From the Departments of § Vascular Biology and
Biochemistry, Holland Laboratory, American Red Cross,
Rockville, Maryland 20855, the ** Department of Biochemistry and
Molecular Biology and Institute for Biomedical Sciences, George
Washington University Medical Center, Washington, D. C. 20037, and
the ¶ Department of Chemistry, Carnegie Mellon University,
Pittsburgh, Pennsylvania 15213
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ABSTRACT |
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The 39-kDa receptor-associated protein (RAP) is
an endoplasmic reticulum resident protein that binds to the low density
lipoprotein receptor-related protein (LRP) as well as certain members
of the low density lipoprotein receptor superfamily and antagonizes
ligand binding. In order to identify important functional regions of RAP, studies were performed to define the domain organization and
domain boundaries of this molecule. Differential scanning calorimetry
(DSC) experiments revealed that the process of thermal denaturation of
RAP is highly reversible and occurs in a broad temperature range with
two well resolved heat absorption peaks. A good fit of the endotherm
was obtained with four two-state transitions suggesting these many
cooperative domains in the molecule. A number of recombinant fragments
of RAP were expressed in bacteria, and their domain composition and
stability were characterized by DSC, circular dichroism, and
fluorescence spectroscopy. The results confirmed that RAP is composed
of four independently folded domains, D1, D2, D3, and D4, that
encompass residues 1-92, 93-163, 164-216, and 217-323,
respectively. The first and the fourth domains preserved their
structure and stability when isolated, whereas the compact structure of
the fragment corresponding to D2 seems to be altered when isolated from
the parent molecule. Isolated D3 was partially degraded during
isolation from bacterial lysates. The isolated D4 was capable of
binding with high affinity to LRP whereas neither D1 nor D2 bound. At
the same time a fragment containing both D1 and D2 exhibited high
affinity binding to LRP. These facts combined with the thermodynamic
analysis of the melting process of the fragments containing D1 and D2
indicate that these two domains interact with each other and that the
proper folding of the second domain into a native-like active
conformation requires presence of the first domain.
The 39-kDa receptor-associated protein (RAP)1 was
initially identified while purifying the
low density lipoprotein receptor-related protein (LRP) by ligand
affinity chromatography (1, 2). The amino acid sequence of human RAP
(3) revealed that it contains a putative signal sequence that precedes
a 323-residue mature protein. Comparative sequence analysis reveals
that human RAP exhibits 77, 73, and 65% identity with the mouse
(4), rat (5, 6), and chicken (7) homologue, respectively.
Interestingly, a gene has been identified in Caenorhabditis
elegans which encodes for a 290-amino acid protein (gene accession
number Z75527) that has some similarity to human RAP (8).
RAP binds with high affinity to certain members of the low density
lipoprotein (LDL) receptor family such as LRP (9-11), gp330/megalin (12, 13), and the very low density lipoprotein (VLDL) receptor (14, 15)
and, once bound to these receptors, antagonizes their ligand binding
ability. Despite the presence of a putative signal sequence on RAP,
this molecule is not secreted but remains cell-associated (3).
Immunoelectron microscopy revealed that in human glioblastoma U87
cells, RAP is contained primarily within the endoplasmic reticulum and
Golgi compartments, with only a trace found at the cell surface and
within the endosomal compartments (16). Genetic deletion of RAP in mice
has no effect on LRP mRNA levels but does lead to incomplete
processing of LRP in the brain and liver resulting in decreased LRP
antigen levels in these organs (17, 18). These studies support the
notion that RAP resides in the endoplasmic reticulum and functions as a
molecular chaperone by binding to newly synthesized LRP, gp330/megalin,
and VLDL receptor, thereby preventing their association with ligands.
To understand how RAP functions, studies utilizing deletion mutants of
RAP have been conducted (19-22), and the results of these studies
suggest that multiple portions of RAP are capable of interacting with
LRP. However, a detailed understanding of structure-function
relationships is hampered by the fact that the domain organization of
RAP is not known. A possible internal triplication is noticed by
aligning residues 1-100, 101-200, and 201-319 of rat RAP (20), which
led to the suggestion that RAP is composed of three domains. This
initial observation was modified by Ellgaard et al. (22) who
suggested the three putative domains may comprise residues 18-112,
113-218, and 219-323 of human RAP. Although the three-dimensional
structure of RAP is not yet known, the solution structure of fragment
18-112 has been solved by NMR (23). These studies revealed that this
portion of RAP consists of three helices composed of residues 23-34,
39-65, and 73-88. Residues 92-112 were found to be disordered in the
structure, raising the possibility that the domain boundaries deduced
from the primary sequence may not be correct.
A more comprehensive identification of functional regions on RAP
requires the delineation of its domain organization. To accomplish this
we performed studies to define the domain structure of RAP and the
boundaries of its domains by differential scanning calorimetry (DSC).
The results of these studies reveal that human RAP is composed of four
independently folded domains. The first NH2-terminal domain and the second domain appear to interact with each other, whereas the
forth COOH-terminal domain seems to be rather independent within the
molecule. The third domain is proteolyzed during isolation resulting in
destabilization. Binding studies reveal that the first and second
domain cooperate to form a high affinity binding site for LRP and that
the isolated fourth domain of RAP also binds with high affinity to LRP.
