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Binding of Longer Peptides to the H-2Kb
Heterodimer Is Restricted to Peptides
Extended at Their C Terminus: Refinement
of the Inherent MHC Class I Peptide Binding
Criteria
Heidi Hörig, Aideen C. M. Young, Nicholas J.
Papadopoulos, Teresa P. DiLorenzo and Stanley G.
Nathenson
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 1999 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 1999; 163:4434-4441; ;
http://www.jimmunol.org/content/163/8/4434
Binding of Longer Peptides to the H-2Kb Heterodimer Is
Restricted to Peptides Extended at Their C Terminus:
Refinement of the Inherent MHC Class I Peptide
Binding Criteria 1
Heidi Hörig,* Aideen C. M. Young,2* Nicholas J. Papadopoulos,3* Teresa P. DiLorenzo,* and
Stanley G. Nathenson4*†
A
class I MHC molecule is a trimolecular complex formed
by noncovalent association of an MHC-encoded heavy
chain, b2-microglobulin (b2m)5, and a peptide (1). The
cell surface presentation of peptide fragments of cellular proteins
by class I molecules enables CTL to recognize and eliminate virally infected or transformed cells (2). Over the last several years,
the three-dimensional structures of a number of human and murine
MHC class I molecules have shown that a single peptide is bound
in a groove formed by the a1 and a2 domains of the MHC class I
molecule (1, 3). In general, the bound peptides are 8 –10 aa in
Departments of *Microbiology and Immunology and †Cell Biology, Albert Einstein
College of Medicine, Bronx, NY 10461
Received for publication June 1, 1999. Accepted for publication August 5, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the National Institutes of Health Grant 5R37AI07289
(to S.G.N.). H.H. and T.P.D. were supported by postdoctoral fellowships from the
Cancer Research Institute (New York, NY). A.C.M.Y. was supported by a postdoctoral fellowship from the Irvington Institute for Immunological Research (New York,
NY). N.J.P. was supported by National Institutes of Health Training Grant
5T32CA09173.
2
Current address: PPS International Communications Ltd., P.O. Box 4141, International House, Worthing, West Sussex, U.K. BN11 1BZ.
3
Current address: Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road,
Tarrytown, NY 10591.
4
Address correspondence and reprint requests to Dr. Stanley G. Nathenson, Albert
Einstein College of Medicine, Chanin Building 407, 1300 Morris Park Avenue,
Bronx, NY 10461. E-mail address: [email protected]
5
Abbreviations used in this paper: b2m, b2-microglobulin; ER, endoplasmic reticulum; RP-HPLC, reverse phase HPLC; VSV, vesicular stomatitis virus; PC, C-terminal
peptide residue.
Copyright © 1999 by The American Association of Immunologists
length, depending on the particular class I allele examined. For
example, H-2Kb binds peptides of 8 –9 residues, while H-2Db
binds peptides that are 9 –10 residues long (4). However, peptides
longer than 8 –10 residues have been isolated from class I molecules purified from cellular extracts (5– 8).
Analysis of MHC class I three-dimensional structures suggests
that there is little variability in the canonical peptide length of
8 –10 aa due to the fact that the peptide binding cleft is itself of
defined length. In general, the peptide N and C termini are located
in pockets abutting the ends of the cleft, where they are involved
in conserved H-bonding interactions with conserved residues in
the cleft (1, 3). These H-bonding interactions are important in stabilizing the MHC-peptide complex, as demonstrated by studies
substituting either the amino or carboxyl terminus of a peptide
bound to HLA-A2 with a methyl group, thereby abrogating the
H-bonding interactions (9). Peptides with both termini modified
simultaneously did not promote stable binding with HLA-A2, suggesting that at least one peptide terminus must be bound within the
peptide binding groove via the conserved array of H-bonding interactions for a stable complex to be formed. Different length peptides have been shown to be accommodated by a single class I
allele by having the longer peptide bulge or zigzag in the middle,
while still preserving the binding of the peptide termini in the class
I cleft (10 –12). In one case, an additional glycine residue added to
the C-terminal end of an antigenic 9 mer core peptide was accommodated by extension out of the peptide binding groove, while the
binding of the first nine amino acids of this decamer was similar to
that of several nonamers with which HLA-A2 had also been crystallized (13). To our knowledge, there is no structural evidence that
extensions at the amino-terminal peptide end might be similarly
0022-1767/99/$02.00
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MHC class I molecules usually bind short peptides of 8 –10 amino acids, and binding is dependent on allele-specific anchor
residues. However, in a number of cellular systems, class I molecules have been found containing peptides longer than the
canonical size. To understand the structural requirements for MHC binding of longer peptides, we used an in vitro class I MHC
folding assay to examine peptide variants of the antigenic VSV 8 mer core peptide containing length extensions at either their N
or C terminus. This approach allowed us to determine the ability of each peptide to productively form Kb/b2-microglobulin/
peptide complexes. We found that H-2Kb molecules can accommodate extended peptides, but only if the extension occurs at the
C-terminal peptide end, and that hydrophobic flanking regions are preferred. Peptides extended at their N terminus did not
promote productive formation of the trimolecular complex. A structural basis for such findings comes from molecular modeling
of a H-2Kb/12 mer complex and comparative analysis of MHC class I structures. These analyses revealed that structural constraints in the A pocket of the class I peptide binding groove hinder the binding of N-terminal-extended peptides, whereas
structural features at the C-terminal peptide residue pocket allow C-terminal peptide extensions to reach out of the cleft. These
findings broaden our understanding of the inherent peptide binding and epitope selection criteria of the MHC class I molecule.
