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Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21205
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Here we isolate these different aspects to investigate the mechanistic reasons behind the slow peptide binding to MHC. Based on the observations that peptides play an important role in determining MHC structure (9, 10, 11, 12), we first used low affinity peptides to induce conformational changes that may be required before specific binding and then determined the kinetics of binding of a high affinity peptide to MHC in such an induced conformation. We used the well-characterized HLA-DR1 and the influenza hemagglutinin peptide (HA306–318)4 and its variants as our model system. The strong hydrophobic interactions between the tyrosine anchor of HA and the pocket 1 of HLA-DR1 are primarily responsible for the longevity of DR1/HA complex (13). We eliminated this tyrosine to design the low affinity HA variant peptides. By using a new fluorescence assay that enables simultaneous detection of two different peptide complexes, we determined the rate of dissociation of the low affinity peptide and the subsequent rate of association of HA. Our results show that the protein conformational change preceding the stable binding to generate a receptive molecule is the rate-limiting step, while the specific interactions occur extremely rapidly. Furthermore, in the absence of the peptide, the MHC molecule loses its receptive conformation quickly and consequently loses its ability for rapid binding of peptides offered at a later time. Our results suggest different roles for low affinity complexes and HLA-DM in enhancing Ag presentation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Soluble DR1 protein was expressed and purified as originally
described (11). Baculovirus transfer vector containing both the 
and
ß genes with two polyhedrin promoters (5) was provided by Dr.
Lawrence Stern, Massachusetts Institute of Technology (Cambridge, MA).
Baculovirus DNA (BaculoGold, PharMingen, San Diego, CA) and transfer
vector containing the and ß genes were cotransfected into Sf9
insect cells to produce recombinant virions. High-5 cells were infected
with the recombinant virus, and DR1 was purified from the culture
supernatant using anti-DR1 mAb (L243) immunoaffinity chromatography
columns (14). Boiled samples of purified protein migrated in SDS-PAGE
at the expected sizes of and ß subunits (32 and 29 kDa).
Peptide synthesis and labeling
HA peptide (306–318 residues of influenza virus hemagglutinin)
and several HA variants (Cys-HA305–318, Y308A,
Anchorless-HA, and AAK) that were used in this study are listed in
Table I
. The peptides were synthesized and purified to
>95% homogeneity by reverse phase preparative HPLC, and their
identities were confirmed by mass spectrometry. The concentrations of
the peptides were determined by ninhydrin assay. Cys-HA was
fluoresceinated at its N-terminal cysteine using
fluorescein-5-maleimide (Pierce, Rockford, IL). Fluorescein-Cys-HA
(Fl-HA) behaved similarly to 125I-HA labeled at tyrosine
308 (3) as determined by association and dissociation rates with sDR1.
Furthermore, the Fl-HA/DR1 complex showed resistance to SDS-induced
dissociation expected of a DR1/HA complex. The peptides Y308A,
Anchorless-HA, and AAK were labeled at their terminal amines with
7-amino-4-methylcoumarin-3-acetic acid (AMCA) N-hydroxy
succinimide (Pierce). Since the pKa of the N-terminal -amine
(7.6–8.0) is lower than the pKa of the 
-amine of the lysine
(9.3–9.5), we minimized the conjugation of the label to the lysines
compared with the N-terminal amines by performing the labeling reaction
in PBS at pH 7.4. Although we cannot completely rule out the
possibility of the AMCA label also being conjugated to the lysines, the
crystal structure of DR1/HA complex (4) indicates that all the lysines
of the peptide are pointing away from MHC, suggesting that the labels
on these residues would not hinder binding to DR1. This idea was
confirmed by comparing the results of experiments with labeled and
unlabeled peptides. The unconjugated label was removed by Sephadex G-10
(Pharmacia) spin columns, and the labeled peptides were stored in PBS.
The labeled peptides exhibited the same excitation and emission
characteristics as the free label. The concentration of Fl-HA was
determined spectrophotometrically at 490 nm using an extinction
coefficient of 78,000 M-1 cm-1 (15),
which is typical value for fluorescein-labeled peptides (16).
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HA binding to dissociating short-lived complexes.