Proteins--
Human RAP cDNA that was cloned into the
pGex-2T vector (10) was utilized as a template to amplify by polymerase
chain reaction a cDNA encoding the RAP fragments studied. The
following oligonucleotides were used as primers:
5'-CCGCGTGGATCCTACTCGCGGGAA-3' and
5'-CATGAATTCTCAATTGAGGTTGCGTAT-3' for fragment 1-82;
5'-CCGCGTGGATCCTACTCGCGGGAA-3' and 5'-CATGAATTCTCATCCGTCCAG ACCATA-3'
for fragment 1-92; 5'-CCGCGTGGATCCTACTCGCGGGAA-3' and 5'-CATGAATTCTCACACCTGCCGAGCGTC-3' for fragment 1-99;
5'-CCGCGTGGATCCTACTCGCGGGAA-3' and 5'-CATGAATTCTCAAGCCTCAGTGCTGTA-3'
for fragment 1-216; 5'-CCGCGTGGATCCTACTCGCGGGAA-3' and
5'-CATGAATTCTCATTCGGTCCTGCTCAG-3' for fragment 1-164;
5'-CCGCGTGGATCCTACTCGCGGGAA-3' and 5'-CATGAATCCTCACAGGTCCGAGGGGCT-3'
for fragment 1-175; 5'-TTGGCCGGATCCGGTCTGGACGGAAAG-3' and
5'-CATGAATTCTCATTCGGTCCTGCTCAG-3' for fragment 89-164; 5'-TTGGCCGGATCC GGTCTGGACGGAAAG-3' and 5'-CATGAATTCTCAAGCCTCAGTGCTGTA-3' for fragment 89-216; 5'-ATACGCGGATCCAATGTCATCTTGGCC-3' and
5'-GACGGCGAATTCTCAGAGTTCGTT GTGCCG-3' for fragment 82-323;
5'-TGCTGGGATCCCTGAGCAGGACCGAA-3' and 5'-CATGAATTCTCAAGCCTCAGTGCTGTA-3'
for fragment 159-216; 5'-CCGCATGGATCCAGGGTCAGCCAC-3' and
5'-GACGGCGAATTCTCAGAGTTCGTTGTGCCG-3' for fragment 206-323; 5'-TACAGCGGATCCGCTGAGTTCGAGGAG-3' and
5'-GACGGCGAATTCTCAGAGTTCGTTGTGCCG-3' for fragment 216-323. The
sense primer was designed with a BamHI cleavage site and the
antisense primer with an EcoRI cleavage site to facilitate
cloning into the pGex-2T vector. The cleaved pGex-2T vector and the
cDNA of the RAP fragment were isolated after digestion with
BamHI and EcoRI. The cDNA was then ligated into the cleaved pGex-2T vector and then transformed into
Escherichia coli DH5 F'. Clones were selected for growth on
LB plates containing ampicillin. The resultant clones were then
sequenced to determine integrity. This expression vector is designed to
produce a fusion protein of the insert encoded protein and glutathione
S-transferase (GST) from Schistosoma japonicum
and contains a thrombin cleavage site between the GST and encoded
protein. The fusion proteins were purified and digested with thrombin
as described (10) except that the cleavage was performed at room
temperature. In the case of fragment 216-323, digestion was carried
out at room temperature for 8 h using a 1/500 ratio of thrombin to
fragment. Even under these conditions only approximately 40% of the
GST fusion protein was cleaved.
Secondary Structure Prediction--
Secondary structure
predictions for RAP and its fragments was done with PCGene software
using GGBSM method that employs the protocol of Gascuel and Golmard
(24).
Protein Concentration Determination--
Protein concentrations
in all experiments were determined spectrophotometrically using
absorption coefficients (A2800.1%)
calculated from the amino acid composition of RAP and its individual fragments by the following equation:
A2800.1% = (5,690W + 1, 280Y + 120 S-S)/(0.1m), where W, Y, and S-S
represent the number of Trp and Tyr residues and disulfide bonds,
respectively, and m represents molecular mass (25, 26).
Molecular masses of the recombinant fragments were calculated based on
their amino acid composition. The following values of molecular masses
and A2800.1% were used: RAP, 0.926 and
37,915; fragment 206-323, 0.688 and 13,995; fragment 1-216, 1.11 and
25,298; fragment 1-164, 1.38 and 19,408; fragment 1-175, 1.30 and
20,613; fragment 1-99, 1.18 and 11,801; fragment 1-92, 1.29 and
10,975; fragment 89-164, 1.44 and 8,937; fragment 89-216, 0.952 and
14,827; fragment 82-323, 0.84 and 28,246.
CD Measurements--
Circular dichroism (CD) spectra were
recorded on a Jasco J-715 spectropolarimeter with a Peltier PFD-350S
unit for temperature control. Proteins were dissolved in phosphate
buffer (10 mM, pH 8.65), and their concentrations were
optimized as recommended by Johnson (27). Protein thermal stability was
monitored by CD spectroscopy at constant wavelength. For assessing
tertiary structure, unfolding was monitored in the near UV at 268 nm
(ellipticity minimum) and 291 (ellipticity maximum). For secondary
structure unfolding, changes in ellipticity were followed at 222 nm,
the characteristic benchmark for Fluorescence Measurements--
Fluorescence measurements of
thermal unfolding were performed by monitoring the ratio of the
intrinsic fluorescence intensity at 350 nm to that at 320 nm with
excitation at 280 nm in an SLM 8000-C fluorometer. Right angle light
scattering was measured simultaneously in the same fluorometer with
excitation and emission set at 350 nm. Temperature was controlled with
a circulating water bath programmed to raise the temperature at
1 °C/min. Protein concentrations ranged from 0.04 to 0.05 mg/ml.
Titration with urea or guanidinium chloride (GdmCl) was accomplished in
the same instrument at room temperature by continuous addition with a
motorized syringe of a concentrated stock solution of the titrant (8 M GdmCl or 10 M urea) at 20 µl/min to a
stirred cuvette containing 1.6 ml of the protein while monitoring the
fluorescence ratio and right angle light scattering as above. Both the
fluorometer and the syringe driver were controlled by a computer that
automatically corrected the fluorescence intensity for dilution
assuming a linear dependence on protein concentration below 0.15 mg/ml.
