event.
Lipids are essential molecules that serve a variety of roles in biological systems including formation of the primary structural components of membranes, serving as a source and store of metabolic energy, and as messengers for signal transduction. The intracellular trafficking of these poorly soluble molecules has been hypothesized to be mediated by a family of proteins known as the intracellular lipid-binding proteins (1). These proteins are characterized in part by their conserved gene structure, similarity of amino acid sequences, and the ability to bind lipophilic molecules such as fatty acids and retinoids (2, 3). The adipose specific member of the gene family is known as the adipocyte lipid-binding protein (ALBP or aP2)1 (4). This 14.6-kDa protein is found in great abundance in the adipocyte cytosol and has been shown to bind long chain fatty acids and fatty acid analogs with an affinity of 0.1-1.5 µM (4-8).
The crystal structure of ALBP, like all members of the iLBP family, is
relatively easy to visualize (1) and a stereodiagram of the main chain
is given in Fig. 1. The single polypeptide chain is folded such that
the 10 antiparallel -strands hydrogen bond to each other in a
barrel-like fashion. Starting from the N-terminal, the strands have
been referred to in numerous other publications as
A through
J.
The single exception to the inter-strand H-bonding appears between
strands
D and
E. The distance between these two strands is too
large to form normal
-sheet hydrogen bonds but the gap is filled
with side chains maintaining the integrity of the
-barrel. The ends
of the
-barrel are closed off by a helix-turn-helix segment at one
end, and the last few residues of the N-terminal and side chains at the
other. The helix-turn-helix is located between strands
A and
B.
ALBP and other family members have an unusual structural property. The binding site for fatty acids and hydrophobic ligands is an internalized cavity. This cavity is partially hidden from the external milieu in some iLBPs and is nearly totally hidden for ALBP. The internalized cavity is much larger than the ligand and is filled with ordered and presumably disordered water molecules. For most iLBP proteins, including ALBP, the ligand:protein stoichiometry is 1:1. The crystal structures of the apo and holo forms of ALBP are essentially the same, which suggests that a conformational change must accompany both the association and dissociation processes (9).
Members of the iLBP family have dissociation constants for their ligands in the micromolar to nanomolar range. The thermodynamics of the association reaction have been determined by titration calorimetry for ALBP binding to oleic and arachidonic acid (10), and by van't Hoff data for ALBP and a number of other fatty acid-binding proteins (7). These studies reveal that the relatively high affinity of an iLBP for a hydrophobic ligand is influenced by enthalpic factors. This is in agreement with the crystal structures of the apo and holo forms of the protein which suggest that some of the binding energy is derived from the coulombic interactions between the ionized carboxylate of the fatty acid and a binding triad formed by the side chains of two arginines and a tyrosine which are present within the cavity.
Structural studies of ALBP in the presence and absence of various fatty
acid ligands have also lead to the identification of a distinct region
of the protein surface known as the portal, as first described by Xu
et al. (9). The portal is a region physically distinct from
the fatty acid binding cavity and is formed by helix II and turns
between
C-
D and
E-
F. One of the characteristics of the
portal region is the relatively mobile side chain of Phe57.
The position of the side chain of Phe57 is significantly
different in the apo- and holoprotein. The side chain is near the
hydrocarbon tail of the fatty acid and points toward the exterior of
the molecule in the holoprotein. In the apoprotein, the aromatic ring
swings back and appears to block access to the cavity. These
observations suggest the region around Phe57 could be
indicative of a favored connection between the internal binding site
and the environment.
To address the functional role of Phe57 and the portal region, we have used site-directed mutagenesis to introduce the mutations V32D/F57H into the murine ALBP cDNA. The mutant was designed to form a histidine-aspartic acid salt bridge across a region of the ALBP molecule thought to be the preferential pathway for ligand entry into the binding cavity. Furthermore, the region of ALBP mutated is distant from the internalized arginines and therefore would not be expected to interfere with the coulombic binding reaction. Our goal was to test the portal hypothesis by attempting to confer pH dependence to the binding characteristics.
In this report, biochemical characterization of the mutant protein including stability, ligand binding, and kinetic properties is described. To understand the conformational implications of the mutations, the crystal structure of the mutant form of ALBP is compared at two different pH values. Last, in an attempt to further understand how the mutation could affect binding, the electrostatic potential at atomic sites occupied by ligand atoms in the mutant structures were calculated from the crystallographic coordinates, and the resultant values compared with native forms of ALBP.
Restriction enzymes and DNA modifying enzymes were purchased from Promega. Double-stranded DNA sequencing was performed using a sequencing kit manufactured by Life Technologies, Inc. and carried out in a DNA Thermal Cycler Model 480 from Perkin-Elmer. Oligonucleotides were purchased from Microchemical Facility, University of Minnesota. 1,8-ANS was purchased from Molecular Probes Inc. and fatty acids were purchased from Nu Check Prep Inc. Nalidixic acid and all other chemicals used were reagent grade and obtained from Sigma.
