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ugi2,
Immunology and
*
Molecular Biology Programs, Memorial Sloan-Kettering Cancer Center, New York, NY 10021;
Sloan-Kettering Division, Cornell University Graduate School of Medical Sciences, New York, NY 10021;
Howard Hughes Medical Institute, Department of Biochemistry and Biophysics, Columbia University, New York, NY 10168; and
§
National Jewish Center for Immunology and Respiratory Medicine, Denver, CO 80206
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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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106 binding
specificities/allele) and specific (only certain peptides can bind to
each allele). Promiscuity is explained by the fact that the majority of
interactions between the peptide and class I involve the main chain
atoms of the peptide and by the presence of two highly conserved
pockets, named A and F, at each end of the groove, that bind the amino
and carboxyl ends of the peptide, respectively, using conserved
hydrogen bond networks (3). These interactions are relatively
independent of the nature of peptide side chains at the N- and
C-termini of the peptide. By contrast, the binding specificity is
imparted by one or more of the remaining pockets (B–E), located in the
middle of the groove, that vary in size, shape, and importance among
different MHC class I molecules. Each allele possesses, on the
average, two dominant pockets that dictate the nature of the
complementary peptide anchor residues that bind to it. The murine class I molecule H-2Kb (Kb) has two features that distinguish it from all other classical class I molecules. It dominantly binds octameric peptides, although exceptional nonamers can bind as well (4, 5), and it uses a deep C pocket at the center of groove as its dominant anchor binding pocket (6). This pocket interacts with the characteristic amino acids found in Kb-restricted peptides: a phenylalanine or a tyrosine located at P5 in octapeptides. The specificity of the C pocket is determined by medium-sized or small residues, MHC9V, MHC97V, and MHC99S. The only other molecule that uses the C pocket as an anchor pocket is H-2Db, but its C pocket is more shallow, and polar, suitable to accommodate a small, polar P5N (5, 7). Furthermore, the H-2Db molecule binds nonamers (4, 5).
In the present study we sought to investigate the specific influence of the Kb C pocket in selecting peptide motif and length. We generated a Kb variant in which the C pocket is flat and incapable of binding canonical F or Y residues. As a consequence, the new molecule selected peptides containing at least two new strong anchor residues that compensated for the loss of binding energy caused by the lack of pocket C:P5Y/F interaction. The loss of the deep C pocket also forced the molecule to preferentially, if not exclusively, bind nonameric peptides. These results delineate the forces governing the peptide length preference of class I molecules.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Site-directed mutagenesis of the codon 9 (GTC
V
TGGW) was
performed by Kunkel’s method (8) on a 771-bp KpnI fragment
of genomic H-2Kb DNA that had been ligated into pBluescript
II SK (Stratagene, La Jolla, CA). Mutated KpnI fragment was
isolated, sequenced, and religated into the pKbTCF neo-
KpnI construct (9), which contains the 11-kb genomic
H-2Kb sequence without the 0.7-kb KpnI fragment,
as well as the bacterial neomycin resistance gene.
The P815 mastocytoma (H-2d), the TAP-deficient RMA-S lymphoma (H-2b), and a mutant of RMA-S deficient in H-2Kb gene expression (RMA-S-Kb-, provided by Drs. J. Rodgers and R. Rich, Baylor University, Houston, TX), were transfected (200 mV, 960 µF) with constructs encoding wild-type and mutant Kb molecules. Neomycin-resistant colonies were expanded, tested for Kb expression by staining with the anti-Kb mAb Y3, subcloned by limiting dilution, and maintained in RPMI 1640 medium supplemented with 7.5% FBS (10) containing 400 µg/ml neomycin.
Monoclonal Abs, peptides, and the class I stabilization assay
Anti-H-2Kb mAb Y3 (American Type Culture Collection, Rockville, MD) was used in the form of a diluted ascites fluid to detect transfected molecules. The isotype control (mouse IgG2b) and secondary phycoerythrin-conjugated goat anti-mouse IgG2b were purchased from Fisher Biotech (Malvern, PA). Staining and flow cytometric (FCM)3 detection were described previously (10).