Core peptides extended at their N terminus cannot bind, but peptide extensions at the C terminus are tolerated. The Journal of
Immunology, 1999, 163: 4434 – 4441.
The Journal of Immunology
4435
Table I. Names and amino acid sequences of peptides used in this study
Peptide Name
VSV 8merN52–59
C-terminal extended peptidesa
C-12 mer
C-12 mer-P9 mix
C-12 mer-P10 mix
N-terminal extended peptidesa
N-9 mer
N-10 mer
N-11 mer
N-12 mer
N-9 mer-P21 mix
a
b
c
Peptide Sequence
R G Y V Y Q G L
R G Y V Y Q G L K S G N
R G Y V Y Q G L Xb S G N
R G Y V Y Q G L K Xb G N
L
D L
S D L
L S D L
Xc
R
R
R
R
R
G
G
G
G
G
Y
Y
Y
Y
Y
V
V
V
V
V
Y
Y
Y
Y
Y
Q
Q
Q
Q
Q
G
G
G
G
G
L
L
L
L
L
The C- and N-terminal amino acid extensions are according to the natural sequence of the VSV nucleoprotein.
X 5 Q, R, D, T, M, L, F.
X 5 Q, K, R, D, T, M, F.
Materials and Methods
Peptide synthesis and purification
VSV peptide and its variants were synthesized by the solid phase method
using F-moc chemistry on an Applied Biosystems 433A peptide synthesizer (Foster City, CA). All peptides were purified by RP-HPLC to .95%
purity with a Vydac C18 column (2.1 or 4.6 mm 3 25 cm; 300 Å) on a
Hewlett Packard HP-1090 M instrument (Palo Alto, CA). Peptides were
analyzed by electrospray ionization mass spectrometry on a PE-Sciex APIIII instrument (PE Biosystems, Foster City, CA) to confirm their identity.
The peptide variants synthesized are shown in Table I. These include
the C-12 mer peptide, consisting of the core VSV peptide with the LysSer-Gly-Asn (KSGN) extension at the C-terminal end; the C-12 mer-P9
mix, in which the P9 position of the C-12 mer consists of a mixture of the
amino acids Gln, Arg, Asp, Thr, Met, Leu, and Phe; and the C-12 mer-P10
mix in which the P10 amino acid position of the C-12 mer peptide is
substituted by the same mixture of amino acids. N-terminal-extended peptides are the N-9 mer, N-10 mer, N-11 mer, and N-12 mer in which amino
acids Leu, Asp, Ser, and Leu are added sequentially to the N-terminal end
of the core VSV peptide (Table I); and the N-9 mer-P21 mix, in which the
P21 peptide position, amino terminal to Arg at P1 of the core VSV peptide,
consists of a mixture of the amino acids Gln, Lys, Arg, Asp, Thr, Met, and
Phe. In each peptide mixture, equimolar amounts of the different peptide
variants are present.
