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Departments of
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Microbiology and Immunology and
Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 and 2 domains of
the MHC class I molecule (1, 3). In general, the bound
peptides are 8–10 aa in 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, 6, 7, 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, 11, 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 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, 15, 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, 18, 19), some longer peptides might be protected through binding to the MHC molecule itself (20) or to chaperones (21, 22, 23, 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 detergent-free in vitro folding system (29) using highly purified recombinant murine heavy chain (Kb), light chain (ß2m), 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 x 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 API-III 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 Lys-Ser-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-P-1 mix, in which
the P-1 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.
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Details of the cloning and expression of recombinant murine Kb heavy chain and ß2m have been reported previously (30). Briefly, Kb and ß2m 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. ß2m 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/ß2m/peptide complexes
Heavy chain (Kb), light chain (ß2m), 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, ß2m and peptide were added in the presence of 17.5 mM reduced glutathione and enzymatic inhibitors (1 mM EDTA, 10 µM 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/ß2m/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 complex was then separated by RP-HPLC with a Vydac C18 column (2.1 mm x 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 (ß2m), 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.
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 mol-1 Å-2. 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 MilliQ+ apparatus. HPLC solvents were obtained from Burdick & Jackson (Muskegon, MI).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Highly purified recombinant H-2Kb heavy
chain (33 kDa) and ß2m (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, ß2m, 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.
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To determine the molar stoichiometry of the individual class I components (heavy chain, ß2m, 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; ß2m, 17,900; and
Kb, 73,270 (29). Based on 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, ß2m 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
ß2m 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 ß2m were in the predicted 1:1
ratio, we used the relative stoichiometry of peptide and
ß2m 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:ß2m; Table II). This result indicates a less ideal ratio of peptide to
ß2m, which may be explained by the lower
affinity of the C-12 mer, leading to some peptide displacement or some
empty peptide binding grooves.
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). 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
ß2m of 1:3.9. With the N-12 mer peptide (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 ß2m 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 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 ß2m 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.
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) containing various amino
acids (Gln, Lys, Arg, Asp, Thr, Met, Phe) at P-1. 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 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 N-terminal 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
ß2m were found at a molar ratio of 1:1.6, a
ratio close to the model ratio of 1:1 (VSV 8 mer
peptide:ß2m; 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-terminal-extended peptides
(peptide:ß2m = 1:3.9). Substitutions in
the flanking region of the N-terminal-extended peptide
(P-1 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 bond can be accommodated at the end of the
cleft, with no perturbation of the P8 anchor residue in the F pocket
and no steric overlap with residues of the MHC. Obviously, because
there are no apparent constraints on the binding orientation of the
peptide residues that are located outside the cleft itself, the peptide
tail could assume a virtually infinite number of conformations, in any
of which there would be no steric hindrance with the MHC.
We have chosen, for the purposes of the model, to fix the geometry of the core VSV peptide as well as the surrounding residues of the MHC cleft. However, it is likely that the existence of the four-residue C-terminal peptide tail induces subtle conformational changes in the precise three-dimensional structure of the remainder of the peptide and thereby in residues lining the cleft. Indeed, small shifts in the positions of the core anchor residues and/or alterations in the H-bonding pattern between the core peptide and the MHC cleft are probably the cause of the observation that the carboxyl-terminal-extended peptides form complex with only 30% the efficiency of the core VSV peptide. Nonetheless, it remains the case that the core peptide anchor residues must be located in their appropriate cleft pockets for the extended peptide to bind. Thus, it can be assumed that the model presents a reasonable approximation of the actual complex.
These modeling studies are consistent with our data demonstrating the binding of C-terminal-extended peptides in the H-2Kb cleft. Such binding is possible because the side chain of the P8 residue of peptides binding to H-2Kb is buried within the F pocket, leaving an O atom of the terminal carboxyl group exposed on the surface of the complex. Because it appears to be universal that all MHC class I molecules bind peptides with their PC peptide residue buried as an anchor in the F pocket (4), it follows that all MHC class I molecules ought to be able to bind peptides extended at their C-terminal ends.
