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Cutting Edge: HLA-DM−Mediated Peptide
Exchange Functions Normally on MHC Class
II −Peptide Complexes That Have Been
Weakened by Elimination of a Conserved
Hydrogen Bond
Andrea Ferrante and Jack Gorski
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J Immunol 2010; 184:1153-1158; Prepublished online 28
December 2009;
doi: 10.4049/jimmunol.0902878
http://www.jimmunol.org/content/184/3/1153
Cutting Edge: HLA-DM–Mediated Peptide Exchange
Functions Normally on MHC Class II–Peptide Complexes
That Have Been Weakened by Elimination of a Conserved
Hydrogen Bond
Andrea Ferrante and Jack Gorski
critical event in the initiation of an immune response
is the recognition by CD4+ T cells of pathogen-derived peptides bound to MHC class II (MHC II)
molecules and exposed on the surface of APCs. In general,
presentation of specific peptide–MHC II complexes may be
considered the outcome of an intracellular selection process
(1). A key step in the process occurs when the MHC II has
been delivered from the endoplasmic reticulum to a specialized
compartment by the chaperone invariant chain and comes into
contact with internalized Ags. In this acidic compartment, the
invariant chain is hydrolyzed, leaving a peptide in the MHC II
binding groove referred to as CLIP. In most of the cases, the
exchange of CLIP for antigenic peptides requires the interaction with a class II-like molecule called HLA-DM (DM).
In addition to CLIP release, the presence of DM promotes
peptide exchange and repertoire skewing in favor of stable
peptide–MHC II complexes (2). However, the precise mo-
lecular mechanism by which DM promotes both peptide release from and binding to MHC II and affects epitope
selection has not yet been elucidated.
Our approach to investigating DM activity is based on the
analysis of cooperativity in peptide interaction with MHC II.
We have shown that, in the absence of DM, peptide binding to
the human MHC II HLA-DR1 (DR1) is a cooperative event in
that all peptide residues can synergistically contribute binding
energy (3, 4). We interpret cooperativity as evidence of the
folding process involving both peptide and MHC II that results in a stable complex. Kinetic analyses of DM function
have suggested that DM acts as a conformational catalyst to
promote the conversion between the empty and bound conformation of the peptide–MHC II complex (5). Therefore,
we have investigated the effect of DM on the folding-unfolding of the complex and how this may be related to the
mechanism underlying the peptide exchange reaction. We
have shown: 1) the requirement of DM for an exchange
peptide at equimolar or greater concentration than the preformed complex to promote prebound peptide release; 2) the
absence of measurable cooperativity in the release of the
prebound peptide, probably due to a simultaneous disruption
of the interactions between MHC II and peptide mediated by
DM; and 3) the exchange ligand needs to fold into the groove
more efficiently than the prebound peptide to displace it (6).
Breaking of a key source of binding energy may account for
the absence of measurable cooperativity in DM-mediated
prebound peptide release. One likely candidate is the conserved
hydrogen bond (H-bond) between Hisb81 of the MHC II
and the backbone of the peptide at position 21 (b81 Hbond). In specific conditions, this particular H-bond has been
found to be crucial for the stability of the complex both in the
absence and in the presence of DM (7, 8). Absence of cooperativity in intrinsic peptide release from a DR1 molecule
missing the b81 H-bond would argue in favor of the hypothesis that DM targets this specific interaction to destabilize
the complex. Alternatively, cooperative unfolding in the
presence of DM and absence of the b81 H-bond would
Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI 53201
The online version of this paper contains supplemental material.
Received for publication September 3, 2009. Accepted for publication December 2,
2009.
Abbreviations used in this paper: DM, HLA-DM; DR1, HLA-DR1; FP, fluorescence
polarization; HA, hemagglutinin; HAS, hemagglutinin306–318 with a P2 V/S substitution; HAC, hemagglutinin306–318 with a P7 L/C substitution; H-bond, hydrogen
bond; ln, natural log; MHC II, MHC class II; wt, wild-type.
A
This work was supported by National Institutes of Health Grant RO1AI63016.
Address correspondence and reprint requests to Dr. Andrea Ferrante, BloodCenter of
Wisconsin, P.O. Box 2178, Milwaukee, WI 53201. E-mail address: andrea.ferrante@
bcw.edu
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902878
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The mechanism by which HLA-DM (DM) promotes
exchange of peptides bound to HLA-DR (DR) is still
unclear. We have shown that peptide interaction with
DR1 can be considered a folding process as evidenced by
cooperativity. However, in DM-mediated ligand exchange, prebound peptide release is noncooperative,
which could be a function of the breaking of a critical
interaction. The hydrogen bond (H-bond) between
b-chain His81 and the peptide backbone at the 21
position is a candidate for such a target. In this study,
we analyze the exchange of peptides bound to a DR1
mutant in which formation of this H-bond is impaired.