Purification of H-2Kb heavy chain and b2m light chain
Details of the cloning and expression of recombinant murine Kb heavy
chain and b2m have been reported previously (30). Briefly, Kb and b2m
were produced as inclusion bodies in Escherichia coli and solubilized in
8.0 M urea, 10 mM Tris (pH 8.5), and 50 mM reduced glutathione. b2m
was dialyzed (500 molecular weight cutoff) against 10 mM Tris (pH 8.5)
for 24 h and purified by gel exclusion chromatography (Superdex-75, Pharmacia, Piscataway, NJ) in 10 mM potassium phosphate, pH 7.0. Kb heavy
chain was purified by ion exchange chromatography in 4 M urea and 20
mM Tris-HCl (pH 8.5) on a MonoQ column (Waters, Milford, MA). The
protein purity was verified by SDS-PAGE and silver staining.
Assembly of Kb/b2m/peptide complexes
Heavy chain (Kb), light chain (b2m), and peptide were combined in a molar
ratio of 1:3:5, as described in detail previously (29). Approximately 6 nmol
of Kb were precipitated with 10% TCA, washed twice in ethanol, and
air-dried. To this pellet, b2m and peptide were added in the presence of
17.5 mM reduced glutathione and enzymatic inhibitors (1 mM EDTA, 10
mM each of amastatin and leupeptin). The reaction mixture was buffered in
55 mM Tris-HCl (pH 8.5) and incubated at 4°C for 24 h on a rotating
platform.
Analysis of the molar stoichiometry of the individual class I
components
After 24 h, the Kb/b2m/peptide complex (Mr, 45 kDa) was separated by gel
exclusion chromatography using a Superdex-75 column (Pharmacia) equilibrated in 10 mM potassium phosphate (pH 7.0) and 150 mM NaCl operating at a flow rate of 0.75 ml/min. The peak containing the ternary class
I complex was isolated and denatured in 800 mM GnHCl. The denatured
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accommodated. Therefore, structural features of the class I cleft
might limit the binding of peptides with extensions at their
amino-terminal end.
Within the cell, the endogenous peptides available for class I
binding are generally limited to those generated by Ag processing
and transported into the endoplasmic reticulum (ER) by the TAP.
The TAP preferentially transports peptides of 8 –12 aa in length;
however, longer peptides (,24 aa) can also be transported, although at a reduced efficiency (14 –16). The present evidence suggests that final processing of class I ligands may occur in the ER,
but details of such processing are still controversial. Although the
presence of various ER resident proteases has been suggested (8,
17–19), some longer peptides might be protected through binding
to the MHC molecule itself (20) or to chaperones (21–24). Others
have suggested that peptides are directly handed off from the TAP
into the class I binding cleft without ever being free in solution (25,
26). In this model, the newly synthesized class I heterodimers
physically associate with the TAP until the peptide is properly
positioned in the binding cleft, which then leads to the release of
the ternary class I complex (27, 28). Thus, longer peptides would
only be found associated with class I molecules if they fulfilled the
structural requirements for peptide transport and final delivery to
the class I heterodimer.
In the in vitro study described here, we examined the inherent
ability of MHC class I molecules to bind extended peptides without the limitations of a cellular system. We employed a detergentfree in vitro folding system (29) using highly purified recombinant
murine heavy chain (Kb), light chain (b2m), and synthetic variants
of the vesicular stomatitis virus (VSV) 8 mer core peptide, the
immunodominant epitope of the VSV nucleocapsid protein. Synthetic variants of the VSV peptide used in this study included those
with amino acid extensions at the N or the C terminus as well as
extended peptides containing various substitutions. Using stoichiometric analysis based on the molar extinction coefficients of the
individual class I components at 280 nm, we have examined
whether peptides extended at either their N or C terminus can
promote productive formation of the trimolecular class I complex.
We have also examined whether the chemical nature of the extension affects the efficiency of complex formation. The availability of
the three-dimensional structure of the H-2Kb/VSV 8 mer complex
(30) allowed us to use energy minimization studies and three-dimensional modeling to provide a structural explanation for our in
vitro folding results. We propose a revised model of the inherent
peptide binding capabilities of the MHC class I and discuss the
possible implications for peptide delivery to the class I.
4436
BINDING OF EXTENDED PEPTIDES TO H-2Kb
FIGURE 1. Gel exclusion profiles of
MHC class I assembly reactions after 24 h of
incubation. Assembly reactions of Kb/b2m/
VSV 8 mer (solid line) and Kb/b2m/C-12
mer (dashed line) were separated on a Superdex-75 column as specified in Materials
and Methods, and absorbance at 214 nm was
recorded. Putative MHC class I complexes
(;45 kDa) elute at 15.0 min as indicated.
