From the Laboratory of Molecular Genetics, The
Rockefeller University, New York, New York 10021 and the
§ Department of Biology, Brookhaven National Laboratory,
Upton, New York 11973
Received for publication, November 6, 2000, and in revised form, December 22, 2000
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ABSTRACT |
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Vaccination with heat shock protein
gp96-antigenic peptide complexes produces a powerful specific
immune response against cancers and infectious diseases in some
experimental animal models, and gp96-peptide complexes are now being
tested in human clinical trials. gp96 appears to serve as a natural
adjuvant for chaperoning antigenic peptides into the immune
surveillance pathways. A fundamental issue that needs to be addressed
is the mechanism of binding of antigenic peptide to gp96. Here, we show
using scanning transmission electron microscopy that recombinant gp96
binds peptide in stable multimeric complexes, which may have biological
significance. To open the possibility for genetically engineering gp96
for improved immunogenicity and to understand if molecular recognition
plays a role in the binding of antigenic peptide, we mutagenized some specific aromatic amino acids in the presumed peptide-binding pocket.
Replacement of Tyr-667 or Tyr-678 to Ala reduced affinity for
peptide whereas conversion of Trp-654 to Tyr increased peptide binding.
Similarly, changing Trp-621 to Phe or Leu or Ala or Ile negatively
affected peptide binding whereas changing Trp-621 to Tyr or Val
positively affected peptide binding. Probing the peptide microenvironment in gp96-peptide complexes, suggested that hydrophobic interactions (and perhaps hydrogen bonding/stacking
interactions) may play a role in peptide loading by gp96.
The endoplasmic reticulum
(ER)1 is the organelle
responsible for constant peptide trafficking. The most abundant
peptide-binding protein in the lumen of ER is the heat shock chaperone
gp96 (GRP94, Ref. 1). gp96 has drawn the attention of many
investigators for its induction by glucose starvation, estrogen, and
interferons and for its role in ischemia, tumor immunity, calcium, and
peptide binding, as well as its role as a chaperone, a glycoprotein,
and a phosphoprotein (reviewed in Ref. 2). gp96 and heat shock protein
HSP90, its cytosolic paralog, share about 50% sequence homology (3).
Both are dimeric proteins (4-6) and are also found as oligomers
(7-10). Both HSP90 and gp96/GRP94 (1, 11) bind a variety of antigenic
peptides in vivo and in vitro (for reviews, see
Refs. 12-14). Intriguingly, gp96 acts as a chaperone of peptides and
aids in provoking a strong immune response against peptide antigens
(12). Its potential as a natural adjuvant for therapeutic cancer
vaccines makes this protein highly significant for human health. The
proposed role of gp96 is to ferry antigenic peptides into specialized
antigen-presenting cells via receptor-mediated internalization of
gp96-antigenic peptide complexes (15-17). The peptides are presumed to
be transferred to and represented by MHC class I molecules (12,
18-22). Clearly, the primary event in the HSP-mediated immune response
pathway appears to be the recognition and binding of antigenic peptides
by gp96. In this context many questions remain unanswered;
e.g. how are peptides selected, and what are the mechanisms
of peptide binding by gp96? To address this, we developed a highly
sensitive fluorescence-based assay for gp96/peptide binding (9) and
mapped the minimal peptide-binding site to amino acid residues 624-630
in the C-terminal region of gp96 (23). A molecular model of the gp96
peptide-binding site was offered with the suggestion that the antigenic
peptide may bind in a pocket. Here, to further explore the molecular
interactions of peptide and gp96, we used STEM to show that
gp96-peptide complexes exist in higher order multimeric complexes and
used site-directed mutagenesis to understand the role of specific
aromatic amino acid residues in the gp96 peptide-binding pocket.
Protein Expression and Purification--
Wild-type and mutant
His-gp96 proteins were purified from plasmid-bearing Escherichia
coli strain JM109 using published procedures (9, 10). Plasmid
pHisGP96 encodes murine gp96 with an N-terminal His6 tag
(10), and the mutants are described below. The purity and intactness of
the proteins were assessed by electrophoresis in SDS-containing 10%
polyacrylamide gels, silver staining, and immunoblotting (9).