We observe that DM still functions normally. However, as expected of a cooperative model, this H-bond
contributes to the overall energetics of the complex and
its disruption impacts the ability of the exchange ligand to fold with the binding groove into a stable
complex. The Journal of Immunology, 2010, 184:
1153–1158.
1154
CUTTING EDGE: ENERGETICS OF DM-MEDIATED PEPTIDE EXCHANGE
indicate its role in the loss of cooperativity. Consequently, we
investigated the binding properties of a mutant DR1
(b81mut) in which formation of the b81 H-bond is prevented
by His/Asn mutation, and we tested it for cooperativity and
sensitivity to DM action. Our results indicate that in the absence of b81 H-bond, intrinsic release of peptides from DR1
can still be considered an unfolding process, and DM promotes exchange of peptides bound to b81mut with the same
criteria we have observed for wild-type (wt) DR1. However,
the b81 H-bond contributes to the energetics of the complex
with a magnitude dependent upon peptide–MHC II interactions throughout the binding site (distributive model). This
is evidenced, in particular, by reduced cooperativity of the
exchange ligand in DM-mediated displacement of peptides
bound to b81mut as compared with DR1. The implications
of these findings in a possible mechanism for DM action
are discussed.
Peptide synthesis
Peptides derived from the sequence of wt hemagglutinin (HA)306–318 (G)
PKYVKQNTLKLAT have been synthesized as described previously (3, 4, 6)
and are listed in Supplemental Table I and II.
Generation and expression of b81 substituted DR1 molecules
Plasmids encoding truncated forms of the HLA-DRa and DRb*(0101) genes
were the gift of Dr. Lawrence Stern (University of Massachusetts Medical
School, Worcester, MA). Site-directed mutagenesis of DRb*(0101) at position 81 was performed as described (3).
Expression and purification of recombinant soluble DR1 and DM
protein
Recombinant soluble empty (peptide-free) b81mut and soluble FLAG-tagged
DM were produced and immunoaffinity purified from a stably transfected
Drosophila S2 insect cell line essentially as described (3).
Fluorescence polarization dissociation measurements
Dissociation of peptides from DR1 and b81mut was measured with fluorescence polarization (FP) spectroscopy essentially as described (6). The t1/2
values of the various complexes are reported in Supplemental Table I and II.
Competitive peptide binding assay
Peptide KD values for DR1 and b81mut were assessed in equilibrium-based
competition binding assay as previously described (6). Affinity values are
reported in Supplemental Table I.
Calculation of cooperative effects
We used a multiple substitution strategy previously used to identify interacting
partners during protein folding (9, 10). To normalize the t1/2 values of a given
peptide–MHC II complex, we define the effect of each substitution as the ratio
of the substituted measurement over that of the b81/wtHA value (Dt1/2). For
calculating cooperativity, the effect of multiple substitutions is measured directly
(observed value). The expected value for a combination of substitutions is calculated as the product of the individual substitutions [e.g., Dt1/2,exp = (Dt1/2, x)
3 (Dt1/2, y)]. For peptides with three substitutions, the expected value would be
the product of all the different substitutions. The cooperativity is the ratio of the
expected to observed (C = expected/observed) values for Dt1/2. Cooperativity is
evidenced when the ratio of expected/observed is not equal to 1.
Results
Peptides derived from HA by cycle mutation interact cooperatively with
a DR1 molecule lacking the conserved H-bond formed by Hisb81
To test the hypothesis that DM mediates a noncooperative
release of peptides from MHC II by breaking one critical
binding source, we measured the stability of peptides bound to
a mutant DR1 (b81mut) in which the conserved H-bond
formed between His at position 81 of b chain and the peptide
DM affects a noncooperative release of peptides bound to b81mut
The above results indicate that in the absence of b81 H-bond,
intrinsic peptide release is an unfolding event. Because we
observed a noncooperative release of peptides bound to
wtDR1 in the presence of DM (6), we can infer that b81mut
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Materials and Methods
backbone (b81 H-bond) has been disrupted via substitution to
Asn. This substitution has been used in previous studies (3, 8).