All kinetic experiments were performed in PBS buffer at pH 7.4 unless
stated otherwise. The sDR1 was incubated with 100 µM AMCA-labeled
fast dissociating peptides (Y308A, Anchorless-HA, and AAK) at 37°C
for 24 h. Size-exclusion HPLC (Protein-Pak SW300 gel filtration
column, Waters, Milford, MA) was then used to isolate the AMCA-labeled
/ß DR1 complexes. The concentration of the complex, after high
performance size exclusion chromatography (HPSEC), was
determined by absorbance at 280 nm using the extinction coefficient of
77,000 M-1 cm-1 (11). Either 2 or 50 µM
Fl-HA was added to approximately 1.5 µM AMCA-labeled complex and
incubated at 37°C for various times. Then, the excess free peptides
were removed by Sephadex G-50 (Pharmacia, Piscataway, NJ) spin columns,
and the fluorescence intensities of the complexes were measured.
Optimal emission and excitation wavelengths were determined for each of
the fluorescent peptide-DR1 complexes studied here. The concentration
of DR1/AMCA peptide complex was determined by exciting the sample at
350 nm and measuring the fluorescence at 445 nm for all peptides except
AAK. The DR1/AAK complex showed a unique fluorescence spectrum with an
emission peak at 410 nm, and the fluorescence at this wavelength was
used to monitor the complex concentration. The concentration of
DR1/Fl-HA complex was determined by exciting at 490 nm and measuring
the fluorescence at 517 nm. The fluorescence intensities of AMCA- and
Fl-HA-labeled complexes, at their optimal emission wavelengths, were
fit to single exponential curves to determine the rate of dissociation
of the short-lived complex and the rate of formation of the stable HA
complex.
HA binding to purified DR1. To measure the association rate of Fl-HA with insect cell purified DR1, 1 µM DR1 was incubated with 50 µM peptide at 37°C for various times. After the binding reaction, the excess labeled peptide was separated using Sephadex G-50 spin columns. The concentration of the DR1/Fl-HA complex was then determined as outlined above.
In experiments to study the lifetime of the receptive conformation, unlabeled DR1/Y308A complexes were separated by HPLC as described earlier. DR1/Y308A (1.5 µM) was allowed to dissociate for various times at 37°C in PBS (pH 7.4) and in 0.1 M citrate/phosphate (pH 5.5) buffer followed by binding with 50 µM Fl-HA for 1.5 h in the respective buffers. The unbound peptides in both reactions were removed using Sephadex G-50 spin columns equilibrated with PBS, as the fluorescein had significantly reduced fluorescence at pH 5.5. In another set of experiments, DR1/Y308A complexes were allowed to dissociate at 37°C for 6 h and then were incubated with 50 µM Fl-HA for different times. In both sets of experiments, the concentrations of DR1/Fl-HA complexes were determined as described earlier.
SDS-PAGE
The rate of formation of HA complexes, when HA binds to
dissociating DR1/Anchorless-HA complex, was determined using SDS-PAGE.
SDS-PAGE was performed essentially as previously described (17).
DR1/Anchorless-HA complex (0.5 µM; separated by HPSEC) was incubated
with 50 µM HA for various times at 37°C. The reaction samples mixed
with equal volumes of SDS-PAGE sample buffer containing 0.2% SDS
(final concentration) were incubated for 15 min at room temperature.
Samples were run on a 12.5% polyacrylamide gel and silver-stained
according to standard protocols. The gel was scanned on an Agfa
(Ridgefield Park, NJ) Arcus Laser Scanner, and the intensities
of the /ß heterodimer bands were analyzed using the National
Institutes of Health Image program.
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Results |
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Influenza virus hemagglutinin peptide
(HA306–318) binds to DR1 with high affinity and
has a half-life of about 6 days (3). The stability of this binding is
mainly due to the hydrophobic interaction between the first anchor,
Tyr308, and the pocket 1 residues of DR1 (13, 32). Fl-HA binding to DR1, however, is a very slow
process, with a half-time of 12.6 ± 0.9 h (Fig. 1). To investigate whether slow structural
reorganization of protein before specific binding is the cause of the
slow rates, we first formed a short-lived complex with a low affinity
peptide. The low affinity peptides used for this purpose lacked either
just the primary tyrosine anchor (Y308A) or all the anchors
(Anchorless-HA and AAK). Similar to HA binding (3), incubation of DR1
with a 200-fold excess of these low affinity peptides for 10–30 min
indicated the existence of a fast forming intermediate, which
dissociated in minutes (data not shown). Upon further incubation, these
intermediate complexes converted to relatively longer lived terminal
complexes with half-lives ranging from 0.5–3 h (32). We then studied
the kinetics of HA binding to nascent DR1 generated from dissociation
of such low affinity terminal complexes. To facilitate simultaneous
measurement of dissociation of the short-lived complex and the
subsequent association of HA peptide, we labeled the Anchorless-HA
peptide with AMCA and the HA peptide with Fl. These fluorophores were
chosen because of their nonoverlapping spectra. The optimal excitation
and emission wavelengths of DR1/AMCA-Anchorless-HA complexes were
350 and 445 nm, respectively, whereas the optimal excitation and
emission wavelengths of DR1/Fl-HA were 490 and 517 nm, respectively.
