From the Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom
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ABSTRACT |
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Class I major histocompatibility complexes (MHC)
are heterotrimeric structures comprising heavy chains (HC),
2-microglobulin (
2-m), and short
antigenic peptides of 8-10 amino acids. These components assemble in
the endoplasmic reticulum and are released to the cell surface only
when a peptide of the appropriate length and sequence is incorporated
into the structure. The binding of
2-m and peptide to HC
is cooperative, and there is indirect evidence that the formation of a
stable heterotrimer from an unstable HC:
2-m heterodimer
involves a peptide-induced conformational change in the HC. Such a
conformational change could ensure both a strong interaction between
the three components and also signal the release of stably assembled
class I MHC molecules from the endoplasmic reticulum. A peptide-induced
conformational change in HC has been demonstrated in cell lysates
lacking
2-m to which synthetic peptides were added. Many
features of this conformational change suggest that it may be
physiologically relevant. In an attempt to study the peptide-induced
conformational change in detail we have expressed a soluble, truncated
form of the mouse H-2Db HC that contains only the peptide
binding domains of the class I molecule. We have shown that this
peptide-binding "platform" is relatively stable in physiological
buffers and undergoes a conformational change that is detectable with
antibodies, in response to synthetic peptides. We also show that the
structural features of peptides that induce this conformational change
in the platform are the same as those required to observe the
conformational change in full-length HC. In this respect, therefore,
the HC
1 and
2 domains, which together
form the peptide binding site of class I MHC, are able to act
independently of the rest of the molecule.
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INTRODUCTION |
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Glycoproteins encoded by the major histocompatibility complex
(MHC)1 class I locus present
peptide antigens to cytotoxic T lymphocytes. MHC class I molecules are
transmembrane glycoproteins comprising a 45-kDa heavy chain (HC) with
three extracellular domains (1,
2, and
3), noncovalently associated with
2-microglobulin (
2-m). These subunits are
assembled in the endoplasmic reticulum (ER) in association with peptide
epitopes that are either derived from the cytosol and delivered to the
ER by the transporter associated with antigen processing, or generated
in the ER itself (see Refs. 1 and 2 for reviews). Biochemical evidence
shows that when an appropriate peptide binds to the
HC:
2-m heterodimer, the complex is stabilized (3-6) and
that, in this respect, peptide binding and class I assembly are linked
phenomena in so much as the formation of an MHC class I-peptide complex
can be seen as the assembly of a trimolecular complex of HC,
2-m, and peptide. The crystal structures of several MHC
class I-peptide complexes have now been solved (reviewed in Ref. 7) and
support this view, showing that the peptide ligand is deeply buried in
the peptide-binding cleft formed by the
1 and
2 domains. In some cases, as much as 80% of the peptide
is buried (8), and in two cases, a salt bridge forms over the bound
peptide, between side chains of the
1 and
2 domains on either side of the cleft (9). It is
difficult to envisage the diffusion of peptide in and out of this
peptide-binding groove according to the classical image of a
receptor-ligand interaction. Despite this, several attempts have been
made to measure apparent equilibrium binding constants (10) (reviewed
in Ref. 11). Also, dissociation rates have been measured for a variety
of class I peptide complexes (10). In only one report have both kinetic constants (ka and kd) and
thermodynamic constant (Ka) been measured in the
same system, and this concluded that peptide-binding groove to which
peptides bind is different from those from which they dissociate, the
implication being that a conformational change occurs in the class I
molecule upon peptide binding (12, 13). Such a conformational change
has been observed indirectly in another system. In the absence of bound
2-m, free HC undergoes a conformational change that is
detectable with antibodies (4, 14-16), and similarities in the
structural requirements for this conformational change and those for
stable peptide binding to HC:
2-m heterodimers suggest
that this conformational change may be related or identical to that
proposed for the HC:
2-m complex upon peptide binding
(12, 17).
The relevance of such a conformational change is 2-fold. First, it provides a mechanism for the MHC class I binding site to bind a wide variety of peptide ligands with high affinity. It is more usual to observe receptor ligand interactions in which increased affinity is achieved only at the "expense" of greater specificity. Second, it provides a way in which the MHC class I molecule could signal its release from cofactors that are responsible for retaining incompletely assembled class I molecules in the ER.