Calorimetric Measurements--
Differential scanning calorimetry
(DSC) measurements were made with a DASM-4M calorimeter (29) at a scan
rate of 1 °C/min. Protein concentrations varied from 1.0 to 2.0 mg/ml. The DSC curves were corrected for the instrumental base line
obtained by heating the solvent. Deconvolution analysis was performed
as described (29, 30). The software allows an analysis by either
independent or dependent schemes. The former is based on the assumption
that each domain unfolds independently, regardless of the state of the
neighboring domains. The latter assumes an ordered process in which
constituent domains unfold sequentially, implying the occurrence of
interactions between domains such that the unfolding of any given
domain depends on the status of its neighbors. In the absence of
domain-domain interactions the two schemes give essentially the same
results, whereas the dependent scheme gives a better fit if there is
such interaction. Melting temperatures (Tm),
calorimetric ( Iodination of RAP and RAP Fragments--
RAP and all RAP
fragments were iodinated with 125I by IODO-GEN (Amersham
Pharmacia Biotech) as described previously (33) with specific
activities ranging from 3 to 17 µCi/µg.
Solid Phase Binding Assay--
Wells of microtiter plates
(Dynatech Immulon 2, Dynatech Laboratories Inc., Chantilly, VA) were
coated overnight at 4 °C with 100 µl of 1 µg/ml LRP. The wells
were then blocked with 3% bovine serum albumin in 50 mM
Tris, pH 7.4, 150 mM NaCl, 5 mM
CaCl2 for 1 h at 25 °C. Following washing, the
indicated concentrations of RAP, fragment 1-92, fragment 89-164,
fragment 1-164, fragment 89-216, or fragment 206-323 were added to
the wells and also to control wells coated with just bovine serum
albumin. After an overnight incubation at 4 °C, the wells were
washed, and 100 µl of 0.1 N NaOH was added. An aliquot
was removed and counted. Data were analyzed by nonlinear regression
analysis using (Equation 1),
Fluorescence-detected Thermal Denaturation of
RAP--
Fluorescence spectroscopy and light scattering were used to
derive initial information about the conditions for denaturation of RAP
in order to identify those under which the protein unfolds reversibly
and/or without aggregation. Fig.
1A presents several curves
obtained by heating RAP at pH values between 2.9 and 10.5 while
monitoring the ratio of fluorescence intensity at 350 nm to that at 320 nm as a measure of the spectral shift that accompanies unfolding. Under
such conditions the protein exhibited a sigmoidal denaturation
transition whose midpoint (Tm) was sensitive to pH.
The denaturation process was partially or fully reversible in all cases
since the fluorescence parameter returned to a value near the original
upon cooling of the heated sample with the highest degree of
reversibility occurring between pH 4.3 and 8.7. The protein also
exhibited the highest stability in this pH range. At the same time the
denaturation was accompanied by partial aggregation of the denatured
molecules since simultaneous light scattering measurements indicated an
increase in turbidity following denaturation that was not restored upon
cooling (an example is shown in Fig. 1B). The turbidity was
abolished when RAP was heated at pH 8.7 in the presence of 0.5 M urea (Fig. 1B) with 0.25 M GdmCl
producing a similar effect (not shown). Both urea and GdmCl did not
change noticeably the stability of the protein at pH 8.7. These
conditions were selected for studying the denaturation of RAP with DSC,
a technique that gives more information about the structural
organization of the molecule.
DSC-detected Thermal Denaturation of RAP--
The original
endotherm of RAP obtained by heating the protein in the calorimeter
while monitoring the changes in its heat capacity
(Cp) is presented in Fig.
2A (solid curve). The endotherm exhibits two well resolved heat absorption peaks that are
centered at 43 and 63 °C and reflect denaturation of at least two
compact regions with different stability. The denaturation of RAP was
highly reversible since the endotherm was well reproduced when the
protein was heated up to 95 °C then cooled and reheated (Fig.
2A, inset). This is in excellent agreement with the
fluorescence data even though the concentration of the protein used in
the DSC experiment was more than 1 order of magnitude higher. The Cp function of the denatured RAP almost coincides
with the theoretically calculated Cp function after
85 °C indicating that the protein was fully denatured at this
temperature and that the exothermic processes of aggregation are absent
upon denaturation. The high reversibility of the process and the
absence of post-denaturation aggregation enabled for accurate
determination of the Cp function in the studied
temperature region. This allowed us to perform a detailed thermodynamic
analysis of the excess heat capacity function
(
Although the accuracy of the deconvolution analysis depends mainly on
the accuracy of the experimentally determined Cp function, in the case of RAP the main challenge is the proper selection
of the base line for accurate determination of
DSC-detected Thermal Denaturation of the 1-216 and 216-323
Fragments of RAP--
The deconvolution of the RAP endotherm into
three two-state transitions (see above) is in agreement with the
three-domainal composition of RAP proposed on the basis of triplicate
internal sequence similarity with residues 18-112, 113-218, and
219-323 forming three individual domains (22). To test this model we expressed two fragments as follows: 1-216 that was expected to comprise two domains, and 216-323 corresponding to the third predicted domain. Note that we selected slightly different boundaries between predicted domains 2 and 3 (at 216-217) to correlate with the
boundaries between exons encoding corresponding regions (Fig.