The other peaks in the profile are also identified (GSSG, oxidized glutathione; GSH, reduced glutathione).
Energy minimization study of the H-2Kb/C-12 mer complex
The C-12 mer peptide was modeled by building KSGN onto the end of the
core VSV eight-residue peptide in the H-2Kb/VSV 8 mer structure (30)
using the molecular modeling program TOM (31). The backbone torsion
angles of KSGN were adjusted until all obvious steric overlaps with the
MHC were removed, and the extension lay roughly flat along the surface
of the protein. This model was then subjected to 1000 cycles of steepest
descent energy minimization using INSIGHT (Biosym Technologies, San
Diego, CA). All atoms of the MHC were fixed with the exception of those
within 6 Å of peptide residues 7–12. Peptide residues 1–7 were also fixed,
and peptide residue 8 (Leu) was tethered with a force constant of 100 Cal
mol21 Å22. The final minimized structure was analyzed in INSIGHT for
steric overlaps, of which there were none, and the Ramachandran plot
for the peptide was created using PROCHECK (32). All peptide bonds in
the extended peptide were located in allowed regions of the Ramachandran plot.
Reagents
Biochemicals were obtained from Sigma (St. Louis, MO) unless otherwise
stated. Sequanal grade urea and 8 M GnHCl were obtained from Pierce
(Rockford, IL). All water used was passed through a MilliQ1 apparatus.
HPLC solvents were obtained from Burdick & Jackson (Muskegon, MI).
Results
C-terminal-extended peptides allow a higher H-2Kb folding
efficiency than do N-terminal-extended peptides
Highly purified recombinant H-2Kb heavy chain (33 kDa) and b2m
(11 kDa) were assembled together with the VSV 8 mer peptide or
its N- and C-terminal-extended peptides (;1 kDa; see Table I)
using our previously described in vitro folding method (29). After
incubation, the MHC class I complexes (;45 kDa) were purified
by gel size exclusion chromatography (Fig. 1). For the VSV 8 mer
and all the extended peptides, the formation of an ;45-kDa product was observed. A comparison of the integrated area of each
complex peak at 280 nm allowed us to estimate the efficiency with
which each N- and C-terminal-extended peptide promoted folding
of a class I complex (Table II). Folding of Kb, b2m, and the core
VSV 8 mer peptide was used as the standard and was defined as
100%. Although it appears as though all the peptide variants promoted complex formation, none did so as efficiently as the VSV 8
mer. Complex formation with each of the carboxyl-terminal-extended peptides occurred with an efficiency of 30%, while the efficiency of complex formation using amino-terminal-extended
peptides was even lower, ranging from 10 –20%, depending on the
length and composition of the N-terminal extension (Table II). As
previously reported, folding of an H-2Db-binding peptide (ASNENMETM) from influenza virus did not lead to the formation of
a 45-kDa product, presumably due to the lack of anchor residues
that fulfill the Kb motif requirement (29). Similarly, a 45-kDa
product was never observed in the absence of peptide.
Stoichiometry of H-2Kb/peptide complexes reveals ternary
complex formation with C-terminal-extended peptides, but not
with N-terminal-extended peptides
To determine the molar stoichiometry of the individual class I
components (heavy chain, b2m, and peptide) within each of the
purified MHC class I complexes, the isolated 45-kDa products
were denatured and subjected to RP-HPLC analysis, and the
280-nm peaks corresponding to the individual components were
integrated.