Peptides were obtained from the Memorial Sloan-Kettering Cancer Center Microchemistry Core Facility or Research Genetics, Inc. (Huntsville, AL). Original references for the peptides OVA-8 (SIINFEKL), HSV-8 (SSIEFARL), VSV-8 (RGYVYQGL), SEV-9 (FAPGNYPAL), SVT-9 (VVYDFLKCL), and HIV-10 (RGPGRAFVTI) are given in Ref. 10. Sequences of the other peptides are given in the text and figure legends. Substituted peptides are named to indicate the position and the amino acid replacement in the sequence, relative to the original peptide (e.g., P3A denotes replacement by alanine at the third residue of the peptide).
Class I stabilization assay was performed exactly as described previously (11, 12). Briefly, transfected RMA-S-Kb- or wild-type RMA-S indicator cells were incubated overnight at 29°C, washed, and incubated in HBSS in the presence of the indicated concentration of peptides at 29°C for 30 min. Samples were then diluted with 40 vol of HBSS prewarmed to 37°C and incubated for 4 h at 37°C. Cells were washed three times, stained with Y3, and analyzed by FCM, as described. Results are shown as the mean Kb or KbW9 fluorescence intensity (MFI) of the indicator cells at indicated peptide concentration or, where direct comparisons between Kb and KbW9 stabilization was necessary, as the percent maximal stabilization, calculated as (experimental MFI - control MFI/maximal MFI - control MFI) x 100. The control MFI was obtained from cells incubated without peptide at 37°C, while the MFI of cells at 29°C was taken as maximal.
Peptide elution, purification, and structural analysis
Peptides were eluted from Kb and
KbW9 molecules as described previously (13). Briefly, the
manufacturer’s (Pharmacia, Piscataway, NJ) instructions were
followed for coupling 10 mg of purified Y3 (anti-Kb,
specific) or 11.5.2.1.9 (anti-I-Ak, irrelevant) mAbs to
3 g of dry Sepharose. Swelled, mAb-coupled Sepharose 4B beads were
stored at 4°C in PBS/sodium azide, and 1 ml was transferred to
columns for affinity chromatography. P815-KbW9 or E.G7
(Kb-expressing) cells were expanded in 2 l roller
bottles, counted, collected, and washed once with 100 vol of
cold PBS. Cell pellets were lysed by shaking on ice for 15 min using 10
vol of the ice-cold lysis buffer (PBS containing 1% Nonidet P-40, 0.1
mM PMSF, 1 mM EDTA, 10 mM iodoacetamide, 1 µg/ml each of leupeptin
and aprotinin, and 0.2% sodium azide), and further procedures were
conducted strictly at 4°C or on ice. Cell lysates were centrifuged at
30,000 x g for 30 min, supernatants were collected,
and recentrifuged at 150,000 x g for 2 h. These
supernatants were stored at -70°C until a total of 1 x
1010 P815-KbW9 or 1 x 109
E.G7 cells had been processed. For affinity chromatography, 1 ml of
mAb-coupled Sepharose 4B was transferred to glass columns. Cell lysates
were precleared by passing over an irrelevant column
(anti-I-Ak specific), and the flow-through was applied
to the specific (anti-Kb) columns. After washing with
10 ml of ddH2O, the beads in each column were resuspended
in 2 ml of ice-cold 0.1% trifluoroacetic acid, and rotated for 10 more
min at 4°C. The trifluoroacetic acid extract solutions were spun
through Centricon-3000 columns as indicated by the manufacturer
(Amicon, Beverly, MA), and the flow-through fractions were dried by
vacuum centrifugation (Speed-Vac, Savant, Farmingdale, NY).
Peptides were fractionated by microbore reverse phase HPLC. Solvents
and system configuration were previously described (14), except that a
1.0-mm Reliasil C18 (Column Engineering, Toronto, Ontario,
Canada) column was eluted at a flow rate of 30 µl/min. Mass analysis
of individual peptides was conducted using a model Voyager RP
matrix-assisted laser desorption ionization time-of-flight
instrument (PerSeptive, Framingham, MA) in the linear mode, with
-cyano-4-hydroxy cinnamic acid (Linear Science, Reno, NV) as the
matrix; a 30-kV ion acceleration voltage (grid voltage at 70%; guide
wire voltage at 0.1%) and a -2.0-kV multiplier voltage were
used.