3A). The fragments were
expressed in a bacterial system as fusion proteins (see "Experimental Procedures"). Digestion of GST-RAP1-216 generated
fragment 1-216 in high yield. In contrast, the
GST-RAP216-323 fusion protein was not readily cleaved by
thrombin (see "Experimental Procedures"), most likely due to the
presence of acidic residues near the cleavage site. To avoid this
complication we expressed a longer version of this fragment starting at
residue 206, and we obtained high yields of the final 206-323
fragment. This fragment exhibited a CD spectrum typical for
Denaturation of the 1-216 fragment occurred at higher temperature with
an enthalpy of denaturation of 119 kcal/mol (Fig. 5 and Table I). The
combined enthalpy of the two fragments, 1-216 and 206-323, was very
close to that of the whole RAP ( Further Study of the Domain Structure of the 1-216 RAP
Region--
To explore further the domain structure of the 1-216
region of RAP, several smaller fragments were expressed (Fig.
3C). The selection of the size of these fragments was based
on the following assumptions. First, based on the deconvolution results
(see above), we roughly divided the 1-216 region into three portions
1-72, 72-144, and 145-216. Then, taking into account that the border of the COOH-terminal domain (216-323) coincides with the intron position, we selected the nearest intron position at 82-83 and 163-164 as possible boundaries between domains. Finally, we analyzed the probability of secondary structure formation by analysis of the RAP
sequence to check if the selected boundaries coincide with the
disruption in the regular structure (Table
II). The accuracy of this prediction was
confirmed by comparing the predicted results with the three-dimensional
structure of the NH2-terminal fragment (18-112) determined
by NMR (23). The comparison gave excellent correspondence of the
When melted in the calorimeter, recombinant 1-99 fragment exhibited a
symmetrical endotherm that was readily fitted by a two-state transition
with
The other fragment, 1-164, including the NH2-terminal
domain and the putative second domain, exhibited an endotherm that was readily fitted with two two-state transitions indicating the melting of
two domains (Fig. 5 and Table I). The dependent scheme (see "Experimental Procedures") gave a better fit suggesting that these domains interact with each other. It should be mentioned that the
longer version of this fragment, 1-175, exhibited similar DSC-detected
curve that was fitted with two similar two-state transitions (Table I)
suggesting that extra 11 residues in this fragment are not involved in
the formation of the compact structure. Thus the results obtained with
smaller fragments that represent more simple systems in terms of domain
composition allow us to conclude unambiguously that RAP consists of
four domains with approximate boundaries at 92-93, 163-164, and
216-217 and that deconvolution of the endotherm of RAP into 4 two-state transitions (Fig. 2B, lower curve) closely
describes the denaturation process of this complex molecule.
Assignment of the Individual Transitions in RAP--
Comparison of
the enthalpies and melting temperatures of the individual transitions
in RAP and its various fragments (Fig. 5 and Table I) allows one to
assign these transitions to the melting of certain domains. For
example, the first transition in RAP undoubtedly reflects the melting
of the COOH-terminal domain D4 (217-323) since its
Tm and Preparation and Characterization of Other RAP Fragments--
To
clarify further the relationship between the first and the second
domains in RAP we expressed an 82-323 fragment lacking the first
domain as well as some shorter fragments (Fig. 3D). When
melted in the calorimeter the 82-323 fragment exhibited an asymmetrical endotherm that was fitted with two two-state transitions whose enthalpies and Tms were similar to that of the first two transitions in RAP (Fig. 6 and
Table I). Since in RAP the first and the second transitions correspond
to the melting of domains D4 and D3, respectively, the transitions
observed in the 82-323 fragment most probably reflect melting of these
domains. A shorter fragment containing residues 89-216 that includes
domains D2 and D3 exhibited a symmetrical heat absorption peak that was fitted with a single two-state transition. The enthalpy and
Tm of this transition were similar to that of the
second transition in RAP, suggesting that it reflects melting of D3. No
observable transition corresponding to the third transition in RAP was
observed by DSC in both fragments suggesting that their regions
corresponding to the second domain (93-164) do not form a meltable
structure. To test these suggestions, we expressed the 89-164 and
159-216 fragments that correspond to the second and third domain,
respectively, and characterized their stability and folding status by
different methods.
The 159-216 fragment was partially degraded during isolation from
bacterial lysates. Mass spectroscopic analysis revealed several
fragments resulting from cleavage at Arg-205, Arg-203, Arg-195, and
Lys-193. When the mixture of these shorter fragments was analyzed by
CD, a spectrum typical for a random coil was observed in the far UV
region (not shown) suggesting that the truncated domain is destabilized
and unfolded. In contrast, fragment 89-164 (D2) was isolated intact.
When it was heated in the calorimeter, no heat absorption peak was
observed, in agreement with the above suggestion (Fig. 6A, lower
curve). However, at low temperature the heat capacity of the
fragment was significantly lower than the theoretical heat capacity of
the unfolded fragment suggesting compact packing of its hydrophobic
residues (compare curves 1 and 3 in Fig.
6B). The heat capacity increased in a sigmoidal manner upon
heating and at high temperature reached the value calculated for the
unfolded fragment. When the fragment was heated to 90 °C, cooled,
and heated again to 130 °C, the sigmoidal change of the heat
capacity was highly reproducible (compare thin and bold curves 1 in Fig. 6B). When the fragment was
then heated again to 130 °C, cooled, and the process repeated
several times, its heat capacity function was noted to approach that
calculated for the completely unfolded fragment in a broad temperature
range (compare curves 2 and 3 in Fig.
6B). Comparison of the temperature-induced heat capacity
changes of the native fragment (curves 1) with that of the
unfolded fragment (curves 2 and 3) suggests that
the hydrophobic core of the fragment becomes exposed to the solvent in
a sigmoidal manner. This process is accompanied by a very low change of
the enthalpy (compare the 3rd transition in RAP in Fig. 6A
with the heat capacity change of the native fragment in Fig.
6B). This suggest that fragment 89-164 contains some
compact structure that unfolds cooperatively upon heating, but the
structural changes occur with a much lower enthalpy in isolated D2 than
D2 in the parent RAP or fragments that containing D1 along with D2. To
characterize further compact structure of the isolated D2, we performed
CD experiments.