As a means of producing MHC class I molecules in a soluble form that could allow a study of the peptide-induced conformational change using high resolution techniques, we chose to make only the portion of a class I molecule that contains the peptide binding site. Given the apparent structural independence of this peptide-binding platform, our strategy was to express residues 1-193 of the murine MHC class I molecule H2-Db in Escherichia coli and to renature it after solubilization in 8 M urea. We show here the successful expression of this fragment and its ability to undergo a peptide-induced conformational change.
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EXPERIMENTAL PROCEDURES |
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Vectors, Cells, and Antibodies--
pGMT7 is a 3.1-kilobase
pair, pAR2067-based plasmid containing T7 promoter and terminators
flanking a polylinker. E. coli strain BL21 DE(plys c)is
ampr incorporates an
isopropyl-1-thio--D-galactopyranoside-inducible gene for
T7 RNA polymerase, allowing high level, inducible expression of
transfected DNA. The monoclonal antibodies B22.249 and 27-11-13 (4, 18)
recognize the native conformation of the H2-Db heavy chain.
Rabbit antiserum T18 was raised against
D1-193b inclusion bodies. It recognizes
epitopes on both Db and Kb heavy chains, which
are lost upon
2-m binding but remain when heavy chains
bind to peptides in the absence of
2-m.
Cloning and Expression of D1-270b and
D1-193b--
Full-length Db heavy chain
corresponding to nucleotides 1-874 (amino acids 1-270,
D1-270b), and a fragment of the
Db heavy chain corresponding to nucleotides 64-642 (amino
acids 1-193, D1-193b) were amplified
by a polymerase chain reaction from cDNA made from the
Rausher-transformed thymoma cell line RMA-S (19, 20). The amplification
incorporated a 5'-BamHI and 3'-HindIII
recognition site and added nucleotides coding for four additional
histidine residues at the C terminus of the fragment. The polymerase
chain reaction products were cloned into the vector pGMT7, and
sequenced by the dideoxy method before being transferred into the BL21
strain of E. coli for expression. Transformed bacteria were
grown in L broth containing 100 µg/ml ampicillin to a density of
0.3-0.5 A600 units before adding 0.5 mM isopropyl-1-thio--D-galactopyranoside for
5 h to induce expression. Clones expressing over 20 mg of protein/ml of culture were isolated.
Purification and Refolding-- Both D1-270b and D1-193b were expressed as inclusion bodies and were purified from protein-expressing BL21 transformants as follows. A washed cell pellet was lysed in a minimum volume of 50 mM Tris, pH 8.0, 25% sucrose, 1% Nonidet P-40, 0.1% sodium deoxycholate, 5 mM EDTA, 2 mM dithiothreitol by sonication (14 µm on ice until the decrease in viscosity indicated that all DNA had been sheared). Following cell disruption, and the removal of cell debris by centrifugation, inclusion bodies were pelleted (5 min, 10,000 rpm). Inclusion bodies were then washed twice in 25 mM Tris, pH 8.4, 2 M NaCl, 2 M urea, 2 mM dithiothreitol, and resuspended in 25 mM Tris, pH 8.4, 8 M urea. D1-193b refolding was carried out by dilution into 100 mM Tris, pH 8.0, containing 2 mM EDTA, 0.5 M arginine as a stabilizer, and a redox couple consisting of 0.5 mM oxidized, 5 mM reduced glutathione to encourage appropriate formation of the intramolecular disulfide bond between Cys101 and Cys164. After concentration in a Centriprep C10 (Amicon), the renatured product was purified by gel filtration on a Superdex 75 FPLC column equilibrated with TBS, using FPLC (Pharmacia Biotech Inc.). Fractions containing soluble, monomeric D1-193b were collected, pooled, and concentrated further by membrane filtration. Protein concentrations were determined by the method of Lowry.
Small, experimental refolding reactions were carried out for analysis by enzyme-linked immunosorbent assay in which 50 µg of D1-193b or D1-270b inclusion bodies resuspended in 10 µl of 8 M urea were refolded in 2 ml of refolding buffer containing different concentrations of peptide for 48 h at 4 °C. Where appropriate, the refolding buffer also contained 1.36 µM recombinantImmunoprecipitation-- 1-ml samples of D1-193b containing 2-10 µg of protein were incubated at 4 °C with 50 µM of either the H2-Db-restricted influenza A nucleoprotein epitope residues 366-374 (ASNENMDAM), or the H2-Db and H2-Kb-restricted Sendai virus nucleoprotein epitope residues 324-332 (FAPGNYPAL) for 1 h. D1-193b was then immunoprecipitated by adding the antibodies (10 µg/ml for the monoclonal antibodies, 1/500 dilution for the antiserum), incubating for 90 min at 4 °C, then precipitating immune complexes with 50 µl of 10% w/v protein A immobilized on Sepharose 4B (Sigma) for 30 min. The precipitates were washed four times in TBS, then analyzed by SDS-polyacrylamide gel electrophoresis.