Automated chemical sequencing of either unfractionated or purified
peptides was performed using a model 477A instrument from Applied
Biosystems (Foster City, CA). Stepwise liberated phenylthiohydantoin
amino acids were identified using an on-line 120A HPLC system (Applied
Biosystems) equipped with a phenylthiohydantoin C18
(2.1 x 220 mm; 5-µm particle size) column (Applied Biosystems).
Instruments and procedures were optimized for femtomolar level
phenylthiohydantoin amino acid analysis as previously described (15, 16). Peak areas were integrated to calculate the recovery of all amino
acids after every cycle, background subtracted, and plotted for each
residue individually. Dominant amino acids were then identified as
those scoring above the background and were reported hierarchically
(Table I
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To generate a Kb variant devoid of a deep pocket
C, we replaced the triplet encoding the wild-type valine (V) at the MHC
position 9 (Fig. 1) with a triplet
encoding tryptophan (W), the natural amino acid with the largest side
chain. The construct carrying the mutation as well as the wild-type
Kb control construct were used to transfect P815
(H-2d) mastocytoma cells. The expression and conformation
of KbW9 on the surface of transfected cells were evaluated
by FCM following staining of transfected cells with the
Kb-specific mAb Y3. Binding of this Ab to H-2Kb
is strongly dependent on the correct conformation of the molecule, but
is not affected by the variations in the Kb-bound peptides
(17, 18, 19). Y3 strongly stained both Kb and KbW9
transfectants, but did not stain mock-transfected P815 cells (Fig. 2). However, the staining intensity of
the mutant molecule was 6- to 8-fold lower than that of the wild-type
molecule. Nearly identical results were obtained using five other
Kb-specific mAb; each stained Kb and
KbW9 well, but the staining intensity of KbW9
indicated 5- to 10-fold lower expression of each mAb epitope compared
with that of Kb (A. Molano and J.
Nikoli-ugi, manuscript in preparation). These
results are consistent with the correct folding and conformation, but
an overall lower expression, of KbW9.
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-ugi, manuscript in preparation). Recently,
Ribaudo and Margulies (20) have shown that differential affinity of
murine class I alleles for ß2m can be traced to the
nature of the side chain of MHC residue 9. Therefore, although the side
chain of this residue points directly to the peptide binding site (6, 21), the introduction of a bulky W side chain can affect the
association with ß2m. This could be due to a
rearrangement of this portion of the molecule, inasmuch as the side
chain of W9 appears unlikely to adopt the preferred rotamer orientation
without rearrangement of the side chains of the adjacent residues (Fig. 1
). We conclude that lower expression of KbW9 may be caused
by impaired ß2m association, but our results, at present,
do not exclude limited peptide availability as an alternative
explanation. KbW9 cannot bind Kb-binding peptides bearing the characteristic P5F/Y anchor
The KbW9 mutation was generated with the idea to
disrupt the binding of Kb-specific peptides. To test
peptide binding, we generated tap-2-
KbW9 cells by transfecting KbW9 into
RMA-S-Kb- cells (a Kb loss variant of RMA-S
(12). Once generated, these cells (named RS-W9) as well as the
Kb+ RMA-S cells were used in an MHC class I stabilization
assay (11, 12) that gives a simple and accurate measurement of
peptide:MHC interaction. Class I molecules that reach the cell surface
in tap- cell lines are unstable at 37°C. They
fall apart rapidly, yielding 20-fold lower class I surface fluorescence
upon FCM analysis compared with that of wild-type cells. Exogenously
added peptides can bind to and stabilize class I molecules on RMA-S,
resulting in a dose-dependent increase in surface class I fluorescence.
Figure 3 shows an example of this assay,
performed with three typical Kb binding peptides, HSV-8,
OVA-8, and SEV-9, on Kb- and KbW9-expressing
cell lines. While these peptides bound very well to Kb
(Fig. 3), even at concentrations as low as 10-9 M (
Figs. 5–8 and not shown), neither of them bound to the KbW9
mutant, even at peptide concentrations as high as 3 to 5 x
10-4 M. Virtually identical results were obtained with the
VSV-8 and SVT-9 peptides (not shown).
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demonstrates an exquisite binding specificity of the eluted peptides;
the Kb-eluted peptides stabilized only Kb
molecules, while the KbW9-eluted peptides stabilized only
KbW9 molecules. A control eluate from the
anti-I-Ak-specific column did not stabilize either
molecule, confirming the peptide specificity of the assay. Together
with the data from Figure 3, these results demonstrate that the
KbW9 mutation sterically prevented the interaction of this
molecule with the Kb binding peptides. Furthermore, these
results indicate that the peptide repertoires of Kb and
KbW9 must be largely nonoverlapping in vivo, an issue
discussed further below.