Preparation of Nanogold-labeled Peptide--
Peptide
RGYVYQGLKSGLRRASLGRS is a known ligand of gp96 that contains a
vesicular stomatitis virus-derived core sequence (23). It was
synthesized (Alpha Diagnostic, San Antonio, TX) and purified by HPLC.
The peptide was covalently labeled with nanogold using the
amine-reactive reagent sulfo-N-hydroxysuccinimido nanogold according to the manufacturer's protocol (Nanoprobes, Yaphank, NY). A
10-fold molar excess of peptide over nanogold was used to maximize the
coupling efficiency. To purify nanogold-peptide from free peptide and
nanogold, the reaction was subjected to ultrafiltration (10 kDa
molecular mass cut-off) and washed repeatedly with 0.1 M triethylammonium acetate-water. The retentate was
filtered (0.45 micron) and the nanogold-peptide conjugate was purified by C18 reverse phase HPLC. The following HPLC gradient was
used: 10 min of 0.1 M TEAA-water and a linear ramp from
100% 0.1 M TEAA-water to 100% MeOH in 30 min. The
nanogold-peptide-containing fractions were identified using inline
absorption spectra (A280,
A420) collected using a diode array detector
(Hewlett-Packard 1090 series HPLC) and identified by polyacrylamide gel
electrophoresis. Fractions containing nanogold-peptide were dried
in vacuuo and dissolved in water. Portions were separated on
SDS-containing 15% polyacrylamide gels, which were stained with LI
silver enhancement solution (Nanoprobes) to detect gold-containing bands.
Visualization of Nanogold-Peptide-gp96 Complexes by STEM--
A
binding mixture consisting of 5 µM gp96 and 5 µM nanogold-peptide in 10 mM
Na+/HEPES, pH 7.8, 150 mM NaCl, 1 mM MgCl2 was incubated for several hours at
room temperature. Assuming that the Kd for the modified peptide was equivalent to that of the unmodified peptide (10), greater than 90% of the nanogold-labeled peptide would have been bound to gp96. The STEM analysis was carried out as described previously (10, 24).
Construction of Mutants--
Mutations were introduced into the
gene for gp96 by oligonucleotide-directed site-specific mutagenesis of
pHisGP96 (10). Deoxyuracil-containing single-stranded pHisGP96 DNA was
extracted from phagemids grown in an E. coli host
with dut ung mutations (25). The mutagenic oligonucleotides
used are listed in Table I. Sequenced DNA
intervals containing the mutations were reselected and cloned into
naïve pHisGP96 to ensure that no clones had spurious mutations.
Circular Dichroism--
WT and mutant His-gp96 proteins (1 mg/ml) were dialyzed versus 20 mM sodium
phosphate buffer, pH 7.8, and the spectra were acquired and data
processed as described before (10).
Fluorescence Assays--
Peptide-pyrene conjugate was prepared
and purified by HPLC as described previously (9). Equilibrium
peptide-pyrene binding to WT and mutant His-gp96 proteins was done in a
final volume of 12 µl of binding buffer (20 mM
HEPES/Na+, pH 7.8, 200 mM NaCl, 2 mM MgCl2). A fixed concentration of gp96 (typically 5 µM) was mixed with different concentrations
of peptide-pyrene (400 pM to 5 µM). Binding
mixtures were prepared at 25 °C and incubated at room temperature
for ~3 h. Control reactions were prepared with matched concentrations
of peptide-pyrene alone. Binding buffer was added to a final volume of
650 µl. The steady-state fluorescence intensity of the samples was
measured and corrected for background buffer signal. All steady-state
fluorescence spectra were acquired at 23 °C using JOBIN YVON/SPEX
fluorolog3
Intensity-average lifetime (< Binding of Peptide to gp96 as seen by STEM--
Recently, we used
STEM to show that gp96 forms higher order multimers larger than dimers
(10). It was shown that native mouse protein also forms higher order
structures (10). We wished to know if peptide could be bound to gp96 in
these large multimeric structures. Therefore we prepared antigenic
peptide conjugated to 1.4 nm-nanogold and used STEM to analyze the
peptide-gp96 complexes. STEM is particularly useful for studying higher
order structures because one observes bare, unstained molecules and can
obtain estimates of masses and dimensions. For this purpose STEM is
superior to solution-based techniques and other types of electron
microscopy. Moreover, using STEM one can directly visualize and count
nanogold-peptide particles (28, 29). Nanogold-peptide-gp96 complexes
were prepared at concentrations approaching intracellular gp96
concentrations (~5-10 µM, Ref. 1) and the complexes
were diluted just prior to visualization by STEM (Fig.