Enzyme-linked Immunosorbent Assay-- 96 flat-bottomed well, UPVC plates were coated overnight with 200 µl of 1 µM soluble D1-193b-ASNENMDAM complex in phosphate-buffered saline. Excess sites were blocked with 2% bovine serum albumin in phosphate-buffered saline at 4 °C for 2 h. 100 µl of unpurified refolding mix were preincubated at 4 °C for 2 h with BB7.2 at a final concentration of 250 ng/ml in phosphate-buffered saline, 2% bovine serum albumin, after which time the whole mixture was transferred to wells of the coated plate and incubated at 4 °C for 1 h. After removal of the mixture, the wells were washed three times in 200 µl TBS, 0.5% (v/v) Tween 20, 0.4% bovine serum albumin, and 200 µl of alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma) were added (1/1000 dilution in TBS) and incubated for 1 h at room temperature. Following a further three washes, 200 µl of substrate containing 1 mg/ml p-nitrophenyl phosphate in 200 µl of TBS were then added to each well and incubated at 37 °C for 20 min before reading A405 on a Titertek Multiskan plate reader.
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RESULTS |
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Cloning, Expression, and Purification of the H2-Db
Peptide-binding Platform D1-193b--
The
193-amino acid fragment amplified from cDNA lacks the signal
sequence (residues 1-22), most of the 3 domain, the
transmembrane, and cytoplasmic domains and is called
D1-193b. In addition, the C terminus of
the fragment was tagged with four additional histidine residues, which
along with the two naturally occurring histidine residues at the C
terminus of this fragment, could be used to purify the protein by
nickel chelation chromatography. We chose this fragment rather than a
shorter one encompassing only the
1 and
2
domains in order to preserve a conserved salt bridge between
Arg181 and Asp183, which we felt could be
important in stabilizing the platform. Previous attempts to express
slightly shorter fragments of other alleles have been unsuccessful in
producing a soluble antigen binding
platform.2
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Biological Activity of D1-193b--
We have
previously shown that, in the absence of 2-m, short
peptides that specifically bind to Db can induce a
conformational change in the heavy chain in cell lysates (4, 14, 15).
We therefore investigated the ability of Db-binding
peptides to induce a conformational change in
D1-193b in solution. In TBS (and
TBS/arginine), the soluble monomer was unreactive with two monoclonal
antibodies that recognize epitopes present in the native conformation
of H2 Db
1 and
2 domains (B22
and 27-11-13). It was, however, recognized by antiserum T18 which was
raised to D1-193b (see "Experimental
Procedures"). Fig. 3 shows that, when
either of the Db binding peptides (FAPGNYPAL or ASNENMDAM)
were added to a final concentration of 50 µM, the
conformation-sensitive epitope is recovered, and
D1-193b could now be recognized by mAb
B22.249. This is more readily observed for the latter peptide, which
has a higher apparent Ka for
H2-Db,3 and
strongly suggests that the peptides induce a conformational change in
D1-193b upon binding. No such
conformational change is observed when a control peptide (the
H-2Kb-binding peptide SIINFEKL) was added to a
concentration of 100 µM (Fig. 3, c and
d). These results are consistent with those obtained for
full-length and truncated heavy chains studied in detergent lysates of
mammalian cells that do not express
2-m (4, 14, 15).
These chains, unlike D1-193b, are
N-glycosylated. Thus the absence of N-linked
carbohydrate does not appear to influence the ability of the
1 and
2 domains to undergo this
conformational change in response to optimal peptides.
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DISCUSSION |
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This is the first description of a soluble fragment of an MHC
class I molecule comprising the two N-terminal domains of the protein
that form the antigenic peptide-binding site. In the absence of
peptides, this 193-amino acid fragment is reasonably stable in
physiological buffers, but the two domains exist in a non-native conformation as judged by their inability to be recognized by monoclonal antibodies raised against the native whole molecule. A
similar situation is seen for full-length heavy chains synthesized in
mammalian cells that lack 2-m. Here, although the
immunolglobulin-like
3 domain appears to be in a native
conformation, the
1 and
2 domains do not.