MutagenesisThe V32D/F57H mutant was created in a
sequential manner. PCR-mediated site-specific mutagenesis was used to
construct the single V32D mutation in the murine ALBP cDNA. The
plasmid pGSTALBP (5) was used as a template for two polymerase chain
reactions using either the V32D oligo (5-ACAAGGAAAGACGCAGCCATG-3
) and an oligo complimentary to the 3
end of the cDNA
(5
-TTGATGGATCCTTCCATCCAGGC-3
) or an internal ALBP oligo
(5
-GGTGTTTTTATGAGTACTCTC-3
) and an oligo complimentary to the 5
end
of the cDNA (5
-AGGACGGATCCTCCTCGAAGGTT-3
). Two products were
obtained, a 350-bp V32D product which corresponds to nucleotides
131-476 of the cDNA and a 208-bp fragment corresponding to
nucleotides 12-227. The PCR products were mixed, denatured at 95 °C
for 10 min, and subsequently allowed to cool to room temperature over a
60-min period. The heteroduplexed DNA was then used as the template for
a second round of PCR utilizing the 5
- and 3
-oligonucleotides (above)
that bracket the ALBP cDNA and which contain BamHI
restriction sites to facilitate cloning. The 450-bp PCR product was
subcloned into BamHI digested, dephosphorylated, vector
3Zf(
). Ligation products were transformed into Escherichia coli strain JM109 and recombinant plasmids were selected on the basis of blue/white color selection. Plasmids were recovered and V32D
mutants were verified by double-stranded DNA sequencing.
The V32D/F57H double mutant was created by single-stranded mutagenesis (11). Plasmids were isolated and screened via restriction digest with the enzyme DraI. Successfully mutagenized plasmids were then verified via DNA sequencing. By analogous procedures, the individual F57H mutation was generated using single-stranded DNA prepared from wild-type cDNA.
To obtain large quantities of recombinant mutant proteins, the
individual cDNAs were subcloned into the prokaryotic expression vector pJMB100A. PCR was used to engineer novel XbaI
restriction sites into cDNA flanking regions to facilitate
subcloning. A 27-bp 5-oligonucleotide
(5
-GGTTTACACCATGGGTGATGCCTTTGT-3
) and a 25-bp 3
-oligonucleotide
(5
-ATTCCTCTAGATCCTTCCATCCAGG-3
) (Genosys Biotechnologies Inc.) were
used to produce a 447-bp PCR product that could be directly subcloned.
The PCR product was subcloned into XbaI-digested pJMB100A.
Recombinant plasmids were identified via diagnostic restriction enzyme
analysis with HindIII, and sequenced to verify the integrity
of the nucleotide sequence.
During the molecular manipulation of the various cDNAs, it became
apparent that the use of the NcoI site at the 5 terminus of
the cDNA would facilitate the subcloning procedures. The
incorporation of the 5
NcoI site resulted in the
conservation of the initiating methionine residue but a subsequent
mutation of cysteine 2 to a glycine residue. Previous work in our
laboratory (12) had shown that E. coli expressed wild-type
ALBP contains a post-translational modification of Cys2 such
that the sulfhydryl group becomes modified and is unreactive to
group-specific reagents. As shown by crystallization studies, Cys2 is found on
A, quite far from the ligand binding site
or the portal. Consequently, we anticipated that the C2G mutation would have no effect on ALBP properties. To ensure that the C2G mutation had
no adverse effects upon the folding of ALBP, intrinsic tryptophan fluorescence was measured and compared with that of wild-type ALBP. The
excitation and emission maxima of all forms were essentially identical
to wild-type ALBP (results not shown) and concluded that the C2G
mutation would have negligible effects upon ALBP structure and
function. All protein described within this report contains the C2G
mutation.
The V32D/F57H ALBP cDNA was transformed into E. coli JM 101 and protein expression induced by the addition of nalidixic acid as described previously (12). Proteins were purified as described previously (9) except that cation exchange chromatography was used as the terminal step, replacing immobilized metal affinity chromatography. Concentrated protein solution was dialyzed into 50 mM sodium acetate, pH 5.2, and fractionated utilizing a Mono S HR 10/10 cation exchange column. Recombinant ALBP forms were eluted with a NaCl gradient to 500 mM salt and fractions containing the protein were identified via SDS-polyacrylamide gel electrophoresis. During purification of all forms, the pH of the extract was reduced to 5.0 for 8-12 h. Under these conditions, any potentially bound endogenous ligand is released from the binding site. All preparations of ALBP were found to be apoprotein as revealed by Cys117 titration. For ALBP, fatty acid binding blocks Cys117 modification and conversely, Cys117 modification blocks fatty acid binding (13), lowering the pH to 5.0 releases bound fatty acids and allows the titration of the sulfhydryl group.
Denaturation StudiesEquilibrium unfolding as a function of increasing concentration of guanidine HCl was carried out as described previously (13). Briefly, V32D/F57H ALBP or wild-type ALBP were dialyzed into either 50 mM MES, pH 5.5, or 50 mM Tris-HCl, pH 8.0, and then diluted to 0.5 µM in the appropriate buffer at 25 °C. The emission spectrum of each protein was recorded in the presence of increasing guanidine HCl with a Perkin-Elmer 650-10S fluorescence spectrophotometer with an excitation wavelength set to 285 nm. The emission wavelength maximum was plotted versus the concentration of denaturant for each point in the unfolding profile. The Gibbs free energy was then calculated for each point in the unfolding range and replotted versus guanidine HCl concentration. Line fitting by linear regression was used to determine the concentration of denaturant at the midpoint of the transition.