In Fig. 2, RP-HPLC chromatograms are shown for the denatured
H-2Kb/VSV 8 mer (Fig. 2A) and H-2Kb/C-12 mer (Fig. 2B) complexes. The VSV 8 mer and C-12 mer peptides were identified by
their unique retention times (Fig. 2), and these assignments were
confirmed by mass spectrometric analysis and protein microsequencing (data not shown). In the case of trimolecular complex
formation, the individual class I components should be present in
equimolar ratio. To estimate the molar ratio of the class I components, we used their molar extinction coefficients at 280 nm: VSV
8 mer peptide, 2,560; b2m, 17,900; and Kb, 73,270 (29). Based on
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complex was then separated by RP-HPLC with a Vydac C18 column (2.1
mm 3 25 cm, 300 Å, 0.2 ml/min, 1%/min increase in acetonitrile (0.1%
trifluoroacetic acid)) on a Hewlett Packard HP-1090 M instrument, and its
individual components were assayed via their absorbance at 214 and 280
nm. Integration of the individual 280-nm peaks corresponding to heavy
chain (MHC class I), light chain (b2m), and peptide allowed for the calculation of the molar stoichiometry of the three components in each complex. The integration of the heavy chain peak area was difficult to assess,
as Kb eluted in a tailing peak caused by its highly hydrophobic nature, as
previously reported (29). The individual peaks corresponding to each component in the complex were collected and analyzed by mass spectrometry.
The identity of the Kb peak was verified by mass spectrometry across the
tailing peak. Furthermore, the formation of H-2Kb/VSV 8 mer, H-2Kb/
C-12 mer, and H-2Kb/C-12 mer-P9 mix complexes was verified by protein
microsequencing on an Applied Biosystems 477A protein sequencer.
The Journal of Immunology
4437
Table II. Efficiency of complex formation and molar stoichiometry for the various N- and
C-terminal-extended peptides
Peptide
VSV 8 mer
C-terminal extended peptides
C-12 mer
C-12 mer-P9 mix
C-12 mer-P10 mix
N-terminal extended peptides
N-9 mer
N-10 mer
N-11 mer
N-12 mer
N-9 mer-P21 mix
Efficiency of
Complex Formationa
(%)
Integrated HPLC Peak Area
Ratio at 280 nm,b
(peptide:b2m:(Kb)
Estimated
Molar Ratio,
(peptide:b2m)
100
1:7:(19)
1:1.0
25–30
25–30
25–30
1:11:(39)
1:10:(36)
1:10:(37)
1:1.6
1:1.4
1:1.4
15–20
15–20
15–20
10–15
10–15
1:27:(53)
1:27:(73)
1:27:(50)
NDc
ND
1:3.9
1:3.9
1:3.9
ND
ND
a
Using the in vitro folding system, the efficiency of complex formation using the VSV 8 mer peptide was ;20 –30%. To
compare the efficiency of complex formation using VSV peptide analogs, the VSV 8 mer efficiency was set to 100%.
b
Due to the tailing nature of the Kb peak, its value could not be accurately determined and is therefore shown in parentheses.
c
There was no peptide peak identified and, therefore, no ratio determined.
FIGURE 2. RP-HPLC separation of denatured
MHC class I complexes. A, H-2Kb/VSV 8 mer; B,
H-2Kb/C-12 mer. After assembly, putative MHC
class I complexes were purified by gel exclusion
chromatography, denatured as described in Materials and Methods, and the individual class I components were separated on a Vydac C18 column (2.1
mm 3 25 cm) using a flow rate of 0.2 ml/min, and
a gradient of 1%/min acetonitrile containing 0.1%
trifluoroacetic acid. Absorbance at 280 nm is shown.
The retention times of the individual components
are: C-12 mer, 26.8 min; VSV 8 mer, 31.5 min; b2m,
42.7 min; Kb, 46.2–56 min.
(Table II), not even trace amounts of peptide could be identified on
denaturation, even though an ;45-kDa complex had been observed upon gel size exclusion chromatography. The ratio of one
peptide molecule to nearly four b2m molecules for the N-9 mer,
N-10 mer, and N-11 mer, along with the absence of peptide in the
complex formed in the presence of N-12 mer peptide, suggests that
N-terminal-extended peptides could only initiate the folding of
empty class I heterodimers without the peptide being placed into
the class I binding groove. These results support the idea that
N-terminal-extended peptides are unable to productively form trimolecular H-2Kb complexes. As the isolated complexes were immediately subjected to denaturation and subsequent RP-HPLC
analysis, the degradation of complex can be excluded.