CD measurements revealed that fragment 89-164 has a spectrum in the
far UV region typical for
The above results indicate that the 89-164 fragment corresponding to
the second domain in RAP forms a cooperative domain that contains
secondary and tertiary structure and exhibits a typical sigmoidal
transition upon denaturation. However, its structure appears to be
different from that in the parent RAP molecule and in all fragments
containing the first NH2-terminal domain since when
isolated its denaturation occurs with a very low enthalpy (see above
and Fig. 6). In the absence of D1 (i.e. in the 89-216 and
82-323 fragments), D2 also seems to denature with a low enthalpy. The
fact that the 89-216 fragment has a higher Binding of RAP Domains to LRP--
To measure the binding of LRP
with RAP, a solid phase assay was employed. In this assay LRP was first
coated to microtiter wells, and then increasing concentrations of
125I-labeled RAP or its fragments, 1-92, 1-164, 89-164,
89-216, and 206-323, were added. Following incubation and washing,
the amount of bound radioactivity was determined. As a control, LRP was
omitted during the coating step, and the binding of iodinated RAP and RAP fragments to bovine serum albumin-coated wells was measured. In all
cases the amount of radioactivity bound to these wells was less than
1% of the binding to the LRP-coated wells. The results of this
experiment are shown in Fig. 8, and the
apparent KD values derived from non-linear
regression analysis of the data are summarized in Table
III. RAP bound to LRP with an apparent KD of 3.1 nM, a value in excellent
agreement with that previously published (10). Fragment 206-323 also
bound to LRP with a high affinity, but fragment 1-92 and 89-164
failed to bind to immobilized LRP. Interestingly, fragment 1-164 bound
to LRP with high affinity, although the amount bound appears lower than that observed for RAP or fragment 206-323. Thus, the fourth domain of
RAP binds LRP, whereas the first and second domains of RAP are both
required to form a second high affinity site on RAP that is recognized
by LRP.
There are several commonly used approaches for the establishment
of the domain structure of complex multimodular proteins whose
three-dimensional structures are not known. They are based either on
the results of limited proteolysis, on the analysis of the exon/intron
map, or on internal amino acid sequence homology. The latter was used
to predict the existence of three domains in RAP on the basis of the
triplicate internal sequence similarity (20, 22). Although this
approach usually gives accurate predictions, there are a number of
examples indicating that some homologous sequences, often called
modules, can form more than one independently folded domain. For
example, the serine proteinase module in several proteins was found to
consist of two domains (34-37) and the fibrinogen-like module to
consist of three domains (38, 39). Thus the structure predicted by any
of the above approaches requires experimental verification to check if
the predicted domains satisfy folding criterion, i.e. if
they are properly folded into a compact structure that unfolds
cooperatively in a two-step manner. DSC is the most appropriate method
for such testing since it allows one to evaluate the number of
cooperative domains in a protein and its fragments by monitoring their
unfolding process (40, 41). In this investigation we have utilized DSC
measurements to establish that RAP consists of four independently
folded cooperative domains.
The unfolding of RAP occurs in a broad temperature range creating
difficulties in the accurate determination of the excess heat capacity
of the process and its subsequent thermodynamic analysis. To simplify
the analysis we turned to recombinant RAP fragments that represent a
more simplified system. We initiated our studies with two fragments,
1-216 and 206-323. The first fragment (1-216) was predicted to be
composed of two domains, whereas the second fragment (206-323) was
predicted to be composed of a single domain (22). In agreement with the
prediction, DSC measurements revealed that the 206-323 fragment
denatured as a cooperative unit characterized by a single two-state
transition. This fragment was found to be stable as a separate domain
and was unfolded only upon heating as detected by three independent
methods, spectrofluorimetry, CD, and DSC. This is in contrast to the
previous report based on NMR data that a 219-323 fragment from RAP is
largely unfolded when isolated (22). Although the extra 3 residues
present in our 216-323 fragment may account for its proper folding,
the alternative explanation of this discrepancy may result from a
comparatively low thermal stability of the fourth domain. Indeed, this
domain begins to unfold at about 30 °C in neutral buffer and thus
could be unstable under conditions employed in the NMR study. Whichever explanation is correct, the data obtained in our study has provided us
with the background for the selection of the appropriate conditions for
the determination of the three-dimensional structure of the 216-323
fragment by NMR, a study that is currently in progress. Our studies
also demonstrated that fragment 1-216 of RAP contains three domains in
contrast to two predicted by others (22). To confirm that this is not
an artifact of the deconvolution analysis but an internal property of
the system, we expressed two shorter versions of this fragment, 1-164
and 1-92, and demonstrated that they contain 2 domains and 1 domain,
respectively. Thus, the data are consistent with the presence of four
domains in RAP.