Sulfhydryl ModificationModification of Cys117
was carried out as described previously (13). Briefly, protein was
added to a final concentration of 10 µM in 50 mM Tris-HCl, pH 8.5, 100 mM NaCl and reacted
with 80 µM DTNB at room temperature. Sulfhydryl titration
was monitored spectrophotometrically and the amount of
thionitrobenzoate released was calculated from the absorbance at 412 nm
( = 13,600 cm
1 M
1). Protection
assays were performed by mixing protein with 50 µM ligand
(oleic acid or 1,8-ANS) for 3 min before the addition of DTNB.
Stopped-flow analysis was carried out with an Applied Photophysics
apparatus and data analyzed with the X suite of kinetic analysis
programs. Data were fit to a single exponential equation which used a
steady state value to mark the completion of the reaction.
Ligand binding to V32D/F57H ALBP and
wild-type ALBP was assessed using the fluorescent probe 1,8-ANS (14).
The probe was dissolved in absolute ethanol and its concentration was
determined spectrophotometrically (372 = 8000 cm
1 M
1). Final ethanol
concentrations were kept below 1% (v/v). Proteins were dialyzed into
50 mM NaPO4 at either pH 6.0 or 8.0 and added in 1 µM aliquots to 0.5 µM 1,8-ANS. The
samples were mixed for 1 min and their fluorescence was measured in a
thermostatted (25 °C) Perkin-Elmer 650-10S fluorescence
spectrophotometer. All manipulations took place under dimmed lights.
Relative fluorescence was plotted versus increasing protein
concentration and Scatchard analysis was performed to determine binding
parameters.
Oleic acid was used as a competitor for 1,8-ANS binding to either V32D/F57H ALBP or wild-type ALBP. Subsaturating concentrations of V32D/F57H ALBP (2.3 µM) or wild-type ALBP (0.6 µM) in 50 mM NaPO4, pH 8.0, were mixed with 0.5 µM 1,8-ANS at 25 °C and the fluorescence signal was measured. Increasing concentrations of oleic acid (from a 10 mM stock in absolute ethanol) were added and the subsequent loss of fluorescence was observed. Relative fluorescence was then plotted versus increasing concentration of oleic acid and a polynomial equation was used to determine the amount of oleate at the midpoint of the competition (50% loss of initial fluorescence) and the apparent Ki (14).
Crystallization TrialsThe protein was equilibrated in 12.5 mM HEPES buffer, pH 7.5, and concentrated to 10 mg/ml. Crystallization screens were set up using the hanging drop/vapor diffusion method. A pH/phosphate scan was first tried similar to native ALBP crystallization conditions (9). These conditions yielded crystals at 2.4 M phosphate, pH 6.4. Due to the lower quality of these crystals as estimated by the maximum observable resolution, different conditions were screened using the sparse matrix (15) and factorial scan method (16). A different crystal form which diffracted to a higher resolution was found at 2.4 M (NH4)2SO4, 50 mM NaKPO4, and 100 mM acetate, pH 4.5.
X-ray MethodsX-ray diffraction data was collected on a
Siemens multiwire area detector, with CuK radiation from a Rigaku
RU-200 generator implemented with a graphite crystal monochromator.
Data was collected with a crystal to detector distance of 12 cm, and a
crystal rotation of 0.25° between frames. Data collection statistics
are reported in Table I. Neither crystal form was
isomorphous with previously reported crystal forms of native ALBP. The
pH 4.5 form has the space group P43212, with
one molecule per asymmetric unit, and cell dimensions of
a = b = 56.4 Å, c = 80.5 Å. The pH 6.4 form belongs to the space group C2221,
also with one molecule per asymmetric unit, and cell dimensions of
a = 79.5 Å, b = 97.3 Å,
c = 50.7 Å.
|
Phases were obtained using molecular
replacement implemented in X-plor (17), with native ALBP used as the
search model. The refinement was carried out in X-plor, initially using
rigid body and positional refinement, and ending with subsequent rounds of b-factor/positional refinement using a bulk-solvent correction. The
mutated residues were modeled as alanines through the initial stages of
refinement, and were fit to both 2Fo
Fc
density contoured at 1
, and
Fo
Fc
density contoured at 3
. Data was added to the refinements in increments of 0.5 Å or less, starting with data from 8.0 Å to 3.5 Å.
Waters were added to the model when the resolution of the data reached
2.5 Å in the case of the pH 4.5 form, or during final refinement of
the pH 6.4 form. Waters had to meet the following criteria: 1)
presence in 2
Fo
Fc
maps contoured at 1
, and
Fo
Fc
maps contoured at 3
; 2) spherical shape; and 3) within 2.5 to 3.5 Å of a proper hydrogen bond donor/acceptor. The
Rfactor/Rfree was
monitored at all times to ensure proper fitting of the model (18).
Final refinement statistics are presented in Table I. The coordinates at both pH values of the V32D/F57H are deposited in the Protein Data
Bank with the accession codes 1AB0 and 1ACD.