The half-lives for the dissociation of the preformed AMCA-Anchorless-HA
complex (1.5 ± 0.2 h) and the subsequent formation of the
Fl-HA complex (1.8 ± 0.4 h), with the peptide in a 30-fold
molar excess, were determined simultaneously and were similar (Fig. 2A). The binding of HA seemed to occur as
soon as the binding site became available, in contrast to the
previously observed slow HA complex formation. This suggests the
existence of a slow step preceding complex formation, which is
typically reflected in binding rates showing no dependence on peptide
concentration (18). To investigate this, we repeated the above
experiment using a stoichiometric concentration of Fl-HA peptide. The
half-time for the formation of HA complex at this low concentration
still remained at about 1.8 ± 0.3 h (Fig. 2A).
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B.
These relatively rapidly forming HA complexes were indeed SDS stable.
The half-time for formation of these SDS-stable complexes was about
1.5 ± 0.2 h, similar to that obtained with fluorescently
labeled peptides. Furthermore, correlation of results between
experiments with unlabeled and labeled peptides indicated that the
fluorescent labels were not intrusive.
To examine the generality of the observed enhancement in the rate of HA
complex formation when the peptide is binding to dissociating,
short-lived complexes, we used the other fast dissociating complexes,
DR1/AMCA-Y308A and DR1/AMCA-AAK. These experiments were performed
similarly to that with the DR1/Anchorless-HA complex. When Fl-HA binds
to dissociating DR1/Y308A complex, the half-time for the formation of
the HA complex (43 ± 5 min) was similar to that of the
dissociation half-time of the Y308A complex (34 ± 3 min; Fig. 3A). The formation of HA complexes under
these conditions is about 18-fold faster than that observed with
peptide binding to purified DR1. Similarly, in the case of Fl-HA
binding to dissociating DR1/AMCA-AAK complexes, the half-life for the
formation of DR1/HA complexes (3.4 ± 0.2 h) correlated very
well with the dissociation half-time of (3.2 ± 0.4 h) for
the DR1/AAK complex (Fig. 3B).
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Lifetime of peptide-receptive DR1 molecule
The good correlation that exists between the dissociation of the
short-lived DR1 complex and the subsequent association rate of the HA
peptide clearly suggests that the complex that had just lost its
peptide is extremely receptive to the binding of another peptide. We
determined the half-life of this receptive molecule using dissociating
DR1/Y308A, which has the shortest lifetime among the short-lived
complexes used here, as a source for generating nascent, peptide-free
DR1. DR1/Y308A complexes were allowed to dissociate at 37°C in the
absence of any free peptide for various time periods, ranging from
0–26 h at both pH 5.5 and 7.4. The ability of these molecules to
retain maximal binding was then tested by binding to 50 µM Fl-HA for
1.5 h at both pH values. The results indicate that the longer the
newly formed, peptide-free molecule exists in the absence of free
peptide, the lesser its ability to bind the subsequent peptide at
maximal efficiency. The half-time for inactivation of the receptive
molecule is 2 h at pH 7.4 (Fig. 4A) and
1.2 h at pH 5.5 (Fig. 4B). During the incubation for HA
binding (1.5 h), small amounts of preformed DR1/Y308A complexes
continued to dissociate, generating nascent peptide-free DR1. If data
are corrected for this factor, the real half-time for the inactivation
of peptide-free molecule is shorter than the times indicated above and
is 1.5 h for the experiments at pH 7.4. Furthermore, continued
incubation with HA306–318 for a day or longer
showed that a fraction of the MHC molecules had irreversibly lost its
peptide binding ability (data not shown). The inactivation seems to
generate at least two different populations, one binding peptide
significantly below its maximal efficiency and the other completely
incapable of binding. The individual contributions of these two
populations to the observed inactivation cannot be assessed from these
experiments. We further quantitatively evaluated the impact of the time
spent by nascent MHC molecules in the absence of peptides on their
subsequent rate of peptide binding. HPSEC-isolated DR1/Y308A complexes
were incubated for 6 h without the addition of any excess peptide
and were then allowed to bind to 50 µM Fl-HA for various times at
37°C. Here, HA306–318 binding showed a
biphasic trend, with a fast phase having a half-time of 35 ± 5
min, accounting for 20% of the total binding, and a slow phase having
a half-time of 12 ± 1 h (Fig. 5). Strikingly,
the rate of the fast phase correlated very well with the dissociation
of the residual DR1/Y308A complexes (34 ± 3 min; Fig. 3A) and the rate of slow phase correlated well with the
peptide binding rate observed with the insect cell purified sDR1
(12.6 ± 0.9 h; Fig. 1).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have also determined that in the absence of peptides, the nascent MHC molecules remain receptive for only a short period of time, with half-times of 1.5 and <1 h at pH 7.4 and pH 5.5, respectively. Interestingly, in the absence of peptides, these highly reactive nascent molecules lose their rapid peptide binding ability and revert to the slow peptide binding behavior of the recombinant peptide-free sDR1. In short, we observed transformation of the poorly binding, purified MHC molecule into an extremely receptive form and vice versa.