1). Nanogold-peptide was seen as bright
white spots against the background of gray gp96 multimeric structures
resting on a black surface (Fig. 1, A-E). It was
not possible to determine whether a given nanogold-peptide resided on a
distal or proximal surface of a complex because with STEM one sees a
two-dimensional (transmission) projection of the particle. Isolated
nanogold-peptide was also seen in some fields (Fig. 1, F-J) but at a much lower density than observed
with or in close proximity to gp96 (Fig. 1,
A--E). In general, these examples of STEM images
showing massive gp96 multimers are similar to ones seen earlier that
were prepared without peptide (10) except that we did not observe many
smaller complexes of gp96 such as monomer/dimer, tetramers etc. This
suggested that gp96-peptide complexes were assembled in large multimers
and that dilution and washing did not dissociate bound peptide,
consistent with our previous findings (10). We manually counted the
number of nanogold-peptide associated with gp96 complexes and found
that the number of bound nanogold-peptide roughly corresponded to the number of gp96 monomer units in a complex (Fig. 1, below).
With some types of samples, e.g. large viruses,
nanogold-signal from a distal surface might be obscured because of
sample thickness. However, this is unlikely to be the case with gp96
complexes. It is possible that some gp96 molecules in the multimeric
complex failed to bind nanogold-peptide or lost their nanogold-peptide during sample processing. The dimensions of the multimeric complexes suggested no specific geometric configuration, only unordered shapes
(Fig. 1, A-E and lower table),
consistent with previous findings (10). Statistical analysis was not
possible with this limited amount of data. Many of the molecules (not
shown) appeared to be "denatured" because of unknown reasons.
The finding that gp96-peptide complexes are assembled in massive
multimers is potentially important because such a binding mode may
facilitate the transport and receptor-mediated uptake of gp96-peptide
complexes into antigen-presenting cells (15). These large complexes may
sequester peptide more effectively than smaller ones (dimers,
tetramers, etc.), resulting in more efficient delivery of antigenic
peptides to their subcellular destination in the antigen presenting
cell for further processing. Also, higher order complexes may be more
easily bound by the cognate gp96 receptors, because the high local
concentration of gp96-peptide complexes could greatly enhance the
frequency of collisional interaction with gp96 receptors. Thus we
suggest a biological significance for the higher order assembly of
gp96-peptide complexes.
Rationale for Site-specific Mutagenesis--
We recently
identified the peptide-binding site of gp96 based on photochemical
cross-linking studies (23). Because no high resolution structure exists
for the gp96- or HSP90-peptide complex, we constructed a computer model
depicting the peptide-binding pocket of gp96 (for details and
rationalization see Ref. 23). This model has served as a guide for
further exploration of the molecular nature of gp96-peptide
interactions. Crystallographic and NMR studies of other peptide-binding
proteins have revealed that aromatic amino acids such as Tyr and Trp
play an important, although nonexclusive role in optimal
peptide-protein interactions (reviewed in Ref. 30). For example, in the
MHC I molecule-antigenic peptide complex, tyrosyl residues from the MHC
form H-bonds with "anchor" tyrosyl residues in the bound peptide
(31). Also, most of the specificity in the MHC class II molecule
HLA·DR1-peptide complex comes from binding to an N-terminal tyrosine
in peptide. Likewise, the SH2 domains of Src-tyrosine kinase and
Syp-tyrosine phosphatase utilize phosphotyrosines in peptide for their
interactions (32, 33).
To better understand the microenvironment of the peptide-binding pocket
of gp96, we turned to site-specific mutagenesis. The peptide-binding
domain of gp96 contains five Tyr and three Trp residues. Site-specific
mutagenesis was used to alter these codons in expression vector
pHisGP96 (Table I). Each of the Tyr codons was individually changed to
an Ala codon; a mutant with both Y677A and Y678A was also constructed.