Peptides with hydrophobic residues in their C-terminal flanking
regions bind preferentially to the H-2Kb heterodimer
We next investigated whether the amino acid composition of the
peptide extensions might influence peptide binding and proper
folding of MHC class I complexes. To do this, a peptide mixture
(C-12 mer-P9 mix) containing various amino acids (Gln, Arg, Asp,
Thr, Met, Leu, and Phe) at position 9 in the C-terminal extension
of the C-12 mer peptide was synthesized (Table I). The substituted
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these molar extinction coefficients, the ratio of the integrated
HPLC peak areas at 280 nm should be 1:7:29 for a trimolecular
complex in which peptide, b2m and Kb have a molar stoichiometry
of 1:1:1. Experimentally, for the H-2Kb/VSV 8 mer complex, although the integrated HPLC peak areas of the VSV 8 mer peptide
and b2m did reveal the predicted 1:1 stoichiometry, the Kb value
was only 0.6. This is due to the highly hydrophobic nature of the
heavy chain Kb causing a tailing peak eluting throughout a 10-min
period. As the VSV 8 mer peptide and b2m were in the predicted
1:1 ratio, we used the relative stoichiometry of peptide and b2m as
a measure of trimolecular complex formation in our experiments
with the extended peptides. Hence, the molar ratio obtained for the
carboxyl-terminal-extended C-12 mer peptide complex was 1:1.6
(peptide:b2m; Table II). This result indicates a less ideal ratio of
peptide to b2m, which may be explained by the lower affinity of
the C-12 mer, leading to some peptide displacement or some
empty peptide binding grooves.
Considerably less peptide was identified in those complexes
formed by folding with peptides extended at their N termini (Table
II). Complex formation with the N-terminal-extended peptides N-9
mer, N-10 mer, and N-11 mer resulted in an estimated molar stoichiometry of peptide to b2m of 1:3.9. With the N-12 mer peptide
4438
BINDING OF EXTENDED PEPTIDES TO H-2Kb
amino acids were representative in terms of their acidic (Asp),
basic (Arg), polar (Gln, Thr), or hydrophobic (Met, Leu, Phe)
properties. A second peptide mixture (C-12 mer-P10 mix) was also
generated, with the amino acids mixture at position P10 of the
C-12 mer peptide (Table I). Both peptide mixtures gave rise to the
same folding efficiency for H-2Kb complex formation as the C-12
mer peptide (30% of that of the core VSV peptide; Table II). As
before, the 45-kDa complexes were isolated by gel exclusion chromatography, then denatured and subjected to RP-HPLC analysis.
The area under each peptide peak of the peptide stock (Fig. 3, A
and B, upper panels) and of each peptide peak of the denatured
class I complexes (Fig. 3, A and B, bottom panels) was measured.
Then, the relative abundance of each peptide in the peptide stock
and in the complex was compared, and this allowed us to establish
whether certain peptide variants were selected for complex formation. The results show that for the C-12 mer-P9 mix, those peptides
with Met, Leu, and especially Phe at P9 were clearly selected for
binding to the MHC class I molecule over the more hydrophilic
amino acids (Gln, Arg, Asp, or Thr; Fig. 3A). Similarly, for the
C-12 mer-P10 mix, the peptide with the hydrophobic amino acid
Leu at P10 was selected for class I binding (Fig. 3B). In a complex
produced using either the C-12 mer-P9 mix or the C-12 mer-P10
mix, peptide and b2m were found in a molar ratio of 1:1.4 (Table
II), slightly more ideal than the ratio of 1:1.6 obtained using the
original C-12 mer peptide with Lys at P9 and Ser at P10. This
again suggests preferential binding of peptides with hydrophobic
residues in the C-terminal-flanking region.
As described above, the N-9 mer peptide with Leu at its N
terminus could not efficiently form trimolecular H-2Kb complexes.
To determine whether the nature of the N-terminal residue might
alter the ability of an N-terminal-extended peptide to form a trimolecular complex, we synthesized a peptide mixture (N-9 merP21 mix, Table I) containing various amino acids (Gln, Lys, Arg,
Asp, Thr, Met, Phe) at P21. Using this peptide mixture for folding,
an ;45-kDa complex was observed by size exclusion (Table II);
however, denaturation and subsequent RP-HPLC analysis yielded
not even trace amounts of any peptide. Thus, altering the composition of the N-terminal flanking region did not improve the binding properties of the N-terminal-extended N-9 mer peptide. These
results are consistent with the above-mentioned ideas that N-terminal-extended peptides fail to productively form H-2Kb complexes and that the composition of the N-terminal-flanking region
cannot adjust for the binding of the extended N-terminal peptide
end into the corresponding pocket of the binding groove.