The peptides adopted for this study are derived from the
sequence of HA (H3 subtype) residues 306–318. Substitutions
have been introduced according to the mutant cycle technique
(9) at positions for which solvent accessibility is minimal (P1,
P6, and P9) and are postulated to interact with the pockets
lining the binding groove through desolvation (hydrophobicity). We have provided the rationale for these mutated
peptides and have observed cooperativity in their binding and
release (4).
Dissociation curves of the various b81mut–peptide and
DR1–peptide complexes are shown in Fig. 1A and Supplemental Fig. 1A, respectively. Stability values are reported in
Supplemental Table I. Cooperative effects were calculated by
determining the ratio of expected t1/2 to observed t1/2 for each
multiple substituted peptide normalized for the off-rate of the
b81mut–HA complex. Plotting cooperativity against the
observed t1/2 of the various b81mut–peptide complexes (Fig.
1B, closed circles), showed a significant correlation (r2 = 0.97)
with a negative slope (20.65). This indicates that in the absence of a source of binding energy such as the b81 H-bond,
release of peptides from DR1 can still be considered an unfolding process. Moreover, as observed for wtDR1 (Fig. 1B,
open circles), these results also indicate that the negative
contribution of cooperative effects to stability increases exponentially as the complex half-life decreases.
Comparing the slope values relative to wtDR1 and b81mut
data would give indications as to the contribution of the b81
H-bond on the overall folding energy of the complex (3).
However, due to the low binding properties of peptides
containing the P6 T/K substitution, cooperativity in stability could be measured only for four complexes. Applying
a Student t test on regression line slope values, we found that
any differences between the two lines were not statistically
significant. We have previously shown that for the same set of
peptides, the relationships between cooperativity and either
KD or t1/2 are very similar, indicating that the cooperative
effects measured are fundamental to the peptide/DR1 interaction (3). Thus, to increase the statistical significance of
the comparison between the b81mut and the wtDR1 data, we
considered cooperativity in peptide affinity because this can be
measured for nine complexes. Competitive binding curves are
shown in Fig. 1C and Supplemental Fig. 1B); affinity values
are reported in Supplemental Table I and cooperativity plotted
in Fig. 1D. The same combinations of substitutions showed
cooperative effects with both b81mut (Fig. 1D, closed circles)
and wtDR1 (Fig. 1D, open circles). Interestingly, the slope
value of cooperativity for the mutant was 1.3-fold smaller than
for wtDR1, and the affinity value at which cooperativity could
be observed shifted to higher values. However, the steepness of
the regression line also indicates that negative contribution of
cooperativity to complex energetics increases with a smaller
magnitude for the mutant MHC II than for the wt as disruptive mutations are added.
The Journal of Immunology
1155
does not structurally mimic the “post-DM effect” conformation of the MHC II. We also analyzed the effect of disrupting this H-bond on DM activity. If the b81 H-bond is
needed to mediate a noncooperative release of the prebound
peptide by DM, the loss of this H-bond might result in
a cooperative unfolding in the presence of DM. Peptide release from b81mut in the presence of DM and 50-fold excess
exchange peptide was measured. Dissociation rate data for all
the tested complexes are shown in Fig. 2A, and stability values
are reported in Supplemental Table I. Cooperative effects
were calculated as above. When cooperativity was plotted
against the observed t1/2 of the various b81–peptide complexes (Fig. 2B), the data fit a linear function with a slightly
positive slope and intercept of 0. These observations clearly
indicate that in the absence of the b81 H-bond, DR1–peptide
complexes are still a target of DM action. Furthermore, the
release is noncooperative as in the case of wtDR1 (6), indicating that the loss of unfolding observed when the b81 Hbond is present cannot be explained with a disruption of this
specific source of binding energy by DM.
Role of the exchange peptide in DM-mediated peptide release from
b81mut
The data presented thus far indicate that interaction of antigenic peptides with DR molecules lacking the conserved b81
H-bond is a cooperative event, and the presence of DM affects prebound peptide release in a similar fashion to what we
detected for wtDR1. We also expect a role for the exchange
peptide as cofactor in DM-mediated release of the prebound
peptide because such a role was shown in the case of wtDR1
(6). We monitored b81mut–peptide complex depletion over
time without manipulation of the sample by FP, in which
signal is continuously acquired in the reaction well. We
started with a prebound b81mut–peptide complex and observed the accumulation of free peptide over time in the absence of an exchange peptide, DM, or both. The missing
component(s) was then added to the reaction, and the exchange rate was measured.