A peptide-receptive conformation generated by dissociation of low affinity MHC/peptide complex
These data suggest that the MHC molecules exist in at least two
different conformations with respect to their peptide binding ability:
one very receptive to binding, and the other not very conducive to
binding. Here we present a mechanism by which the binding of a low
affinity peptide (Plow) changes the conformation from one
to another. An MHC molecule that had remained peptide free even for a
short period of time is in a conformation not very receptive to
subsequent peptide binding and is represented as (ß)x.
The binding of the low affinity peptide (Plow) to this
molecule involves a rapidly forming, rapidly dissociating intermediate
complex during which the MHC still exists in a nonreceptive
conformation (ß)x. During repeated encounters of the
peptide, the nonreceptive class II molecules, (ß)x,
slowly convert to a receptive conformation, (ß)*,
allowing formation of a more stable terminal complex
(ß)* Plow (Equation 1). This terminal
complex with a low affinity peptide, although relatively more stable
than the intermediate form, has a significantly shorter half-life than
the terminal complex with a high affinity peptide (3).
 | (1) |
).
 | (2) |
ß)x, some fraction of which
irreversibly converts to a form no longer capable of binding peptides
(ß)inactive (Equation 3).
 | (3) |
)
involving such molecules. It is very likely, however, that the
molecules generated upon dissociation of high affinity complexes also
undergo a similar inactivation process in the absence of free peptides. The classical induced fit theory, originally intended to account for the use of binding energy of substrate interacting with an enzyme toward catalysis, may explain the mechanism of peptide binding to class II as well. Peptides capable of only the minimal interactions may provide sufficient binding energy to induce or stabilize a conformation that is critical to subsequent binding of high affinity peptides. An example of an enzyme reaction showing similar characteristics is D-lactate dehydrogenase-catalyzed oxidation of pyruvate by DPNH. Prior incubation of this enzyme with pyruvate eliminates the significant lag period, presumably associated with a slow conformational change, that exists when all the reactants are coincubated at the same time (21).
We observe a dramatic increase in the rates of formation of stable
peptide complexes when the MHC is in the correct conformation. The
formation of DR1/HA complex is enhanced about 18-, 7-, and 4-fold when
HA binds to dissociating DR1/Y308A, DR1/Anchorless-HA, and DR1/AAK
complexes, respectively, compared with when it binds to the purified
DR1 (insets of Figs. 2 and 3). The enhancement in the
formation of HA complexes clearly correlates well with the life-times
of the short-lived complexes. Since the HA binding itself seems
extremely rapid, the enhancement is limited only by the availability of
binding sites. If a short-lived complex that has a shorter lifetime
than the complexes indicated above is used or if accessory molecules
such as HLA-DM are used to accelerate the dissociation, then
conceivably the enhancement can be even more dramatic.
One of the problems that has plagued MHC/class II peptide binding analyses is the heterogeneity of the MHC molecules. Recombinant MHC produced in insect cells aggregate in different forms while the molecules purified from cellular sources are mostly occupied by endogenous peptides. Consequently, MHC molecules in various conformations complicate kinetic analysis. Furthermore, we (3) and others (22) have shown that the empty molecules lose their peptide binding activity over a period of time. The use of class II heterodimers that are dissociating from their peptide ligand overcomes this problem and provides a homogeneous pool of MHC molecules for determination of peptide binding.