All electrostatic calculations were carried out in Delphi, part of the Biosym suite of programs for the study of protein structure. The parameters used in the calculations are listed in Table II. Electrostatic potentials were calculated using the crystal structures of native ALBP, the pH 4.5, and the pH 6.4 mutant forms. Potentials were also calculated using models based on the pH 4.5 form with His57 in the three most common rotamer positions to determine the influence of His57 position on the electrostatic potential. The models for this calculation were built using O (19). To assess the effect of the mutations on ligand binding, it was decided to measure the electrostatic potential near the arginine-tyrosine-binding site and at ligand atom positions throughout the cavity using crystallographic coordinates previously determined for a ligand. The structure of native ALBP bound to hexadecane sulfonic acid (HDS; 1lid in the Protein Data Bank) was used to provide the reference ligand position (20). All structures were superimposed in O, then the coordinates of HDS were used to reference points in the three-dimensional electrostatic potential at each atom position of HDS. Similar calculations were also carried out on hypothetical models of the mutant forms R126L/Y128F and T125Q. The former has a negative effect on binding affinity while the latter increases ligand affinity (21).
|
The V32D/F57H mutant was designed to test the hypothesis that
fatty acids preferentially enter and exit the ALBP cavity via the
portal region. Since the mutations involved surface residues, the
change from hydrophobic to hydrophilic side chains was not expected to
notably affect protein stability but would affect accessibility to the
cavity. The residues which were mutagenized can be visualized in Fig.
1. Note that they are located on a helix, -II (V32D),
and a turn between
-strands C and D (F57H). The best model called
for the creation of the mutations V32D/F57H which based upon distances
and geometry, would create a salt bridge between Asp32 and
His57 at a pH above the pKa of
Asp32 and below that of His57.
To purify V32D/F57H ALBP, a combination of pH fractionation, gel
filtration, and ion-exchange chromatography was utilized. Protein
purity was estimated to be greater than 95% based on
SDS-polyacrylamide gel electrophoresis and typical yields of protein
were 10-20 mg/liter of ferment. The protein was soluble at a wide
range of pH values and ionic strengths and stable when stored at
20 °C. The intrinsic tryptophan fluorescence of V32D/F57H ALBP was
assessed and compared with wild-type ALBP as a measure of its folding.
The fluorescence excitation and emission maxima for V32D/F57H ALBP were
285 and 332 nm, respectively, at both pH 5.5 and 8.0. These values are virtually identical with those obtained for wild-type ALBP, which indicated that the tryptophan residues found in V32D/F57H ALBP are most
likely located in the same environment as the tryptophan residues
within ALBP. Integrity of the tertiary structures was confirmed by
crystallography as described below.
The stability of V32D/F57H ALBP at pH 5.5 and 8.0 was examined by observing the equilibrium unfolding of the protein in response to increasing concentrations of denaturant. The transition profiles for denaturation of both V32D/F57H ALBP and wild-type ALBP at pH 5.5 had essentially the same sigmoidal shape. The denaturation was reversible; dilution of denatured protein with the appropriate buffer resulted in a return to emission maximum of 334 nm for each protein (results not shown). The concentration of guanidine HCl at the midpoint of the folded/unfolded transition at pH 5.5 was 1.6 ± 0.02 M for native ALBP and 1.1 ± 0.04 M for the V32D/F57H mutant. Similar results were obtained at pH 8.0. Midpoints for native ALBP were 1.5 ± 0.05 and 1.2 ± 0.08 M for the double mutant. At each pH, V32D/F57H ALBP was denatured at a lower concentration of guanidine HCl than wild-type ALBP, indicating that the mutant form is somewhat destabilized relative to wild-type ALBP. Analyzing the denaturation curves suggests that the V32D/F57H mutant is destabilized by approximately 3.0 kcal/mol at pH 5.5 and 0.8 kcal/mol at pH 8.0.
Next, the rate of entry of ligands into the cavity of the portal mutant
was compared with the native protein. To do this, stopped-flow kinetic
analysis of the modification of Cys117 by DTNB was carried
out. Previous work from our laboratory had shown that the side chain of
Cys117 is located within the ligand binding cavity of ALBP
(22), but nonetheless could be covalently modified by the thiol
reactive agent 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) (13). DTNB is
itself large and bulky, and cysteine modification would be expected to
be dependent on the accessibility of the reagent to Cys117.
Fatty acids protect Cys-117 from modification and conversely, Cys117 modification blocks fatty acid binding (13). Crystal
structure analysis has revealed that the side chain of
Cys117 resides within 4.5 Å of the second, third, and
fourth methylene carbons of the bound acyl chain in the holo-protein
(22).
The modification of both mutant and wild-type protein was monitored by
the release of the thionitrobenzoate anion as a function of time and
the results are shown in Fig. 2. The progress curves were fit to a rate equation which described the reaction as a single
exponential function. Rate constants of 0.100 and 0.720 s1 were obtained for wild-type ALBP and V32D/F57H ALBP,
respectively, indicating that the Cys117 site in V32D/F57H
ALBP is modified at a rate which is 7-fold faster than the wild-type
protein.
In addition to examining rate effects, changes in binding affinity were assessed using the fluorescent probe 1,8-ANS. Recently, we have developed a sensitive fatty acid binding assay for lipid-binding proteins using 1,8-ANS fluorescence (14). The assay provides a large signal to noise ratio as the fluorescence enhancement of 1,8-ANS upon binding to ALBP is substantial, approaching values that are greater than 100-fold. Therefore, we used the reagent 1,8-ANS to analyze the effects of pH upon V32D/F57H ALBP's ligand binding affinities. In addition, we evaluated the effect of the individual mutations, V32D and F57H, on 1,8-ANS binding.