For this experiment, we used an HA306–318 with an L/C
substitution at P7 (HAC), which had been already used with
wtDR1 (6). The L/C mutation was introduced for possible
labeling, and the affinity and stability of this peptide are
comparable with those of wtHA. FP signals were acquired in
the following three reactions (Fig. 3A): 1) b81mut–HAC
complexes in the presence of 3-fold molar excess of DM
(closed circles); 2) b81mut–HAC complexes in the presence
of 100-fold excess unlabeled HA (open circles); and 3)
b81mut–HAC complexes alone (closed triangles).
After 24 h, there was negligible release in the presence of
excess HA (reaction 2) or in the absence of HA and DM
(reaction 3). Approximately 15% free peptide was observed in
the presence of DM (reaction 1). At this point, 100-fold excess
unlabeled HA peptide was added to reaction 1 (already incubated with DM), soluble DM was added to reaction 2
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 1. Peptide interaction with the b81 mutant DR1 is cooperative. A, Dissociation rates of b81mut–peptide complexes. Data are expressed as the
fraction b81mut–peptide complex remaining relative to t = 0. Reactions were performed in triplicate, and data series represent one of three independent experiments. The lines represent the fit of the data either to a single or double exponential function. Peptide substitutions are reported in the legend. Peptide
positions are numbered considering Tyr308 as P1. B, Natural log (ln) plot of cooperativity (expected/observed t1/2) versus intrinsic dissociation rate for each
multiple substituted peptide interacting with either b81mut (closed circles) or wtDR1. Horizontal error bars represent the SD of the t1/2 measurement. Vertical
error bars represent the error of cooperativity as calculated through SE propagation. Lines indicate the fit of the data to a linear regression. C, Competition
binding analysis of P1, P6, and P9 substituted HA peptide variants to b81mut. Data represent the mean and SD of three independent experiments. Lines indicate
the fit of the data to a logistic equation. D, ln plot of cooperativity versus observed affinity for each multiple substituted peptide interacting with either b81mut
(closed circles) or wtDR1.
1156
CUTTING EDGE: ENERGETICS OF DM-MEDIATED PEPTIDE EXCHANGE
(incubated with excess HA), and incubation was continued.
Five hours after the addition of either peptide or DM to the
respective reactions, we observed an equivalent increase in free
HAC peptide (70% increase over the 24 h time point). We
have previously shown that the reduced release of peptides
from MHC II in the presence of DM and in the absence of
exchange ligand is not due to rebinding of freshly dissociated
peptide (6).
Finally, at 48 h, when both an exchange peptide and DM
were added simultaneously to the incubation with b81mut–
HAC complex alone (reaction 3), we observed a similar
magnitude of peptide release. This indicated that the
b81mut–HAC complex was stable and maintained the ability
to undergo peptide exchange after long incubation periods.
The rate of DM-mediated peptide release during the 5 h
incubation in the presence of excess unlabeled exchange
peptide shows a 4.5-fold increase over the 24-h incubation
without exchange peptide (70% versus 15%). This result
clearly shows the requirement for an exchange peptide to
promote significant DM-mediated release of prebound ligand
from the DRb81mut complex.
We have also analyzed the role of b81 using an HA peptide
with a spin-labeled probe at P7 using electron paramagnetic
resonance. This approach was adopted in our previous analysis of DM activity (6). The electron paramagnetic resonance
analysis confirmed that b81mut is sensitive to DM activity
and that in the absence of an exchange peptide, DM promotes
release of prebound peptide very poorly (data not shown).
FIGURE 3. Analysis of the role and cooperativity of free peptide in DMmediated peptide exchange from b81 mutant DR1. A, Real-time FP analysis
of b81mut–HAC complex stability as described in Results. Initial reaction
conditions are identified in the legend. Data is plotted as the percentage of
bound peptide detected. Reactions were performed in triplicate, and data
points represent mean 6 SD for one of two independent experiments. B,
DM-mediated dissociation of the HAS peptide from b81mut. The nature of
the competing peptide present in excess during the reaction is identified in the
legend. Reactions were performed in triplicate, and data points represent one
of three independent experiments. Lines represent the fit of the data to
a single or double exponential decay function. C, ln plot of cooperativity
versus dissociation rate of b81mut–HAS complex for each multiple
substituted exchange peptide tested. For comparison, data relative to wtDR1/
peptide dissociation is reported (dashed line).
Effect of b81 H-bond disruption on DM-mediated complex folding
Having analyzed the effect of the loss of the H-bond at b81 on
DM-mediated peptide release and the requirement for exchange peptide to accelerate this release, we analyzed the effect
of the b81 mutation on the ability of DM to accelerate
folding of the exchange peptide into the groove.