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To determine the common features shared by the peptides bound to
KbW9, we performed bulk sequencing of peptides eluted
from it and compared these sequences to those of peptides eluted from
Kb. Bulk sequencing provides information on the relative
abundance of each amino acid in each of the degradation cycles,
beginning with the N-terminal position of the peptide in the first
cycle (5). Thus, enrichment for a particular amino acid in, for
example, the sixth cycle, would indicate that the peptides bound to the
MHC molecule from which the peptides were eluted have a preference for
that amino acid at position P6. However, this technique has several
limitations. Because of the amino acid background, it yields ambiguous
results regarding the nature of the amino acid present in the first
cycle (position P1 of the peptide), the side chain of which,
fortunately, is not critical in determining binding to the MHC.
Furthermore, it cannot detect cysteines and tryptophans. Despite these
limitations, this analysis revealed several striking differences
between the peptide repertoires of the two molecules. First, while the
readable sequences from Kb-eluted peptides ceased after the
eighth cycle of Edman degradation, a clearly readable ninth cycle, with
a dominant leucine, was obtained from the KbW9-eluted
material (Table I), suggesting that KbW9 may bind longer
peptides than Kb. Second, instead of the aromatic F/Y in
the fifth cycle, typically found in Kb-bound peptides, the
KbW9-eluted material was strongly enriched in positively
charged amino acids, with an absolute dominance of arginine (Table I).
Third, while we confirmed that Y can frequently be found in the third
cycle in Kb-bound peptides, the third cycle of
KbW9-bound peptides also contained a more prominent proline
signal (Table I). Fourth, an unusual feature of the KbW9
was the presence of negatively charged (E and D) residues at P2 (Table I and not shown). This feature is virtually never found in good
Kb binders, since the negatively charged P2 residues are
bound to clash electrostatically with the negatively charged MHC24E of
Kb (12). Finally, a common feature was the presence of a
C-terminal leucine (Table I) and a weaker methionine in both types of
peptides, the residues likely to occupy the C-terminal F pocket.
Preferred size and the peptide motif of KbW9-binding peptides
Using the information from the bulk sequences, we synthesized a
number of peptides. Of these (Fig. 5A), peptides VAPVRLILL
and VEPVRLILL bound very well to KbW9. VAPVRLILL also bound
quite well to Kb, albeit two orders of magnitude less than
typical Kb binders (Fig. 5B). Not
surprisingly, VEPVRLILL bound barely above the background to
Kb due to the mentioned electrostatic repulsion with MHC24E
of Kb. Notably, the analysis also revealed that none of the
octamers tested to date was capable of binding to KbW9, and
that the molecule bound only nonamers (Fig. 5). A set of
alanine-substituted analogues of the peptide VEPVRLILL was used to
elucidate which of the observed features of the KbW9-bound
peptides were critical for tight peptide binding/MHC stabilization.
This analysis showed that P3P and P5R were the primary anchor residues;
their replacement by alanine led to an average of 3 to 4 orders of
magnitude lower binding (Fig. 6). P3P was
preferred to P3Y at lower peptide concentrations, suggesting that this
amino acid provides an optimal stabilization, probably by interacting
with the D pocket. Replacements with alanine at P6 to P8 played no role
whatsoever; not surprisingly, neither did the replacement at P2, where
alanine can actually be found by bulk sequencing. Finally, the results
of P9 replacement decreased binding only by an order of magnitude,
suggesting that P9L has a relatively modest effect compared with P3 and
P5 anchors. This was in contrast to the major role of P8L in
Kb binding peptides (11, 22, 23). Together, the results
suggested that the motif of KbW9 binding peptides may be
XXPXRXXXL, with the role of P4, which was not tested in this
experiment, uncertain.