In vitro binding studies do not necessarily reflect the intrinsic affinity between the peptide and MHC
The observation that the conformational change is the rate-determining step for HA binding to DR1 indicates that the energy barrier for the conformational change is significantly higher than that for peptide binding. This indicates that the association rates measured using purified molecules will reflect this conformational change rather than the intrinsic affinity of HA for DR1. As a result, the affinity of HA will be vastly underestimated. At the other end of the spectrum, the kinetics of an extremely weakly binding peptide will probably reflect the intrinsic affinity of the peptide. For peptides falling between these two extremes, the measured affinities will contain an inseparable mix of contributions from conformational changes and true binding interactions. However, determination of the dissociation rate of the complex does not involve the same problems as the affinity assay and hence is a more reliable measure of the true interactions between the peptide and MHC. This may explain why certain structural and functional aspects of the MHC/peptide complex, such as SDS stability (23, 24, 32) and sensitivity to DM (20), correlate much better with the dissociation rates than they do with the measured affinities.
Receptive conformation of MHC can be induced at neutral pH
MHC molecules are suggested to have evolved to bind better in acidic conditions, since the peptide loading compartments in the APCs are acidic. In this report we have studied stable peptide binding that occurs solely due to the maintenance of peptide-friendly conformation of MHC in neutral pH. Enhanced in vitro peptide binding under mildly acidic conditions has been reported for many, but not all, class II alleles (9, 23, 24, 25, 26, 27). MHC is suggested to undergo conformational changes at mildly acidic pH, thus facilitating optimal peptide binding (28, 29, 30). Our results, however, show that the receptive conformation of DR1 can be induced and maintained at a neutral pH. Notably, peptide binding to DR1 is fairly independent of pH in the 5–7 range (26). In contrast to the allele-specific pH effects, the suggested mechanism by which stable binding is enhanced by prior binding with weak peptides may be more commonly applicable to all alleles.
In vivo relevance of short-lived peptide/MHC complexes
In this report we have shown that the MHC molecule maintaining a
peptide-friendly conformation, (ß)*, is essential for
extremely rapid binding. Our results suggest an allele-independent
mechanism involving weakly binding peptides by which the MHC is
maintained in such a receptive conformation in the APC. After the
removal of class II-associated invariant chain peptide, if the
MHC molecule does not bind other peptides, it will rapidly lose its
peptide binding ability due to conformational changes and aggregation
in the acidic lysosomal compartments. Nevertheless, the Ag loading
compartment is abundant in peptide fragments, a significant fraction of
which will probably be weakly binding. These peptides can bind and
dissociate rapidly, maintaining MHC in the receptive conformation until
a stably binding Ag is encountered.
We find that the conformation of the MHC is the critical qualitative factor influencing rapid binding of peptides. As is evident from this study, this conformation can be achieved in the absence of DM. DM, with its demonstrated ability to accelerate the dissociation of low affinity peptides, however, can increase the number of available MHC molecules in the correct conformations. Thus, the low affinity peptides in the loading compartments play a qualitative role, while DM may only play a quantitative role, in enhancing Ag presentation.
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Acknowledgments |
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Note.
During review of this manuscript, another paper describing the receptive state of class II MHC has been accepted for publication (31).
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Footnotes |
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2 Present address: Department of Biological Sciences, George Washington University, Washington, DC 20052.
3 Address correspondence and reprint requests to Dr. Scheherazade Sadegh-Nasseri, Department of Pathology, Johns Hopkins School of Medicine, 664E Ross Building, Baltimore, MD 21205. E-mail address:
4 Abbreviations used in this paper: HA306–318, peptide containing hemagglutinin residues 306–318 of influenza virus; Fl, fluorescein; Fl-HA, fluorescein-Cys-hemagglutinin; AMCA, 7-amino-4-methyl-coumarin-3-acetic acid; HPSEC, high performance size exclusion chromatography.
Received for publication September 11, 1998. Accepted for publication December 22, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß heterodimers in the absence of antigenic peptide. Cell 68:465.[Medline]
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). B, Kinetics of SDS-stable DR1/HA
complex formation in dissociating DR1/Anchorless-HA complex.
DR1/Anchorless-HA complex (0.5 µM; isolated by HPSEC) was incubated
with 50 µM HA for various times at 37°C in PBS. Samples were then
mixed with equal volumes of SDS-PAGE sample buffer containing 0.2% SDS
(final concentration), incubated for 15 min at room temperature, and
electrophoresed on a 12.5% polyacrylamide gel. The gel was silver
stained according to standard protocols and scanned on a Agfa Arcus
Laser Scanner. The intensities of the 