Energy minimization studies provide a structural explanation for
the formation of the H-2Kb/C-12 mer complex
To explain our findings at the three-dimensional structural level,
we applied the molecular modeling program TOM to build the
C-terminal-extended C-12 mer peptide and to model it into the
H-2Kb cleft. The model containing the extended peptide was subjected to energy minimization as described in Materials and Methods. In this minimized structure (Fig. 4) the P8 to P9 peptide bond
is accommodated at the end of the cleft, with no perturbation of the
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FIGURE 3. Extended peptides with hydrophobic residues at P9 or P10 bind preferentially
to the H-2Kb heterodimer. MHC class I complexes were assembled using the C-12 mer peptide mixture C-12 mer-P9 (A) or C-12 mer-P10
(B). The ;45-kDa complexes were purified, denatured, and subjected to RP-HPLC as described
in Fig. 2. Absorbance at 214 nm is shown for the
expanded peptide region of the HPLC chromatograms. In A and B the upper panel shows the
peptide stock, whereas the lower panel shows
the denatured MHC class I complexes assembled
with the C-12 mer-P9 mix (A) or the C-12 merP10 mix (B). All peptide peaks were identified
by mass spectrometry.
The Journal of Immunology
P8 anchor residue in the F pocket. The KSGN extension beyond
the P8 peptide position extends out of the cleft and lies flat along
the surface of the molecule, beyond the end of the peptide binding
cleft, where it causes no steric overlap with residues of the MHC.
Thus, modeling and energy minimization studies support our findings that peptides with extensions at the C terminus can lead to the
productive formation of class I complexes. This is possible because the side chain of the P8 residue of the C-12 mer peptide is
buried as an anchor in the F pocket of the Kb binding groove,
leaving the O atom of the terminal carboxyl group exposed on the
surface of the complex where bonding to the adjoining residue
(P9) occurs. This is in contrast to the peptide binding at the Nterminal end. Here, the N terminus of the VSV peptide is completely buried (30). With the side chain of the P1 peptide residue
pointing out of the cleft, the formation of a peptide bond to the
buried N atom is sterically prohibited.
Discussion
Peptides longer than the canonical size of 8 –10 aa have been
eluted from cellular MHC class I molecules (5– 8). In one case,
however, these longer peptides were associated with the heavy
chain alone (5), rather than being part of a ternary class I complex.
Studies designed to measure peptide/MHC affinities have also suggested that extended peptides are able to bind to the class I heterodimer (33–35). However, in such studies, the stoichiometry of
the resulting complexes is not determined, so one must make the
assumption that proper folding of ternary class I complexes has
occurred. It is perhaps due to this potential limitation that peptide
affinity studies have yielded somewhat conflicting results regarding the ability of the class I molecule to accommodate N- and/or
C-terminal peptide extensions. To avoid the possible limitations of
cellular systems and peptide affinity studies, we used a cell-free,
detergent-free in vitro class I MHC folding system to investigate
the inherent ability of the class I MHC heterodimer to bind peptides longer than the canonical size. We specifically addressed
whether core peptides could be extended at their carboxyl or amino
termini. The data presented here provide both biochemical and
stoichiometric evidence that extended peptides can productively
form MHC class I complexes, but only if the peptides are C-terminally extended.
For the C-terminal-extended peptides, a putative class I MHC
complex (45 kDa) was formed during an in vitro folding reaction,
as judged by gel exclusion chromatography. After isolation and
denaturation, this was verified because we could identify each individual component of the ternary class I complex. The C-terminal-extended C-12 mer peptide and b2m were found at a molar
ratio of 1:1.6, a ratio close to the model ratio of 1:1 (VSV 8 mer
peptide:b2m; Table II). In all our experiments, the identity of the
recovered peptide was always verified by mass spectrometry. This
ensured that the input extended peptide was truly in the complex,
rather than a shortened version that could have been generated by
proteolytic clipping during the experimental period. Thus, a peptide extended at its carboxyl terminus is able to induce productive
class I complex formation, although the extent of formation of
such complexes is only 20 –30% of that of the VSV 8 mer. The
lower affinity of the C-terminal-extended peptides might cause
some peptide displacement or some empty class I binding sites. To
evaluate the importance of the chemical nature of the peptide extension, peptides with amino acid substitutions at positions P9 and
P10 of the VSV C-12 mer were tested for binding. The analysis
showed that peptides with hydrophobic amino acid residues at position P9 or P10 were preferentially selected for complex formation compared to peptides with hydrophilic residues at these
positions.