Identification of a naturally processed KbW9-bound self peptide
To identify the peptides naturally associated with
KbW9, we subjected the KbW9-eluted
peptides to reverse-phase HPLC. Individual peaks were analyzed by a
combination of Edman degradation and mass-assisted laser
desorption/ionization and time-of-flight analysis. One such peak,
specifically found in the KbW9, but not in the
Kb, HPLC profile, gave an unambiguous sequence and mass to
charge (m/z) ratio, that corresponded to an -actin peptide (residues
96–104) with the sequence VAPEEHPTL. This peptide and its N-end
elongations or truncations (according to the natural -actin
sequence) were then synthesized and tested for the ability to bind to
KbW9 and Kb in a stabilization assay. Of all
the peptides, only the nonamer appreciably stabilized KbW9;
essentially no binding was detected with longer or shorter peptides
(Fig. 7A). However,
this peptide was at least two orders of magnitude less efficient in
KbW9 stabilization than peptides VAPVRLILL and
VEPVRLILL based on the concentration required to reach 50%
stabilization (data not shown). Furthermore, this peptide bound poorly
to Kb; binding was at least four orders of magnitude lower
than that of a typical Kb binder, OVA (Fig. 7B).
Next, we used the alanine substitutions of the -actin peptide to
elucidate the relative importance of the residues at positions 3 to 7.
These residues were chosen because in nonameric peptides the side
chains of P1 and P8 almost invariably point away from the class I (3)
(Fig. 6) and play little, if any, role in peptide binding; the role of
P9L was assessed in Figure 6; and P2 was already an alanine in the
peptide sequence (VAPEEHPTL). Figure 8 shows the binding of the wild-type and
substituted -actin peptide to KbW9. This analysis
demonstrates that P3P and P6H act as the new anchors for this peptide,
because their replacement with alanine either abrogates or drastically
impairs binding to KbW9. This result is consistent with the
analysis of synthetic peptides based on the bulk sequencing information
(Figs. 5 and 6), in that both peptides absolutely required P3P and a
positively charged residue in the middle of the peptide. P5R appears to
be preferred, but in the -actin peptide, it is replaced by P6H.
Alanine substitutions at P4 and P7 had no effect on binding, while the
substitution at P5 had a slight favorable effect, most likely by
relaxing the repulsion between the P4 and P5 residues, both of which
are negatively charged (E) in the native peptide. These results
indicated that residues P4, P5, and P7 of this peptide did not play a
role in peptide binding and suggested that they probably point away
from the MHC and into the solvent.
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Discussion |
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E or V9R substitutions abrogated presentation
of VSV-8 to some, but not other, T cell clones, suggesting that these
mutations did not interfere significantly with peptide binding, but,
rather, with its conformation (22). Thus, even an amino acid of
considerable size, such as arginine, failed to competitively inhibit
binding of peptides bearing Y or F at position 5 or 6 (P5/6). This
suggested that the relative energetic importance of the anchor binding
may be smaller than the interaction of the N- and C-amino acid main
chain atoms with pockets A and F, consistent with the findings of two
previous studies (24, 25). However, Saito et al. (23) demonstrated that
the effects of the anchor residues are far greater than previously
believed, but that some of them may be most dramatically manifested at
the level of class I stabilization (i.e., are affecting mainly the
off-rates of the peptide), rather than at the level of the peptide:MHC
affinity. (Of note, in the present study we measured cell surface class
I stabilization, which is believed to correspond closely to the
immunologic relevance of the peptide:MHC complexes exposed to T cells.)
Tryptophan is the largest hydrophobic amino acid, predicted to block
the access of another large aromatic residue (F or Y) to the C pocket.
Indeed, the interaction of the canonical H-2Kb-restricted
peptides possessing a tyrosine or phenylalanine at P5/6 with the
modified pocket C in KbW9 is not detectable by FCM (Fig. 3)
or CTL assays (A. Molano and J. Nikoli-ugi,
manuscript in preparation). Furthermore, peptides eluted from
Kb and KbW9 stabilized only the molecule from
which they were eluted, suggesting that the two molecules were occupied
by largely nonoverlapping sets of peptides in vivo.
It was of interest to determine how the KbW9 binding
peptides adapt to the loss of the C pocket. Information obtained using
individual HPLC peak sequencing/mass spectroscopy, Edman degradation of
the bulk-eluted peptides, and peptide:MHC binding assays was internally
consistent, and the two sets of data corroborated each other (
Figs. 5–8 and Table I). These results are summarize and discussed below.