The affinities of both V32D/F57H ALBP or wild-type ALBP for 1,8-ANS at pH 6.0 and pH 8.0 are given in Table III. The results demonstrate that the V32D/F57H mutant protein binds 1,8-ANS with less affinity than the wild-type protein at both pH values. Scatchard analysis of the 1,8-ANS binding isotherms displays a Kd of 4.3 versus 0.47 µM for V32D/F57H ALBP and wild-type ALBP, respectively, at pH 6.0. At pH 8.0, 1,8-ANS affinity of V32D/F57H ALBP is again 10-fold less than that of the wild-type protein (Kd 7.1 versus 0.74 µM, respectively) although the alkaline conditions resulted in a 2-fold decrease in affinity for both portal and wild-type ALBP. These results indicate that independent of pH, 1,8-ANS binding affinity is reduced substantially in the portal mutant. A similar trend in binding affinity was observed for the individual V32D and F57H mutants. In both cases at pH values 6.0 and 8.0, the 1,8-ANS affinity was reduced 2-4-fold relative to wild-type (Table III).
|
The displacement assay, which utilized the ability of fatty acids to compete 1,8-ANS bound to ALBP, was then used to assess the ligand binding affinity of the protein for oleic acid. The resultant apparent inhibitor constants (Ki) are displayed in Table III. The Ki of V32D/F57H ALBP for oleic acid is 1030 nM while the Ki of oleic acid for the wild-type protein is 180 nM. These values mirror the trends seen in the 1,8-ANS binding experiments, with the portal mutant displaying 6-fold less affinity for oleic acid relative to the wild-type. Again, as with 1,8-ANS binding, both the single mutants, V32D and F57H, bound oleic acid more weakly than did wild-type.
The fluorescence properties of bound 1,8-ANS were evaluated for both portal and wild-type ALBP to assess the environment in which the ligand is bound. Table IV summarizes the maximum wavelengths of both the emission and excitation spectra of 1,8-ANS bound to the mutant and wild-type proteins at either acidic or alkaline conditions. The 10-nm red shift of the emission maximum seen for V32D/F57H ALBP at pH 6.0 and 8.0 is particularly noteworthy. Red shifts of 10 nm relative to wild-type ALBP implies that the local environment of 1,8-ANS bound to V32D/F57H ALBP has changed in some manner, possibly involving the polarity of the region or accessibility to water. Alternatively, 1,8-ANS may be bound directly at the portal region and the difference in fluorescence properties represents the microenvironment in that region of the two protein forms. X-ray crystallographic analysis of 1,8-ANS bound to ALBP is in progress to address these issues.
|
With clear evidence of alterations in ligand affinities, crystals were
prepared and analyzed at two pH values. Although not isomorphous with
native crystals, the crystal structures were solved by using the
molecular replacement approach. The structure of apo form native ALBP
solved by Xu et al. (9) was used as the search model. The
top cross-rotation function answers were 3.7 above the mean for the
pH 4.5 form, and 3.0
above the mean for the pH 6.4 crystal form.
Each set of answers refined to one common orientation after rigid body
refinement of all potential solutions. The resulting translation
function answers were 10.2
and 11.0
above the mean for the pH 4.5 and pH 6.4 crystal forms, respectively.
The initial models were then refined as described under "Materials and Methods." The crystal structures of the V23D/F57H mutant have been refined to good R factors of 19.3 and 19.2% for the pH 4.5 and pH 6.4 form, respectively. The average b-factors for all atoms in the structures are 30.8 Å2 and 27.5 Å2. Procheck (23) was run on both structures to assess the quality of the models, and all parameters for both models fall within the accepted limits for protein structures solved to their respective resolutions. Table I summarizes the crystallographic results. Examination of the mutant conformations at both pH values indicate there has been no gross change compared with native ALBP. A summary of the coordinate comparisons after rotation to a common frame is given in Table V. Both crystal structures align well with native apo- and holo-ALBP with root mean square differences similar to expected random errors in the coordinates.
|
As noted earlier, both mutants crystallized in space groups previously not observed for ALBP. To address their possible roles in influencing the conformation of the portal region, crystal lattice contacts were studied for both mutant forms and the native form of ALBP. Symmetry mates of the structures were generated, and the residues around the mutated areas were examined with the program LIGPLOT (24). Native ALBP, in space group C21, shows no symmetry related interactions for the region around residue 32, but shows a fairly large hydrogen bond network between the region around residue 57 and a symmetry related molecule. The mutant structure at pH 4.5, in space group P43212, shows no symmetry-related interactions at either location, while the mutant structure at pH 6.4, in space group C2221, shows one hydrogen bond between Asn59 and a symmetry related Glu54.
Because of the known differences in chemical properties, the refined
models were studied carefully to see if the crystal structures could
explain the changes. The first question is the overall change in
stability. Since the conformations of the mutant are identical except
for the new side chains, the temperature factors for the atoms in each
structure were examined. They are plotted in Fig. 3.
Clear differences between the mutant and wild-type protein are visible
but they are mainly in magnitude. As can be seen in Fig. 3, the region
containing Val/Asp32 has the highest temperature factors in
both the wild-type and mutant structures. In the double mutant at both
pH values, the temperature factors in this part of the structure are
elevated. The second highest segment of b-factors appears at the loop
connecting C to
D. Again they are elevated at both pH values in
the V32D/F57H mutant. Although one could justify these increases in
many ways, the most straightforward explanation is that the mutations
have localized effects on the conformation to produce more disorder. This shows up in the electron density in the regions near
Asp32 and His57, which is poor at both pH
values.