Similarly, the three-dimensional structures of MHC class I complexes reveal why N-terminal-extended peptides cannot bind to an MHC class I molecule, although we did not attempt to verify this through modeling. Because the N terminus of the VSV peptide is completely buried, with the P1 peptide residue side chain pointing out of the cleft, the formation of a peptide bond to the buried N atom is sterically prohibited. N-terminal-extended peptides could only bind in the MHC class I cleft via major rearrangements of the MHC residues at this end of the cleft or if the N terminus of the peptide were not bound within the cleft. Neither scenario is energetically likely, supporting our observation that H-2Kb cannot productively bind peptides extended at their N terminus. Again, we postulate that this finding should be universal for all MHC class I molecules, because they all appear to bind peptides whose P1 residue side chain is directed out of the cleft toward solvent, such that the amino-terminal group is buried in the A pocket. The importance of the proper positioning of the N-terminal peptide end had previously been suggested by Matsumura and colleagues (33) upon consideration of the affinities of extended peptides for purified class I molecules.
The processes involved in peptide delivery from the TAP to the MHC
class I molecule are still being clarified. Recent findings revealed
that the class I heterodimer physically associates with the TAP
(25, 26, 36), and several residues on the 3 domain of
the class I (219–233) have been identified to interact with the TAP
(37). A model by Elliott and colleagues (27, 28) suggested that the anchoring of the C-terminal peptide
residue (PC) in the F pocket of the class I binding groove causes a
conformational switch of the short 2 domain
(residues 139–149) from a peptide-receptive open position into a
non-peptide-receptive closed position. This conformational change would
be responsible for disrupting the interaction between the class I
molecule and the TAP, leading to the 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, 41, 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.
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Acknowledgments |
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Footnotes |
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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:
5 Abbreviations used in this paper: ß2m, ß2-microglobulin; ER, endoplasmic reticulum; RP-HPLC, reverse phase HPLC; VSV, vesicular stomatitis virus; PC, C-terminal peptide residue.
Received for publication June 1, 1999. Accepted for publication August 5, 1999.
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References |
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. 1997. The function. In MHC Ligands and Peptides
Motifs. H.-G. Rammensee, J. Bachmann, and S.
Stevanovi, eds. Springer-Verlag, Heidelberg, Germany, p.
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, J. Brunner, H. G. Rammensee, H. Schild. 1997. Protein disulfide isomerase is the dominant acceptor for peptides translocated into the endoplasmic reticulum. Eur. J. Immunol. 27:1685.[Medline]
. 1998. Physical and functional association of the major histocompatibility complex class I heavy chain 3 domain with the transporter associated with antigen processing. J. Exp. Med. 187:865.2 helix of MHC class I is revealed by the crystal structure of HLA-B*3501. Immunity 4:203.[Medline]
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 S. Jiang, N. J. Borthwick, P. Morrison, G. F. Gao, and M. W. Steward Virus-specific CTL responses induced by an H-2Kd-restricted, motif-negative 15-mer peptide from the fusion protein of respiratory syncytial virus J. Gen. Virol., February 1, 2002; 83(2): 429 - 438. [Abstract] [Full Text] [PDF]
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T. M. C. Hornell, S. M. Martin, N. B. Myers, and J. M. Connolly Peptide Length Variants p2Ca and QL9 Present Distinct Conformations to Ld-Specific T Cells J. Immunol., October 15, 2001; 167(8): 4207 - 4214. [Abstract] [Full Text] [PDF]
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G. Geginat, S. Schenk, M. Skoberne, W. Goebel, and H. Hof A Novel Approach of Direct Ex Vivo Epitope Mapping Identifies Dominant and Subdominant CD4 and CD8 T Cell Epitopes from Listeria monocytogenes J. Immunol., February 1, 2001; 166(3): 1877 - 1884. [Abstract] [Full Text] [PDF]
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 P. J. R. Goulder, Y. Tang, S. I. Pelton, and B. D. Walker HLA-B57-Restricted Cytotoxic T-Lymphocyte Activity in a Single Infected Subject toward Two Optimal Epitopes, One of Which Is Entirely Contained within the Other J. Virol., June 1, 2000; 74(11): 5291 - 5299. [Abstract] [Full Text]
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