In contrast, for the N-terminal-extended peptides, the putative
class I complex was formed at an even lower efficiency than was
found for the C-terminal-extended peptides (Table II). Further, after denaturing of those putative complexes, the N-terminal-extended peptides could be detected just over background. Thus, no
substantial ternary complex formation occurred for the N-terminalextended peptides (peptide:b2m 5 1:3.9). Substitutions in the
flanking region of the N-terminal-extended peptide (P21 for the
VSV N-9 mer) did not improve the folding efficiency.
A consideration of the three-dimensional structure of trimolecular MHC class I complexes, in conjunction with modeling of
H-2Kb with the C-12 mer peptide, allowed us to provide a structural basis for our findings concerning peptide binding. Three-dimensional structures of MHC class I complexes have shown that
peptide binding in the class I cleft is dependent upon two or three
anchor residues within pockets in the cleft, as well as an array of
H-bond interactions between conserved residues of the cleft and
mainly main chain atoms of the peptide (1, 3). As it has been
demonstrated that both the anchor residues and the H-bond interactions are important for the stability of the complex, we have
assumed that if any extended peptide can bind to an MHC class I
molecule, it will do so while maintaining the binding pattern of the
core peptide. Hence, in the modeling studies performed here, we
have preserved the geometry of the core 8 residues of the VSV
peptide and the surrounding MHC cleft residues (30); consequently, this preserved the interactions between peptide and cleft
that have been seen to be critical for binding, while accommodating the extra four residues beyond the end of the cleft. The residues
in the extension could either lie along the surface of the molecule,
as shown in Fig. 4, maintaining contact with the surface of the
H-2Kb molecule or, alternatively, they could form a free and disordered tail. What is central to the binding of this extended peptide
is how the peptide bond between the main chain O atom of P8 (PC
in the core VSV 8 mer peptide) and the N atom of P9 is accommodated. In this minimized structure (Fig. 4) the P8 to P9 peptide
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FIGURE 4. Final energy minimized model of the C-12 mer peptide superimposed on the solved H-2Kb/VSV 8 mer structure. The VSV 8 mer
(RGYVYQGL) is shown in yellow, with its N-terminal -NH2 group (green)
pointing down into the P1 pocket, and its C-terminal-COOH group (pink)
pointing out of the PC pocket of the MHC class I molecule. The C-terminal
extension of the C-12 mer (KSGN) is shown in blue.
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release of loaded class I complexes (28, 38). Such a mobile region
impinging upon the F pocket may also facilitate the binding of
C-terminal-extended peptides, as long as the side chain of the PC
anchor residue of the core peptide is sufficiently anchored into the
F pocket of the binding groove, as was observed in the crystal
structure of HLA-A2 with a C-terminal extended core peptide (13).
Our modeling data of the Kb/C-12 mer peptide also suggest that
the binding of a C-terminal-extended core peptide still preserves
sufficient anchoring into the F pocket, and thus would allow the
final release of the Kb/C-12 mer peptide complex by the TAP.
Class I complexes containing C-terminal-extended core peptides
may serve a number of important functions in vivo. The extended
peptides might subsequently be cleaved and may therefore serve as
precursors for antigenic core peptides. Perhaps more importantly,
however, class I complexes containing extended peptides, due to
their relative instability, may be part of a pool of class I molecules
having readily exchangeable peptides in their peptide binding
grooves. For example, in the ER, high affinity peptides derived
from pathogens can exchange with host-derived peptides during an
infection (39). Peptide exchange may also occur at the cell surface
and in the endocytic compartment (40 – 42). Therefore, some class
I complexes containing extended peptides might be sufficiently
stable to be transported to the cell surface, where the extended
peptide could be exchanged with exogenous class I epitopes. Alternatively, endocytosis of such complexes might provide a source
of class I molecules that can readily exchange their peptides in the
endocytic compartment with peptides derived from exogenous
Ags. Thus, the ability of MHC class I molecules to bind exchangeable C-terminal-extended peptides is likely to have important functional implications.
Acknowledgments
We thank Dr. B. Birshtein for critical reading of the manuscript, Dr. R. H.
Angeletti for advice and technical help, and E. Nieves for all mass spectrometrical analysis.
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BINDING OF EXTENDED PEPTIDES TO H-2Kb
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