Alanine was selected as the replacement amino acid for Tyr because it
lacks the large bulky hydrophobic side chain proposed to interface with
peptide ligand but retains some hydrophobicity. Alanine is the most
common amino acid in proteins, does not introduce new hydrogen bonding
or charge and is found in both buried and exposed regions of proteins
and in all types of secondary structure (27). In the case of Trp-codon alterations, Trp-485 was replaced with Tyr because nearly all HSP90
proteins have Tyr at the equivalent position. Trp-621 was substituted
with Ala, Ile, Leu, and Val, because all of these residues can be found
at this position in various HSP90 homologs. Mutants with Phe-621 and
Tyr-621 were also tested, in case gp96 structure or function might
require a large hydrophobic side-chain at this position. In place of
Trp-654, only conservative replacements to Phe, Leu, and Tyr were made;
Trp is the only amino acid found in gp96 homologs at this position.
Characterization of Mutant gp96 Proteins--
gp96 mutant
proteins, which had N-terminal His6 tags, were purified
using Ni2+-affinity chromatography and analyzed by SDS-gel
electrophoresis and Western blotting (Fig.
2). Silver staining of the gel showed that the mutant proteins migrated as single bands similar to the WT
gp96 protein (Fig. 2A). Western blotting with a C
terminus-recognizing anti-KDEL antibody indicated that the proteins
were full-length and intact (Fig. 2B and not shown). The
results indicated that the mutant protein preparation was >95% pure
gp96.
To find out if single mutations in the gp96 protein affected secondary
structure, we used CD spectroscopy. CD is a sensitive method for
measuring changes in protein conformation. An example of the CD data is
shown in Fig. 3, top panel.
Data for the other mutants are not shown because they were similar to
Fig. 3 (top panel) within experimental error. The CD data
showed that global secondary structure content was not appreciably
affected by the changes in single amino acids. However, we cannot
exclude the possibility that local conformation of small segments in
the protein may have been affected by the point mutations, which may
affect peptide binding.
Peptide Binding by WT and Mutant Proteins--
To assay for
peptide binding in solution and to quantitate the differences in
peptide loading efficiencies of gp96 mutant proteins, we used a
pyrene-labeled peptide ligand. The detailed procedure for synthesis of
this fluorescent peptide and method of assay for peptide binding by
gp96 have been described elsewhere (9, 10). Briefly, the fluorescent
group pyrene was attached to the unique lysine residue in the peptide
(SLSDLRGYVYQGLKSGNVS) via an amide linkage. Here the pyrene serves as
an optical sensor of the changes in the environment of the
peptide-binding site. When peptide-pyrene binds to gp96 there is an
increase in the excited-state lifetime of pyrene because it is in a
hydrophobic environment (10). By measuring the fluorescence intensity
and the intensity-averaged lifetime of pyrene, we can gauge peptide binding affinities as well as the extent of changes in hydrophobicity as previously demonstrated (9, 10). This assay has several advantages
over STEM for the present purpose. 1) The fluorescence assay allows for
"sensing" the microscopic changes in pyrene environment (which may
translate into peptide environment) brought about by alterations in
amino acid side chains. 2) The fluorescence assay is quantitative;
therefore potentially a hierarchical gradation of effects of the
mutations is possible; and 3) it is much more rapid than STEM and is
solution-based.