The selection of the boundaries between domains is not always a simple
task even when the boundaries appear to be rather obvious from the
analysis of the primary structure. In this work, we expressed fragments
with alternative boundaries and compared their stability with that of
the corresponding domains in the whole molecule. This approach is
illustrated by our analysis of the first domain of RAP. Among the three
variants expressed, both 1-92 and its longer version, 1-99, exhibited
two-state transition in the same temperature range as in the parent
protein. On the other hand, the shorter version, 1-82, was
dramatically destabilized clearly indicating that the missing 83-92
residues are important for its stability. Indeed, the three-dimensional
structure of the first domain shows that residues 83-88 are involved
in the formation of the third The 216-323 fragment, encompassing domain 4, has the same stability as
that found in the intact molecule, suggesting that it does not interact
with the neighboring domains. Domain 4 can bind directly to LRP with
high affinity and therefore represents a functionally important region
of RAP. The low thermal stability of this RAP domain is of interest and
may be physiologically important. This portion of RAP is likely to be
more flexible and, if so, may more readily interact with LRP and other
members of the LDL receptor superfamily. Although the isolated first
domain, the most thermally stable in the parent molecule, melts at the
same temperature as D1 in RAP, our data provide evidence for its
interaction with D2. The deconvolution analysis of the DSC profile of
1-164 fragment (including D1 and D2) suggests some interaction between them. Additional evidence for an interaction between D1 and D2 was
obtained when D2 was expressed as an isolated fragment. Several lines
of evidence obtained by CD, spectrofluorimetry, and DSC indicate that
it has a high content of secondary structure, a hydrophobic core, and
some tertiary structure that undergo disruption in a cooperative
manner, and thus it forms a compact independently folded domain. At the
same time its enthalpy of denaturation was much lower than that of D2
in the parent molecule. In fact we were unable to determine it reliably
due to a very low endothermic effect in the region of its denaturation
(Fig. 6B). This suggests that an interaction between D1 and
D2 influences the folding status of the second domain. Interestingly,
in solid-phase binding assays, neither D1 nor D2 fragment bound to LRP,
whereas 1-164 fragment including these domains exhibited strong
binding. Thus, it appears that the isolated second domain has a
significantly altered structure from that present in D1D2 fragment or
in RAP and that the presence of the first domain is important for its
proper folding into a native-like active conformation.
In summary, our results indicate that human RAP consists of four
independently folded domains corresponding to residues 1-92, 93-163,
164-216, and 217-323. The first and second domain interact to form a
high affinity binding site for LRP, and the fourth domain also contains
a high affinity binding site for LRP indicating that RAP contains
multiple, independent binding sites for LRP. Defining the domain
organization of RAP and the folding status of its isolated domains will
now allow for structural studies of individual domains and for a more
comprehensive understanding of the functional properties of RAP.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-helix. For temperature scan
experiments, the protein ellipticity was monitored at constant
wavelength in the near and far UV. Spectra were recorded in the
temperature range of 5-95 °C at 0.5 °C/min in steps of 0.1 °C
with an instrument response of 4 s. The protein solution was
contained in a 1-cm optical path length cell, under magnetic stirring.
For secondary structure analysis, a 1-mm path length cell was used, and
CD spectra data points were recorded every 0.5 nm for the wavelength
range 300-180 nm at 5 and 95 °C with 20 nm/min scans and a 2-s
response time. Four scans were accumulated per spectrum. Secondary
structure was estimated via the variable selection method using the
VARSLC1 program (28).
Hcal), and van't Hoff
(
HvH) enthalpies were determined from the DSC
curves using the same software. The values of the theoretical heat
capacity of the denatured RAP and its fragments were calculated based
on the amino acid composition of the protein as described (31, 32). The
values of the partial molar heat capacities of amino acid side chains in aqueous solutions that were used for the calculation were taken from
Privalov and Makhatadze (31).
where B represents the amount of ligand bound,
Bmax is the amount of ligand bound at
saturation, [L] is the molar concentration of free ligand, and
KD is the dissociation constant.
(Eq. 1)
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Fluorescence-detected thermal denaturation of
RAP. A represents fluorescence-detected melting curves
that were obtained upon heating (solid lines) and cooling
(thin broken lines) of RAP in 20 mM Gly buffer,
pH 2.9, 3.2, 8.7, and 10.5, 20 mM sodium acetate buffer, pH
4.3, and in 20 mM Tris buffer, 0.15 M NaCl, pH
7.4. The curves obtained at pH 8.7 with and without 0.5 M
urea essentially coincide. All curves were arbitrarily shifted along
the vertical axis to improve visibility. B
represents turbidity changes upon heating (solid lines) and
cooling (thin broken lines) of RAP at pH 7.4 and at pH 8.7 in the presence and absence of 0.5 M urea. The
turbidity changes were registered simultaneously with
fluorescence changes that are presented in A.
Cp(exc)), i.e. the observed heat absorption on melting of the protein, that provides more
definite information on the number of the independently folded domains
in RAP. In this analysis one can deconvolute the
Cp(exc) function into simple
constituents corresponding to the heat effect of individual two-state
transitions that reflect melting of individual domains (see
"Experimental Procedures").
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Fig. 2.
Original differential scanning calorimetry
curves of RAP and their deconvolution analysis. A shows
DSC curve obtained upon heating of RAP in 20 mM Gly buffer,
pH 8.7, with 0.25 M GdmCl (curve 1); curve
2 (dotted line) represents the theoretical heat
capacity function for the denatured RAP calculated as described under
"Experimental Procedures." Inset shows the original DSC
trace upon first (solid line) and second (broken
line) heating of RAP. B shows the results of the
deconvolution analysis of the alternatively determined excess heat
capacity functions of RAP as indicated in A by broken
lines a and b (see text). Thin solid
lines represent the component two-state transitions obtained by
deconvolution and the best-fit curves that essentially coincide with
the experimental curves (thick solid lines) that are the
same as presented in A.
Cp(exc). Despite the well defined
beginning and ending of the process, the occurrence of the denaturation
in a broad temperature range (25-85 °C) results in difficulties in
the accurate determination of this parameter
(
Cp(exc)). When the base line was drawn arbitrarily as presented in Fig. 2A (dashed
lines, a and b), the total enthalpies of the
process were found to be slightly different, 152 and 170 kcal/mol. Of
significance, the deconvolution analysis resulted in three two-state
transitions in the first case and four transitions in the second case
(Fig. 2B) reflecting the melting of that many domains,
respectively, in the protein. To select between these two schemes an
additional study with individual RAP fragments was performed.
-helical
proteins (Fig. 4A, inset).