The P3 side chain, which can operate as an auxiliary anchor in
Kb binding peptides, has assumed the role of a major anchor
in KbW9 binding peptides. The replacement of proline with
alanine at this position yielded a peptide 1000-fold less efficient in
stabilizing KbW9. Bulk sequences of the
Kb-bound peptides at P3 contain a dominant Y (or F) and a
weaker P; in KbW9-bound peptides, the roles are reversed,
and P is more abundant than Y (Table I). Functionally, P3P also
performs better than P3Y in the KbW9 stabilization assay in
the context of the optimal synthetic peptide VEPVRLILL. P3P/Y would
be likely to contact the D pocket, analogously to the situation in
Kb, and both its main chain and side chain would be
relatively exposed to solvent, as found for P3 of Kb
binding peptides (7). The better performance of P3P over P3Y could be
specific for the peptide used, may be specific for all nonamers (this
residue is found in one of the exceptional nonamers that bind to
Kb, SEV-9), or may be a consequence of an indirectly
altered architecture of the D pocket in KbW9, which now
prefers P over Y/F. Furthermore, the presence of a proline may
facilitate peptide kinking over the raised floor of the groove. Another
possibility, that P3P may operate best in collaboration with P5R,
similar to the case of the preferred P2/P5 pairing in
Kb-bound peptides (23, 26), is difficult to reconcile with
the Kb model, but cannot be excluded at present.
As expected, P5 is no longer occupied by F/Y in KbW9-bound
peptides, and the presence of these residues at P5 is deleterious to
KbW9 binding (Figs. 3, 4, and 6–8). Instead, P5 is
invariably positively charged in KbW9 binding peptides,
with the dominance of the strongly positively charged arginine (a
variation of that rule is seen in the naturally processed -actin
peptide, which has P6H instead of P5R). How does P5R contribute to the
stabilization of the peptide:KbW9 complex, and how can it
be oriented with regard to the MHC? P5R could potentially form a salt
bridge to compensate for the energy lost by the loss of Kb
C pocket:P5F/Y. Of the negatively charged residues in KbW9,
several could participate in this type of interaction, and it is
impossible to predict with certainty which one would do so. The
candidates (Fig. 1A) include MHC24E (B pocket, S2
sheet), MHC77D (E pocket, 1 helix), and MHC152E (E
pocket, 2 helix). Kb and, by extension,
KbW9 have a shallow B pocket that is, in fact, contiguous
with the C pocket (6, 21, 26). In KbW9, the C pocket is
filled by W9. This amino acid is also likely to sterically block the
access of P5R to the B pocket, as MHC9 is located at the ridge between
the B and C pockets (Fig. 1B). We propose that P5R
extends into the E pocket. Indeed, the E pocket would be the only
pocket easily accessible to a large side chain of P5R, and, as this
pocket is constitutively occupied by water molecules in all three
MHC:peptide complexes crystallized to date, P5R could assume the
position of these water molecules. P5R could then form a salt bridge
with either of the two glutamic acids, although the contact with
MHC152E would have to be facilitated by the movement of MHC116Y,
analogous to that described in a human class I molecule (27).
Regardless of which amino acid participates in the salt bridge, the
implication of this arrangement is that the backbone of P5R and a good
part of its side chain would be simultaneously exposed to the solvent
(TCR). In addition to its charged tip, P5R could contribute to the
binding energy via the hydrophobic interactions of the rest of its side
chain. Experiments are in progress to test this hypothesis.
The side chain of C-terminal leucine (P9L), although apparently conserved in KbW9-bound peptides, somewhat surprisingly does not seem to play a major role in MHC stabilization. This is reminiscent of the effects of P8L in the Kb binding peptides on peptide:Kb binding affinity (23); however, in the three studies addressing the role of the P8 anchor, P8L had a major influence on the stability of the peptide:MHC complex (11, 22, 23), being 100 to 200 times more potent than the alanine-substituted analogue. By contrast, we observed only a 10-fold effect of the alanine substitution at P9 on the peptide:MHC stability. Three factors, individually or combined, could reduce the importance of the side chain of P9L in MHC stabilization. First, the abundance of the hydrophobic residues at positions P6 to P8 (L, I, and L, respectively) in the peptide used to test the role of the C-terminal leucine (VAPVRLILL) could compensate, via hydrophobic interactions, for the presence of a suboptimal alanine at P9. Second, the increase in peptide length from eight to nine amino acids would be able to bridge the elevated MHC floor and place the N- and C-termini into the A and F pockets as well as provide a longer main chain for hydrogen bonding. Finally, the development of two strong anchors at P3 and P5 could provide the bulk of the binding energy, making the contribution of the side chain of P9L less critical.