In detergent lysates, these molecules undergo a conformational change
when they bind to antigenic peptides in the absence of
2-m (4, 14-16), such that the
1 and
2 domains acquire epitopes present in the native
structure. We have also suggested that peptides bind to the non-native
conformation of heavy chain and in doing so, initiate the
conformational change (15). This conformational change does not require
the presence of the
3 domain (14), an observation which
led us to speculate that it might be possible to observe the same
conformational change in the isolated peptide-binding platform formed
by the
1 and
2 domains. Our results show
that this is indeed the case and open up the possibility of studying
the conformational change by conventional biophysical techniques.
Indeed, D1-193b is of a size
which is compatible with two- dimensional NMR spectral analysis (22, 23).
We have suggested that the conformational change which we observe for
free heavy chain, and now the isolated peptide-binding platform, may be
related to a conformational change, which is thought to occur in the
heavy chain when peptides bind to the assembled HC:2-m
heterodimer in vivo. This has been observed indirectly by
fluorescence transfer between two MHC class I-bound mAb (17), and by
immunoprecipitation of peptide receptive and peptide-bound
H2-Ld molecules with mAb which discriminate between the two
forms (24). A more persuasive, but no less indirect, indication of the
conformational change arose from a measurement of the kinetic and
thermodynamic binding constants of H2-Db for
Db-binding peptides (12, 13). This study showed that, for
an N-terminally extended peptide ligand, the measured association rate
constant corresponded to that predicted from a simple one-step binding
model in which Ka = ka/kd. However, for the optimal
length peptide, the measured association rate is two orders of
magnitude higher than that predicted from the kd and
Ka. This led to the proposal that a conformational
change in the class I molecule was responsible for the observed the
mismatched kinetics of peptide binding, and that only peptides of the
optimum length can bring it about. It is interesting to note that the
conformational change seen in free heavy chains and
D1-193b is also observed only in
response to optimal length peptides, and that these appear to
induce the conformational change rather than simply
stabilize the native conformer preferentially (15).
Since the solution of the first MHC class I structure (25),
immunologists and structural biologists have been puzzled by the
observation that the peptide ligand is so deeply buried in the
peptide-binding groove as to be considered part of the MHC class I
structure, and have found it difficult to visualize how peptides might
diffuse into the binding site as it appears in the crystal structures.
This is made all the more difficult when the relatively rapid
association rates that have been measured are taken into account. A
peptide-induced conformational change from a more "open" or
"receptive" peptide-binding groove to the "closed" structure
seen by x-ray crystallography would be consistent with these
observations. The exact molecular dynamics that constitute this
conformational change are entirely unknown at the present time, but a
testable model has recently been proposed that involves a movement in
the short helix of the
2 domain (26).
A peptide-induced conformational change in the MHC class I molecule
might also explain why newly assembled HC:2-m
heterodimers are retained in the endoplasmic reticulum until they
become loaded with peptides of an appropriate length and sequence. It
is possible that a cofactor in the ER with an ER-retention signal is
able to bind to peptide-receptive but not peptide-loaded MHC class I
molecules and that the ability to discriminate between the two forms is
due to a peptide-induced conformational change. Indeed, in a
nonphysiological experimental system, invariant chain (a cofactor
molecule that is normally involved in the biogenesis of MHC class II
molecules and not class I) has been shown to bind to H2-Db
in a peptide-sensitive manner (27) in vivo. Other,
physiologically relevant candidates are the calcium-binding chaperones
calnexin and calreticulin (28, 29) and the transporter associated with antigen processing (30, 31).
The production of D1-193b and the demonstration that in the presence of specific optimal peptides it is recognized by conformation-sensitive mAb, provides, for the first time, a means of studying the peptide-induced conformational change by direct biophysical methods.
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FOOTNOTES |
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* This work was supported in part by the Wellcome Trust and the Nuffield Foundation.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.
Wellcome Senior Fellow in Basic Biomedical Science. To whom
correspondence should be addressed. Tel.: 01865 221949; Fax: 01865 22901.
1
The abbreviations used are: MHC, major
histocompatibility complex; HC, heavy chain; 2-m,
2-microglobulin; ER, endoplasmic reticulum; FPLC, fast
protein liquid chromatography; TBC, Tris-buffered saline; mAb,
monoclonal antibody; ELISA, enzyme-linked immunosorbent assay; WT, wild
type.
2 P. Bjorkman, personal communication.
3 T. Elliott, unpublished observation.
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REFERENCES |
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