Comparison of its melting properties with that of the 216-323
fragments by fluorescence spectroscopy revealed that both fragments
exhibit similar sigmoidal transitions in the same temperature range
(Fig. 4B) indicating that they both form cooperative
structure with similar stability. The CD-detected melting curve of the
206-323 fragment (Fig. 4A) also revealed a cooperative
unfolding transition with a similar Tm as that
measured by fluorescence. When melted in the calorimeter the 206-323
RAP fragment exhibited a symmetrical heat absorption peak in the same
temperature range that was readily fitted with a two-state transition
with a denaturation enthalpy (
H) of 45 kcal/mol and a
Tm of 43.1 °C (Fig. 5 and Table
I). These results indicate that the
216-323 region of RAP forms a stable cooperative domain and suggest
that the extra 10 residues in 206-323 fragment most probably are not a part of this domain.
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Fig. 3.
Schematic representation of RAP and its
various recombinant fragments. A, exon-intron
composition of RAP with intron positions indicated by
arrows; B, domain composition of RAP proposed by
Ellgaard et al. (22); C and D, various
recombinant fragments of RAP used in this study; E, domain
composition of RAP revealed in this study. Numbers indicate
positions of introns (A), boundaries of the individual
domains (B and E), and boundaries of the
recombinant fragments (C and D).
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Fig. 4.
Fluorescence- and CD-detected thermal
denaturation of COOH- and NH2-terminal recombinant
fragments of RAP. A shows changes in ellipticity at 222 nm of the COOH-terminal 206-323 fragment; the CD spectra of this
fragment at 25 (a) and 95 °C (b) are presented
in the inset. The experiment was performed in 10 mM phosphate buffer, pH 8.7. B and C
illustrate fluorescence-detected melting curves of different
variants of the COOH- and NH2-terminal domain,
respectively. Numbers indicate boundaries of the
individual fragments. The experiments were performed in 20 mM Gly buffer, pH 8.7, with 0.25 M GdmCl.
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Fig. 5.
Original differential scanning calorimetry
curves of RAP and its recombinant fragments and their deconvolution
analysis. All melting curves were obtained in 20 mM
Gly, pH 8.7, 0.25 M GdmCl. Original DSC curves are
presented by thick solid lines; broken lines
indicate the manner in which the excess heat capacity function for each
curve was determined; thin solid lines represent the
component two-state transitions and the best fits obtained by
deconvolution. Domain compositions of RAP and its fragments are
presented schematically at the right; the domains, D1, D2,
D3, and D4, are numbered from NH2 to COOH terminus.
Thermodynamic parameters of melting of RAP and its fragments
H) are given in kcal/mol; transition
temperatures (Tm) are given in °C.
H = 170 kcal/mol)
indicating that all of the compact structure is present in the
fragments. The deconvolution analysis of the endotherm of the 1-216
fragment revealed three two-state transitions in contrast to the
expected two suggesting that this fragment of RAP contains three
cooperative domains. Thus results using these two fragments of RAP
suggest that RAP may consist of four independently folded domains,
three of which are formed by a region containing residues 1-216 and
the remaining fourth one by a region containing residues 217-323. They
are in agreement with the alternative four-domainal model of RAP (Fig.
2B, lower curve, and Fig. 5, upper curve).
-helices and random coil distribution in this region and also forced
us to shift the COOH terminus of the first putative domain from the
intron position at 82-83 to the end of the theoretically predicted and
NMR-determined third
-helix (73-88). The other putative boundaries
at 164-165 and 216-217 were found to be in regions predicted to exist
as random coils, 160-167 and 208-221, that are excellent candidates
for the interdomainal connectors.
Secondary structure content in RAP and its various fragments predicted
by the GGBSM method and determined from the CD data
H = 56 kcal/mol (Fig. 5 and Table I) indicating that this fragment forms an independently folded compact domain. The
shorter recombinant version, 1-92, exhibited a comparable endotherm
with similar enthalpy (Table I) indicating that the extra 7 residues do
not contribute to the stability of this domain; in fact the 1-99
fragment was even destabilized by about 2 °C (see Table I and Fig.
4C) suggesting that these extra residues may perturb the
structure. Overall, these findings are in a good agreement with the NMR
data (23) demonstrating that the COOH-terminal 20 residues of the
18-112 fragment used in these experiments are not in a well defined
conformation. The other shorter version, 1-82, did not exhibit a
noticeable transition in the calorimeter (not shown). When this
fragment was heated and its fluorescent properties monitored, a broad
transition with a Tm much lower than that of 1-92
or 1-99 fragments was detected (Fig. 4C) indicating that
the missing residues from position 83-92 of RAP are important for
stabilization of this domain. Thus amino acids 1-92 of RAP represents
the minimal sequence to form a stable compact NH2-terminal domain.
H are similar to that in the
corresponding fragment 206-323. This domain is the less stable in the
protein and seems not to interact with the other domains since its
separation does not influence its stability. Similarly, the fourth
transition in RAP, the third transition in the 1-216 fragment, and the
second transition in the 1-164 fragment all have their
Tm and
H similar to that in the 1-92
fragment and reflect the melting of the first NH2-terminal domain D1 (1-92). This domain is the most stable, and its stability is
also preserved upon separation from the other domains. At the same
time, as mentioned above, it may interact with the neighboring second
domain D2 (93-164). Most likely, the latter melts in the third
transition of RAP, in the second transition of 1-216 fragment, and in
the first transition of 1-164 fragment whose enthalpies and
Tms are similar. This leaves the second transition in RAP and the first transition in 1-216 fragment to reflect the melting of the third domain D3 (164-216).
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Fig. 6.
Original differential scanning calorimetry
curves of various recombinant fragments devoid of the first
NH2-terminal domain and their deconvolution analysis.