As seen in all other classical MHC class I molecules that do not
possess a prominent C pocket, nonamers clearly emerged as optimal
peptides for KbW9, as a consequence of closing the large
hydrophobic C pocket. Matsumura et al. (28) have speculated that the
preference for nonamers in other class I molecules could be caused by
an elevated middle portion of the groove, which would force the middle
of the peptide to bulge out and would require extra peptide length to
keep the peptide ends fixed in pockets A and F. As mentioned,
Db is the only other class I molecule that uses the C
pocket for anchor residue binding, but its pocket is shallow, accepting
small, polar P5N. Young et al. have described a prominent ridge in the
Db cleft (caused by the residues MHC73W of the
1 helix, and MHC156Y and MHC147W of the 2
helix) that forces the peptide to arch over it to reach the F pocket
(7). Thus, despite the presence of the C pocket, this ridge prevents
octameric peptides from simultaneously reaching pockets A and F and is
responsible for the lack of binding of such peptides to Db.
In KbW9, not only is the C pocket closed, but MHC9W could
form an obstacle over which the peptide backbone has to arch, which
would explain the inability of octamers to bind to this molecule.
A surprising finding was the abundance of the negatively charged residues at P2 that are not tolerated in Kb binding peptides. How would this residue coexist with the MHC24E, which normally precludes tight binding of peptides bearing P2E/D? One likely possibility is that in KbW9, the amide of the MHC9W acts as the hydrogen bond donor to either MHC24E or P2E/D, thus reducing the electrostatic tension between the two residues of the same charge. Alternatives could include a reorientation of the P2 residue to point away from the B pocket or the salt bridge of P5R to MHC24E in the B pocket, both of which would also eliminate the electrostatic repulsion.
We conclude that the peptides bound to KbW9 exhibit two strong anchors at P3 (P or weaker Y/F) and P5 (R) and a weaker one at P9 (L or, less frequently, M) and are predominantly (and, most likely, exclusively) nonamers. Based on the obtained data, we speculate that P3P and P9L would contact pockets D and F, respectively, just as in Kb, but that P5R would reorient itself compared with the position assumed by P5F/Y in Kb and would now contact the E pocket, where it would be likely to make a salt bridge to MHC77D or MHC152E. The above speculations can only be solved by further structural and functional experiments, some of which are currently in progress.
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Acknowledgments |
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ek-Szabo for advice, help, and
reagents; Dr. P. D. Jeffrey for initial insights regarding crystal
structure modeling; Ms. D. Nikoli-ugi for
performing flow cytometry; and Drs. R. Dyall, and S.
Vukmanovi for critically reading the manuscript. |
Footnotes |
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.), a grant from the Society of Memorial Sloan-Kettering Cancer Center (to J.N.-.), National Science Foundation Grant DBI-9420123 (to P.T.), U.S. Public Health Service Comprehensive National Cancer Institute Cancer Center Core Support Grant CA08748, and Pew Scholarship in Biomedical Sciences (to J.N.-.). 
2 Address correspondence and reprint requests to Dr. J. Nikoli-ugi, Box 98, Immunology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021.
3 Abbreviations used in this paper: FCM, flow cytometry; OVA-8, ovalbumin peptide257–264, SIINFEKL; SEV-9, Sendai virus nucleoprotein peptide322–331, FAPGNYPAL; MFI, mean fluorescence intensity.
Received for publication August 19, 1997. Accepted for publication November 20, 1997.
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-ugi. 1995. CD4-independent in vivo priming of murine CTLs by optimal MHC class I-restricted peptides derived from HIV and other pathogens. Int. Immunol. 7:1205.-ugi. 1996. T cell receptor (TCR) recognition of MHC class I variants: intermolecular second-site reversion of an MHC mutation by substituted peptides provides evidence for peptide/MHC conformational variation. J. Exp. Med. 184:253., S., A. G. Grandea, S. J. Faas, B. B. Knowles, M. J. Bevan. 1992. Positive selection of T-lymphocytes induced by intrathymic injection of a thymic epithelial cell line. Nature 359:729.[Medline]
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