For these experiments, we needed a peptide with sufficient
affinity for the b81mut to allow complex formation but also
one with a greater dissociation constant compared with wtHA
to permit ligand exchange. We decided to use HA306–318 with
a P2 V/S substitution (HAS), which was already shown to
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FIGURE 2. DM nullifies cooperative effects on dissociation of peptides
bound to b81mut. A, Dissociation rate of peptides from b81mut in the
presence of DM. Data is plotted as the fraction of b81mut–peptide complex
remaining relative to t = 0. Reactions were performed in triplicate, and data
points represent one of three independent experiments. Lines fit the data to
a single or double exponential decay function. B, ln plot of cooperativity versus
DM-mediated dissociation rate for each b81mut–peptide complex tested.
The Journal of Immunology
Discussion
In our past investigation of DM activity, we have shown that
the release of prebound peptide from wtDR1 in the presence of
DM appears to be different from the typical unfolding process
observed in the absence of DM (6). A possible explanation for
this observation would consider DM destabilizing one critical
source of binding energy within the MHC II–peptide complex and consequently promoting a dramatic and simultaneous disruption of all the interactions across the binding site.
Moreover, one of the current models accounting for DM
action proposes that DM destabilizes the complex by altering
transiently and repeatedly the b81 H-bond through a hit-andrun mechanism. As corollary, the b81mut would structurally
mimic the “post-DM effect” conformation of the MHC II
(8). Thus, we decided to measure the magnitude of cooperative effects for a panel of peptides derived from HA via
cycle mutation while interacting with the b81mut. Our results indicate that exchange of peptides bound to b81mut,
either in the absence or in the presence of DM, occurs in
a similar fashion to what is observed for wtDR1, and therefore
this H-bond does not constitute a preferential source of
binding energy nor is it a special target of DM activity.
However, the loss of the b81 H-bond does affect the overall
energy of the complex, as could be expected of a distributive
model of peptide interaction with the binding groove. This is
particularly evident in the reduced cooperativity measured for
exchange peptide during DM-mediated ligand displacement.
Because the exchange peptide is required to establish one less
interaction with the MHC II, multiple substituted peptides
can fold the binding groove more successfully in the absence
of the b81 H-bond.
The present results are consistent with our previous analysis
of cooperativity in the b81mut–HA complex as well as reports
investigating DM action on DR1–HA complexes where formation of the conserved H-bond network was impaired by
mutating either the peptide (5) or the DR1 (11). These reports indicated that the b81 H-bond does not play a major
role in stabilizing DR1–peptide complexes nor does it reduce
DM potency in promoting peptide dissociation. Indeed, removing any subset or single H-bonds did not prevent DM
activity, and, in some cases, it amplified its action. In light of
these observations, our data suggest that the presence of
equimolar or higher concentrations of exchange peptide
promotes a short-lived intermediate involving the MHC II–
prebound peptide complex and the exchange peptide. An
enhanced DM activity toward complexes unable to form the
H-bonds between the peptide main chain at the N terminus
and MHC residues a51–53 may indicate that one or more of
these interactions are disrupted in the state recognized by DM
(5, 11). DM binds this intermediate and puts the MHC II
molecule in an exchangeable conformer, promoting a widescale disruption of the interactions throughout the binding
groove and an extremely rapid (noncooperative) release of the
prebound peptide. Our data argue against the possibility that
DM mediates a noncooperative release of prebound peptides
from wtDR1 by destabilizing the b81 H-bond, as we observe
a similar phenomenon when this H-bond is absent. Rather,
DM might generate this effect by inducing a structural rearrangement of the a50–59 region, leading to local exposure
to solvent. This region is thought to be flexible, as evidenced
during peptide binding and the shift between peptide averse
and peptide receptive conformation of the binding groove
(12), and it has been implicated in DM/DR interaction. Once
destabilized, the prebound peptide remains in the complex,
while DM maintains the MHC II in an energetic state sensitive to the folding properties of the exchange peptide. In the
absence of productive folding of the exchange peptide, the
original prebound peptide can rebind to the groove. The end
result of this compare-exchange routine (6) is that in the
presence of DM, MHC II selects for exchange peptides with
the best chance of binding based on the ability to fold with
the groove into a stable low-energy conformation.
Acknowledgments
We thank Trudy Holyst for peptide synthesis, Dr. Lawrence Stern for DR1expressing S2 cells, Dr. Dennis Zaller for DM-expressing S2 cells, and Dr.
Matthew Anderson for helpful discussion.
Disclosures
The authors have no financial conflicts of interest.
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