Fig. 3 (bottom panel) shows examples of pyrene emission
spectra of peptide-pyrene-gp96 complexes. Spectra for other mutant gp96-peptide complexes are not shown because the basic changes were
similar to those in Fig. 3. In all cases, binding of peptide-pyrene resulted in an increase in the intensity of emission bands at 378 and
396 nm compared with the free peptide-pyrene (Fig. 3, green
line). To help describe the molecular interactions that the pyrene
fluorophore experiences and to understand the local environment of the
fluorophore we measured, in parallel to the emission spectra, the
lifetime parameters (Table II). The intensity-average lifetime
(<
Mutation of Tyr-667 or Tyr-678 to Ala caused a marked decrease in
steady-state fluorescence and intensity-average lifetime (Fig. 4
top panel and Table II, <
Next, we examined the potential role of another set of aromatic amino
acids: the tryptophanyl residues. Earlier, we speculated based on
resonance energy transfer between tryptophan and pyrene in
peptide-pyrene-gp96 complexes that one or more Trp residues were close
to the bound peptide (9). To examine the role of Trp residues in
peptide loading, we focused on the three tryptophanyl residues
(Trp-485, Trp-621, and Trp-654) closest to the peptide in the presumed
peptide-binding pocket (23). Trp-654 is a highly conserved residue in
gp96 paralogs, and HSP90 family of proteins and Trp-621 is closest to
the minimal contact region (residues 624-630) that was identified by
cross-linking peptide to gp96 (23). Mutation of Trp654 to Phe or Leu
did not significantly alter gross peptide binding nor did these
mutations affect the microenvironment as indicated by the relatively
little change in the intensity-average lifetimes (Fig. 4, bottom
panel; Table II, <
Because Trp-621 was predicted to be closest to the bound peptide (23)
we wanted to examine in greater detail the effects of mutation at this
position (Fig. 5). Mutation of Trp-621 to either Ala, Phe, Ile, or Leu caused a reduction in peptide binding compared with WT gp96 (Fig. 5, top panel). The
intensity-average lifetime decreased somewhat, suggesting that these
mutations moderately affect binding pocket environment (Table II,
<
The studies described here attempt to decipher the molecular nature of
the interaction of antigenic peptide with gp96 by using site-specific
mutagenesis of gp96. It is important to note that because a high
resolution crystal structure is unavailable, a model-based approach was
used for design of experiments. The finding that gp96 binds peptide in
very large molecular complexes, as shown here (Fig. 1), could explain
some of the difficulties in obtaining crystallographic models. The
peptide-gp96 complexes appear to be organized in no specific geometric
lattices, perhaps adding to the difficulty.
Steady-state fluorescence intensity and intensity-averaged lifetime of
the pyrene probe are used to measure peptide affinity and changes in
local environment of the peptide. It is likely that the affinity of
peptide is altered by changes in the internal milieu (hydrophobicity)
of the binding pocket. Indeed, these two properties viz.
peptide affinity and hydrophobicity of the binding pocket, may be
related and are simply two sides of the same coin. To get a better
perspective, data from titration experiments were used to make our
conclusions regarding the difference in gross peptide affinity.
Differences in intensity-averaged lifetime may reflect changes in both
affinity and hydrophobicity because the fluorescence quantum yield is
related to the excited-state lifetime (26).
Our work suggests that hydrophobic interaction and hydrogen bonding
and/or stacking interactions could play an essential role in peptide
stability in the binding pocket. Some substitutions in the presumed
peptide binding pocket that introduce Tyr residues (W654Y and W621Y)
appear to positively influence peptide binding whereas changes to other
residues such as Trp-621 to Phe, Leu, Ala, or Ile had a negative
effect. This may suggest an important role for Tyr in peptide binding
by gp96 (as seen with other peptide-binding proteins Ref. 31)). The
observation that some specific substitutions positively or negatively
modulate peptide binding could potentially be a useful way to engineer
new mutant gp96 proteins and to investigate their immunogenic
potencies. Such a study might help provide insights into the long
standing and unresolved issue of whether peptide binding can be
correlated to immunogenicity of gp96-peptide complexes. Furthermore,
this work points to the possibility that there may be peptide
selectivity by gp96 based on molecular recognition because specific
alterations in key amino acids in gp96 do appear to affect peptide
affinity for better or for worse. The fact that there are gp96 mutant
proteins that appear to bind peptide better than WT suggests that the
WT gp96 peptide-binding pocket may have evolved in a way that balanced
peptide binding with the potential need to exchange/transfer bound
peptide to other molecules such as co-chaperones and MHC.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Mutagenic oligonucleotides
instrument (Instruments, S.A. Inc., NJ) as previously
described (9). The excitation was performed at 340 nm and the emission
was measured at either 376 or 396 nm with a 5-nm bandpass set for both monochromators.
>) measurements were
acquired by using the phase-shift and demodulation method (9, 26). The
standard-lifetime compounds were: p-Terphenyl (lifetime 1.0 ns) and 1,4-bis[5-phenyl-2-oxazolyl]benzene (lifetime 1.3 ns). A
scattering agent, LudoxTM in water (0 ns lifetime), was
used to correct for baseline fluctuations during lifetime acquisition.