Despite the uncertainty in the positioning of Asp32 and
His57, the final refined positions were examined to check
their interactions. Strong ionic interaction via the ion pair,
Asp32-His57, was not observed in the mutant
crystal structures at either pH. In fact at pH 4.5, there appears to be
a local displacement of the C of His57 away
from Asp32, pushing the side chain further away from the
region thought to be the site of preferred ligand entry. This
displacement is approximately 3.5 Å away from the conformation adopted
by Phe57 in the native crystal structure. The orientation
of the mutated residues at both pH values can be seen more clearly in
Fig. 4. In the pH 4.5 structure, the temperature factor
for Asp32 is very high and it was assumed that it is
pointing into the solvent as is illustrated in Fig. 4.
At pH 6.4, regions near the mutational sites have somewhat lower temperature factors. As is also shown in Fig. 4, His57 refines to a position somewhat closer to Asp32. However, both mutated residues would be closer to the methyl end of the hydrophobic chain belonging to a bound fatty acid. This then could be one of several explanations for the observed reduction in binding affinity. The mutant conformation puts two hydrophilic residues at the portal and because of the positions they have adopted, they would be relatively close to the methylene end of a bound fatty acid. The crystal structures show no evidence that a strong polar interaction forms at either pH between the mutated residues as was originally designed.
Two other structural features related to ligand affinity were then
examined in both the pH 4.5 and 6.4 crystal structures. The first
feature is the bound water molecules within the cavity; the second was
the electrostatic potential in the cavity at the normal positions of
ligand atoms. The latter was done by homologous positioning of
hexadecanesulfonic acid at the binding site in the V32D/F57H mutant
structure. The position of water molecules is done by comparing those
refined positions from the crystallographic studies of the mutant
protein. Previously, a network of 10 crystallographically conserved
waters had been identified within the ALBP binding cavity (20).
Comparison of these waters with the higher resolution mutant form, pH
4.5, shows that most appear to be conserved. Seven of the core cavity
waters previously reported are again observed in the electron density
maps, as well as other surface waters in the D-
E turn which
appear necessary for proper folding. Cautious comparisons of the water
sites is necessary since the crystal structures were not all obtained
with x-ray data to the same resolution. The native ALBP crystal
structure was solved to 1.6-Å resolution, while the pH 4.5 form of the
V32D/F57H was obtained at 1.9 Å.
At pH 6.4, only 2.7 Å data was used. The water molecule which is shown to assist in ligand binding by bridging Arg106 and the ligand is not seen in either apo-mutant structure, having been presumably displaced into bulk solvent. Nonetheless, the seven waters were placed in homologous sites in the mutant structures. At least with the present structural data on the V32D/F57H mutant, major perturbations of the water structure within the binding cavity seems not to have occurred.
With no obvious differences in the structure of the binding cavity or the bound waters, another possible explanation for the reduction in ligand binding affinity was that it was the result of local charge changes. To test whether or not a ligand would sense the charge differences between the V32D/F57H mutant and wild-type protein, the electrostatic potential was monitored at positions in the binding cavity where ligand atoms are normally located. To make these calculations, the crystallographic coordinates of native ALBP and the V32D/F57H mutant structures and modeled coordinates of T125Q and R126L/Y128F were used. Positions marking the location of atoms belonging to a fatty acid ligand correspond to those of hexadecanesulfonic acid (20). The R126L/Y128F mutant of ALBP is known to have a marked decrease in affinity for fatty acids (20), and the T125Q mutant has been shown to have a slightly increased ligand affinity (21).
The results of the electrostatic calculations are shown in Fig.
5. Note that the potential at X0 on the curve marked
"native" in Fig. 5 corresponds to that normally experienced by the
carboxylate of bound fatty acid in the wild-type or T125Q ALBP. Not
surprisingly, the mutant with known decreased affinity for fatty acid,
R126L/Y128F, showed a marked decrease in positive electrostatic
potential near the carboxylate group. When the electrostatic potential
was calculated for the V32D/F57H form using the coordinates from both
pH studies, the electrostatic potential was slightly higher than native
ALBP near the carboxyl binding position, and lower near the proposed entrance. To assure the higher potential was not an effect of the
rotamer selection of His57, potentials were calculated with
His57 in the three most favored rotamer positions for
histidine. While the potentials differed slightly (data not shown),
they were all still above that of native. Therefore, the electrostatic
calculations suggest that the decrease in binding affinity of V32D/F57H
ALBP for fatty acids is not due to any general electrostatic effects near the carboxyl binding position.
The earlier analysis of the crystal structure of apo-ALBP has
revealed that the interior ligand binding site is poorly accessible to
the external milieu. Close examination of the oleate-ALBP crystal structure suggested that a small opening might exist in a region bounded by helix -II and the turns connecting strands
C-
D and
E-
F as defined in Figs. 1 and 4. We and others have called this region the ligand binding portal. Important to this location is the
residue Phe57 which is disordered in some crystal
structures and may undergo a conformational change upon binding fatty
acid. To examine the portal region as a site for ligand entry/exit into
the cavity, we designed the site-specific mutant V32D/F57H which was
predicted to form a pH-dependent, steric barrier at this
site. The attractiveness of such a system was that the barrier would be
formed by the electrostatic interactions between Asp32 and
His57 at low pH, but would be less effective at higher
pH.