A, all melting curves were obtained in 20 mM
glycine, pH 8.7, 0.25 M GdmCl. Original DSC curves are
presented by thick solid lines, while broken
lines indicate the manner in which the excess heat capacity
function for each curve was determined; thin solid lines
represent the component two-state transitions and the best fits
obtained by deconvolution. The transitions in fragments are numbered
according to their appearance in RAP whose DSC curve, which is
essentially the same as presented in Fig. 5, is shown for comparison.
B, DSC-detected changes of the heat capacity function of
fragment 89-164 in 20 mM glycine buffer, pH 8.7, upon
several heating-cooling cycles. The fragment was heated first to
90 °C (thin curve 1), cooled to 1 °C, and heated again
to 130 °C (thick curve 1); thin solid and
broken lines 2 that almost coincide represent the
heat capacity changes of the fragment upon two consecutive heating
after several heating-cooling cycles. Dotted curve 3 represents the expected heat capacity profile calculated for the
denatured fragment obtained as described under "Experimental
Procedures."
-helical proteins (Fig.
7A, inset) with an
-helical
content of 25%. Heating of the fragment while monitoring ellipticity
at 222 nm produced a sigmoidal transition indicative of cooperative
unfolding for the secondary structure (Fig. 7A).
Furthermore, the fragment exhibited a near UV CD spectrum with a
negative and positive maxima at 268 and 291 nm, respectively, reflecting an asymmetry in the environment of its aromatic groups, Tyr
and Trp, i.e. the presence of the tertiary structure (Fig. 7B, inset). The intensity of these maxima decreased in a
sigmoidal manner upon heating (Fig. 7B) suggesting
cooperative unfolding of the tertiary structure. In agreement the
fluorescence spectrum of the fragment had a maximum at 341 nm
indicating hydrophobic environment of its Trp residue(s). Upon heating
(Fig. 7C) or titrating with GdmCl or urea (data not shown)
the maximum was shifted toward a longer wavelength in a sigmoidal
manner indicating cooperative unfolding of the fragment with the
exposure of its Trp residue(s) to the solvent.
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Fig. 7.
Heat-induced unfolding of 89-164 fragment
detected by circular dichroism (A and B) and
fluorescence (C). A, changes in ellipticity
at 222 nm of the 89-164 fragment; the CD spectra of this fragment in
the far UV region at 5 and 95 °C are presented in the
inset. B, changes in ellipticity at 268 and 291 nm of the 89-164 fragment; the CD spectra of this fragment in the near
UV region at 5, 25, 50, and 95 °C are presented in the
inset. C, solid curve and thin
broken curve represent changes of the fluorescence parameter of
the fragment upon its heating and cooling, respectively. The
experiments were performed in 10 mM sodium phosphate
buffer, pH 8.7 (A), and in 20 mM Gly buffer, pH
8.7 (B and C).
-helical content than
one could expect if its D2 region would be in a random coil conformation (compare the value of 47% determined by CD measurement with the predicted value of 41% in Table II) is in agreement with this
speculation. Thus, the presence of the first domain is required for the
second domain to be folded properly into a native-like conformation
suggesting that these two domains interact in the parent molecule. To
test the functional importance of these interactions, we investigated
the binding properties of the individual domains and combinations
thereof to LRP.
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Fig. 8.
Binding of RAP and RAP fragments to
immobilized LRP. Microtiter wells coated with LRP were incubated
with increasing concentrations of 125I-labeled RAP and RAP
fragments. After incubating at 4 °C overnight, the wells were
washed, the radioactivity was solubilized in 0.1 N NaOH,
and counted. Each point represents the average of triplicate
determination. The curves represent the best fit of the data
to Equation 1 as determined by non-linear regression analysis.
Filled circles, RAP; filled triangles, 206-323;
filled squares, 1-164; filled diamonds, 89-216,
open squares, 1-92; inverted open triangles,
89-164.
Apparent KD values determined for the binding of LRP to RAP and
RAP fragments
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-helix (23). A similar situation was
observed with the fourth domain, 216-323, where fragments 216-323 and
206-323 have the same stability as the corresponding domain in the
parent molecule (this work), whereas the 219-323 fragment may be
substantially destabilized (23). The above results indicate
unambiguously that the boundaries between D1 and D2 and D3 and D4 are
at 92-93 and 216-217, respectively. The boundary between D2 and D3 at
164-165 proposed in this paper is less well defined since we were
unable to prepare variants of D2 and D3 domains and test their boundary in the same manner. The 164-216 fragment (D3) was partially degraded upon expression and purification, and isolated 89-164 fragment (D2)
seems to adopt the conformation that is different from that in the
parent molecule. These complicate direct comparison of their stability
with that in the parent molecule. Meanwhile, the fact that the 1-164
and 1-175 fragments which include D1 and D2 exhibited similar
stability (Table I) suggests that the boundary of D2 does not extend
beyond residue 164.
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FOOTNOTES |
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* This work was supported in part by Grants HL50784, HL54710 (to D. K. S.), HL56051 (to L. V. M.), and HL29409 (to M. L.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of National Research Service Award individual
Fellowship 5F32HL09752-02.
To whom correspondence should be addressed: Dept. of Vascular
Biology, J. Holland Laboratory, American Red Cross, 15601 Crabbs Branch
Way, Rockville, MD 20855. Tel.: 301-738-0726, Fax: 301-738-0465; E-mail: strickla{at}usa.redcross.org.
The abbreviations used are: RAP, receptor-associated protein; LRP, low density lipoprotein receptor-related protein; LDL, low density lipoprotein; VLDL, very low density lipoprotein; GST, glutathione S-transferase; DSC, differential scanning calorimetry; GdmCl, guanidinium chloride.
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