Lifetimes of the experimental samples were measured against
1,4-bis[5-phenyl-2-oxazolyl]benzene in MeOH. Excitation was performed
at 340 nm and emission was measured by using the T-detector fitted with
a neutral density filter (KV340, Schott) to cutoff stray light below
340 nm. The AC/DC signals were balanced for the reference and
experimental samples. Twelve modulated excitation frequencies ranging
from 1 to 10 MHz were synthesized. A minimum of 5 and a maximum of 10 repeat measurements of phase and demodulation were taken.
Intensity-average lifetime was calculated from the fractional amplitude
(
) and lifetime (
) using both the
phase-shift and demodulation data. Post-experiment iterative lifetime
modeling program version 2.1 (SPEX) was used to fit the data to a
multiexponential decay model, giving fits with good
2
values (Table II). Fitting the lifetime
data to a single-exponential model gave unacceptable
2
values.
Lifetime parameters for gp96-peptide-pyrene complexes
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
STEM analysis of higher
order complexes of the nanogold-peptide-gp96 complex. Panels
A-E show images of gp96 multimeric complexes with
bound nanogold-peptide, whereas panels F-J show
isolated free nanogold-peptide. The table below gives a
semiquantitative analysis of the complexes. Total masses were computed
using a PC mass program as described before (10). Visible nanogold
particles were counted manually from onscreen images. A collective mass
for nanogold-peptide (Au-pep) was estimated using a value of
20 kDa each. This value was subtracted from the total mass of the
nanogold-peptide-gp96 complex, and the remainder was divided by the
molecular mass of gp96 monomer (94.010 kDa) to estimate the number of
gp96. The dimensions of the complexes were estimated by counting the
number of pixels in enlarged views of images and were corrected for the
appropriate magnification factor used to obtain the different
images.
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Fig. 2.
SDS-polyacrylamide gel (10%) of purified WT
and mutant gp96 proteins after silver-staining (panel
A), and immunoblot of purified WT and selected mutant
proteins with an anti-KDEL (C-terminal) antibody (panel
B). The two mutant proteins not shown here (W654F,
Y677A+Y678A) gave results identical to these.
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Fig. 3.
CD spectra of WT and selected mutant gp96
proteins (top panel) and selected emission spectra of
WT and mutant protein-peptide-pyrene complexes (bottom
panel). The CD values were normalized as
described before (10). All other mutant proteins not shown here showed
similar results with these techniques.
>) of pyrene increased when WT gp96 bound to peptide, indicating
that the pyrene moiety had moved into a hydrophobic environment in the
peptide-binding site in gp96-peptide complexes (Table II). The increase
in <
> is because of sequestration of pyrene from dynamic solute
quenching of the excited state. Binding of peptide-pyrene to mutant
gp96 proteins either increased or decreased the steady-state intensity
and lifetime of pyrene compared with the WT gp96-peptide complex.
Changes in steady-state intensities were quantitatively expressed by
constructing binding isotherms, which reflected gross affinity of
peptide to gp96 (Fig. 4). Here the
steady-state fluorescence intensity at 378 nm was plotted against
peptide-pyrene concentration. Intensity-averaged lifetime was used as a
measure of changes in the peptide microenvironment (hydrophobicity) and
affinity (see below for more
discussion).2
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Fig. 4.
Binding of peptide-pyrene to WT and mutant
gp96 proteins with changes in Tyr and Trp residues. The curves
were constructed from data sets (n = 3) obtained by
titrating a fixed concentration of gp96 with increasing concentrations
of peptide-pyrene (400 pM to 5 µM) as
described under "Experimental Procedures." In a given panel, the
fluorescence from mutant-peptide complexes is plotted relative to the
fluorescence value of WT complexes using the same scale.
>) compared with the WT.