Examination of the crystal structures of V32D/F57H ALBP at two pH
values indicates that unexpected changes occurred. Ion pair formation
between Asp32 and His57 appears to be only
weakly present in the pH 6.4 crystal structure and unlikely at all at
pH 4.5. At both pH values, electron density near the mutational sites
is not definitive, and the refinement indicated a high degree of
thermal motion in this region of the protein. While electron density is
poor in this region, the main chain can be traced fairly effectively in
both forms, and indicates that the conformation of the mutant is still
very similar to that of the wild-type protein. The C
model of V32D/F57H ALBP was shown in stereo in Fig. 4 and the close
agreement between the crystallographic model at two pH values is
clearly visible. Within the usual limitations of crystal/solution
conformations, the V32D/F57H mutation had little or no effect on the
conformation that forms the cavity binding site. As shown in Fig. 5,
within the limitations of electrostatic calculations, the double
mutation did not have a major effect on the electrostatic potential at
the position normally occupied by the carboxylate of a bound fatty
acid.
In terms of their crystal structures, is there any difference between
the mutant and wild-type proteins? Two -helices cover the opening of
the barrel-like structure. In the orientation shown in Fig. 1, the lid
of the barrel would be hinged to the right of the molecule and the
helix,
II, is close to the
C-
D and
E-
F turn. The contact
region between the lid and the
-barrel is crudely represented by the
dotted band. The mutations V32D and F57H were in the fourth residue in
from the COOH-terminal end of
II and on the very tip of the
C-
D turn, respectively. Because of the high crystallographic
b-factors in the vicinity of the changes, one could argue that the
cavity is more accessible in the V32D/F57H mutant. This would coincide
well with chemical modification studies which showed that
Cys117 on the interior of the cavity is modified 7-fold
faster in V32D/F57H than in native ALBP.
Could the poor electron density and high crystallographic b-factors be a result of crystal lattice contacts, and not the mutations? To address this, the crystal lattice contacts around the portal region were examined in both mutant forms and native ALBP. Native ALBP did show more crystallographic contacts around the portal region than the mutant at either pH. However, the structure of another mutant of ALBP has recently been solved in our laboratory, E72K, which is isomorphous with the pH 6.4 form, and thereby has the same crystal contacts. This structure exhibits low crystallographic b-factors and good electron density around the portal region, implying that extensive crystal lattice contacts are not needed for good electron density and low b-factors. While the best evidence that the local instability and flexibility is induced by the mutations would be to crystallize the native form in a space group isomorphous to either mutant, this has not been accomplished to date.
To attempt to explain why the structural changes produce a change in fatty acid affinity we first considered if the electrostatic potential near the R126/Y128 binding site was altered. Ligand interaction with these internalized polar groups provides a major part of the binding energy. However, based on the crystal coordinates and the calculated electrostatic potentials, V32D/F57H ALBP should actually bind fatty acids better because the potential is higher in the carboxylate region of a bound ligand. In fact, ligand binding affinity is actually reduced. Insofar as the electrostatics using crystal coordinates apply to the solution studies, the mutations should have had a small opposite effect to what was observed in the binding of ANS.
In the absence of explanations related to electrostatics of the region near the R126/Y128 binding site, three hypotheses are reasonable to consider. The first is a relatively simple idea which suggests that stability of ALBP or mutant forms is coupled to affinity. That is, mutant forms which stabilize the apo form will result in lower affinity and mutants which stabilize the holo form will increase affinity. To assess this possibility, we evaluated the stability of the portal mutant in comparison to the wild-type protein.
Under both acidic and basic conditions, V32D/F57H ALBP reached the midpoint of denaturation at lower concentrations of guanidine hydrochloride than the wild-type protein. The mutant protein was destabilized by 3.0 kcal/mol at the low pH and 0.8 kcal/mol at the high pH. At these two pH values, the affinity for 1,8-ANS was reduced identically. Therefore, while the change in free energy between native and denatured for wild-type and mutant was affected by pH, there was no evidence for a pH-dependent change in binding affinity. These results suggest that in general, stability of the apo- or holoprotein is not coupled to ligand affinity. Consistent with this interpretation, Herr et al. (8) have recently demonstrated with chemically modified ALBP that when all surface lysine residues are acetylated, the stability of the protein is affected (destabilized) while the affinity is unaltered. Similarly, in studies of cellular retinoic acid-binding protein 1 (25), substitution mutations of critical arginine residues rendered the protein unable to bind retinoic acid but stabilized the molecule to thermal denaturation. Again, the interpretation is that in general, affinity of a lipid-binding protein for hydrophobic ligands is not formally coupled to protein stability.
Second, while the changes in binding affinity did not appear to correlate with alterations in electrostatic potentials near the triad, one cannot rule out some effects introduced in the portal region. Referring to Fig. 5, the reader will notice that the electrostatic potentials near the portal region (X15 and X16), are lower than native ALBP, and are even lower than the R126L/Y128F mutant. If this is the preferred site of ligand entry, and assuming the fatty acid is presented to ALBP with a negatively charged head group, the negative electrostatic potential introduced by V32D could serve as a negative factor in the binding energy. As mentioned earlier, some C18 fatty acid ligands have been observed to make van der Waals contacts with Phe57 in the bound state. The introduction of these two hydrophilic residues for formerly hydrophobic residues would eliminate any such favorable interaction.
A third reasonable hypothesis is that the lipid binding proteins exist
in "open" and "closed" conformations as described by Cistola
et al. (26). A simple conformational difference of this sort
would mean there are really four forms of the protein since one would
expect open/closed types of both the apo- and holoproteins. A diagram
of the proposed states is shown in Fig. 6.
The crystal structures reported here would be in the so-called closed
conformation. In the iLBP family, so far only crystal structures of the
apo closed and holo closed forms have been observed except in the case
of cellular retinoic acid-binding protein I where both an open and
closed form of the apoprotein appears to be present (27). The cellular
retinoic acid-binding protein contained a conformation where the tip of
the C-
D loop moved away from helix
II making the cavity more
accessible. It is this loop which is the most destabilized in terms of
the crystallographic b-factors as was shown in Fig. 3. In this model,
the open and closed forms would be in rapid equilibrium. However, the
rather limited accessibility of a fatty acid through the portal in the apoprotein suggests that the preferred ligand binding pathway may be
from apo-closed to apo-open followed by lipid association to yield
holo-open. Mutations that stabilized the open form would be expected to
reduce the apparent affinity of the protein for fatty acids. However,
as described earlier, mutants that are unstable are not necessarily in
the open conformation because stability does not appear to be linked to
affinity. The solution structure of the holo-open form of a member of
the iLBP family would be an invaluable tool in the assessment of this
hypothesis.
It is clear that the process of ligand binding requires the internalized ligand form of the ionic association with the reactive triad of Arg106/Arg126/Tyr128. In addition, van der Waals contacts between the lipid acyl chain and cavity side chains are established as a consequence of ligand binding. The cavity of the protein is roughly 3-4 times the size of the ligand suggesting that a ligand may be internalized without association with Arg126 or Tyr128. This has been shown most clearly for the binding of 12-(9-anthroyloxy)oleic acid to the R126L/Y128F mutant of ALBP where binding occurred within the cavity without triad association (21). These results imply that although the model describes the binding events in simple terms, multiple equilibria contribute to the binding energy and differentiate between ligand internalization and triad association.
Recent observations by other groups are consistent with the model for a
multiple equilibria binding process. For example, in the intestinal
fatty acid-binding protein 2 (IFABP 2) from the Pima Indian population,
a polymorphism at position 54 results in an alanine to threonine
substitution. This polymorphism increases the in vitro
binding affinity 2-fold, and correlates with greater in vivo
lipid oxidation and insulin resistance (28). Position 54 in this
protein is found at the IFABP 2 portal region and most likely affects
the opening or closing equilibria controlling ligand access into the
cavity. Similarly, Prinsen and Veerkamp (29) report on several
mutations in human muscle FABP (e.g. T40E) which decrease
binding affinity by affecting either the electrostatic triad or the van
der Waals association. Kleinfeld and colleagues (30) have examined the
kinetics of fatty acid binding to ALBP, heart FABP, and intestinal FABP
and concluded that the rate-limiting step in the binding process is the
entry/exit of ligand through the portal into the cavity. This is
consistent with our interpretation that the opening and closing of the
portal are central to the binding event and that equilibria other than
those related to formation of the ionic triad are critical for high
affinity fatty acid association. Consistent with this, Cistola, Frieden
and co-workers (26, 31) have developed a mutant of intestinal FABP
lacking the two helices which connect A to
B and form some of the
components of the portal region. The mutant IFABP, termed
17-SG, is
essentially an all
-barrel protein and binds palmitate with an
affinity 20-100-fold lower than wild-type. The affinity of
17-SG is
virtually identical to that for the R106T IFABP mutant. As evaluated by
heteronuclear two-dimensional NMR, the interaction of the bound
palmitate with protein side chains is essentially identical for
17-SG and wild-type protein. However, there are dramatic differences
in the kinetics of ligand association and dissociation. Since
17-SG
lacks helix
II, which contributes to formation of the portal, it is
reasonable to conclude that the equilibrium of opening and closing are
affected, biasing the mutant toward the open conformation. The IFABP
studies, like our own, indicate that mutant proteins with altered
ligand binding affinity can arise via effects on any of several
equilibrium events.
In summary, our biochemical and structural results describe a portal
mutant of ALBP and confirm the hypothesis that changing the region
bounded by helix II and turns between
C-
D, and
E-
F affects fatty acid affinity. In addition, we present a description of
fatty acid binding equilibria that suggests ligand association is more
complex than that described within a single equilibrium event. The
thermodynamic contributions of the various binding steps to the overall
binding free energy remains to be determined.
The atomic coordinates and structure factors (codes 1AB0 and 1ACD) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
We gratefully acknowledge the contributions of Dr. Judith Lalonde for helping in the initial design of the mutant and Ed Hoeffner for his tireless effort in maintaining computers and x-ray diffraction equipment. We also thank Judy Bratt for assistance in purification of the mutant protein and Amy J. Reese-Wagoner for lending us the E72K ALBP structure. We acknowledge discussions with Drs. Cistola and Frieden at Washington University regarding their hypothesis of open and closed structures of IFABP. We thank the Minnesota Supercomputer Institute for their support of the project through use of the supercomputing facilities. We also thank Dr. Michael Raftery and Dr. Susan Dunn for assistance in performing the stopped-flow experiments and analysis. Finally, we thank the members of both the Bernlohr and Banaszak laboratories for many helpful discussions and suggestions.