However, Y575A, which has an altered residue that was predicted to be
farther away from the peptide (23) showed a lesser reduction in peptide binding (Fig. 4, top panel) and a corresponding lack of
significant effect on the microenvironment, as indicated by the
intensity-average lifetime parameter (Table II, <
>). Similarly,
mutant proteins Y652A and Y677A did not show appreciable changes in the
intensity-average lifetime (Table II, <
>). It is interesting to
note that whereas Y678A affected both peptide binding and the pyrene
environment, Y677A affected neither. Furthermore, with the double
mutant protein, Y677A + Y678A, the intensity-average lifetime decreased
to a similar extent as seen for the single mutant protein Y678A (Table
II). This clearly shows that the two neighboring tyrosines (Tyr-677 and
Tyr-678) are nonequivalent and that Tyr-678 is perhaps closer to pyrene
and may interact with peptide. These findings highlight the crucial
role that tyrosines may play in the peptide-binding site and
demonstrate that the environment of the bound peptide can be affected
in such a way as to decrease peptide binding. Tyrosines closest to the
peptide (at least based on our model Ref. 23) did indeed affect the
peptide environment. In the MHC class I molecule-peptide complex,
tyrosyl residues serve as anchors to restrict size-selectivity of
antigenic peptide (31). Whereas any parallel between MHC class I
molecules and gp96 is debatable, it is intriguing to note that
tyrosines serve an essential role in peptide loading by gp96. The Tyr
may be involved in stacking/hydrophobic interactions with peptide as
observed in other types of protein-peptide interactions (30).
>). The same was true for W485Y, which
presumably lies farther away from the peptide-binding site (23). W654Y
mutant gp96 appeared to bind peptide better than WT (Fig. 4,
bottom panel) and the peptide-binding environment appeared
to be affected as well, i.e. increased hydrophobicity
because the <
> increased (Table II). These data suggest for the
first time that mutagenesis can be used to engineer gp96 for greater
peptide binding affinity. Furthermore, addition of a hydroxyl group (in
W654Y, note that W654F has no affect) may facilitate new interaction
such as H-bonding, which may have increased peptide affinity. The
finding that the W654Y mutant protein (and others, see below) binds
peptide better than WT gp96 opens the door to envisioning an improved
gp96-peptide vaccine if one assumes that peptide affinity is correlated
to immunogenicity, an assumption that needs further investigation.
>). Interestingly, change to either W621Y or W621V significantly
increased peptide binding relative to WT protein (Fig. 5, bottom
panel). Concomitantly, the peptide-binding environment of the
pocket also changed to greater hydrophobicity because the
intensity-average lifetime of pyrene increased significantly (Table II,
<
>). The fact that substitution of Trp-621 with Ile or Leu
negatively affected peptide binding whereas substitution of Val
affected peptide binding positively suggested that both hydrophobicity
(which is similar for Ile, Leu, and Val) and the length of the side
chain influenced peptide binding. Because substitution with Ala had a
negative effect, the extra "greasy" methyls were important. The
substitution of Trp-621 with Tyr affected peptide binding positively
(Fig. 5, bottom; Table II). Putting all these results
together, we suggest roles for hydrophobic interactions and hydrogen
bonding (because W621F negatively affected peptide binding) in peptide
binding to gp96.
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Fig. 5.
Binding of peptide-pyrene to Trp-621 mutant
gp96 proteins. The curves were constructed from data sets
(n = 3) obtained by titrating a fixed concentration of
gp96 with increasing concentrations of peptide-pyrene (400 pM to 2.2 µM) as described under
"Experimental Procedures." The fluorescence from mutant-peptide
complexes is plotted relative to the fluorescence value of WT complexes
using the same scale.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Michael Goger (spectroscopy facility at Rockefeller University) for making available the CD spectrometer. We are grateful to Dr. Joseph S. Wall (Brookhaven National Laboratory, BNL) for the PC mass program for STEM image analysis. The BNL STEM is an NIH Supported Resource Center with additional support provided by United States Department of Energy, Office of Biological and Environmental Research.
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FOOTNOTES |
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* This work was supported in part by grants from the Cancer Research Inst., NY, Antigenics L.L.C, NY, Hewlett Packard Foundation (to S. S.), and Grant P41-RR01777 from the National Institutes of Health and the United States Department of Energy Office of Biological and Environmental Research (to M. N. S. and J. F. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Laboratory of Molecular Genetics, Box 174, The Rockefeller University, 1230 York Ave., New York, NY 10021. E-mail: sastrys@rockvax.rockefeller.edu.
Published, JBC Papers in Press, January 8, 2001, DOI 10.1074/jbc.M010059200
2
Significant differences in <>, generally an
increase or decrease of
40 ns, compared with that in the WT complex
were used for making conclusions.
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ABBREVIATIONS |
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The abbreviations used are: ER, endoplasmic reticulum; MHC, major histocompatibility antigen; HPLC, high performance liquid chromatography; WT, wild-type.
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