| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Gáspár, Jr.2,*
si*zsér
Departments of
*
Biophysics and Cell Biology and
Biochemistry and Molecular Biology, and
Biophysics Research Group of the Hungarian Academy of Sciences, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary; and
Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Conflicting data were reported about the critical number of MHC-peptide
ligands necessary to trigger T cell activation. Monomers of soluble
class I MHC-peptide ligands were shown to trigger calcium responses in
CD8
+, but not in
CD8- T cells (4), suggesting that
the presence of CD8 coreceptor and its interaction with both TCR and
MHC-I molecules are also prerequisite of an efficient T cell
activation. In contrast, Boniface et al. (5) reported that
only dimers or tetramers, but not the monomer of the soluble murine MHC
(IEk)-peptide ligand could stimulate T cells with
a sufficient efficiency. Formation of supramolecular activation
clusters in T cells upon Ag stimulus was also found to be dependent on
the arrangement of MHC-peptide ligands (3). Thus, the
question arises whether a proper supramolecular organization of
MHC-peptide ligands on APCs is also critical to an efficient T cell
stimulation. While molecular organization of the TCR-related signaling
components has extensively been investigated recently, not much
attention has been paid to MHC-peptide organization on APCs.
Fluorescence resonance energy transfer (FRET)3 measurements of molecular proximities on the nanometer scale and lateral diffusion measurements by single particle tracking both can directly probe clustering/association of proteins in the plasma membrane of live cells (6, 7, 8, 9, 10). Spontaneous homo-association of class I HLA molecules has been detected earlier in proteoliposomes (11), on activated T and B lymphocytes, and on a number of transformed human cell lines by FRET and long-range electron transfer (12, 13, 14, 15, 16). The existence and spatial arrangement of such class I HLA oligomers on APCs may have important functional consequences; therefore, it should also be considered in modeling the MHC-TCR interaction.
X-ray crystallographic structures of HLA molecules and their complexes with peptides (17, 18, 19, 20) are sufficiently well resolved and provide detailed three-dimensional (3D) structural information to localize Ab binding sites (epitopes) on these molecules. However, they do not allow steric positioning (orientation) of the molecule relative to the plane of the plasma membrane, since the crystals are usually developed from digested extracellular domains of the proteins; therefore, the influence of the missing domains (intramembrane helix and cytoplasmic tail) on their conformation and orientation cannot be taken into account.
In the present work, we outline a new approach of computer modeling based on docking epitope-specific Ag-binding fragments (Fab) of mAbs to high resolution x-ray structures combined with their steric positioning on the basis of experimental results from FRET-based epitope mapping performed on membrane-bound intact HLA-I molecules in live cells. Fitting the x-ray-resolved structures to the in situ FRET distance estimates helped to generate 3D models of the possible supramolecular organization of class I HLA molecules, reflecting close to physiological conditions. These 3D models could also consider possible dimerization/oligomerization of these proteins.
In this study, we show that only a limited group of sterically allowed, computer-generated 3D molecular superstructures could be fitted to the FRET data measured on live cells. These include both dimers and tetramers of class I HLA. Furthermore, we show a model of a possible supramolecular cluster built from these HLA-I oligomers, CD8 and TCR molecules, that is sterically allowed and matches FRET data, as well. The importance of such analysis is accentuated by the existing contradiction of views on the necessity of a monomeric (4) or oligomeric peptide-MHC complex (5) in the course of initiation of efficient signal transduction through the TCR. The presented 3D models can be considered as possible in situ arrangements of supramolecular HLA clusters on APCs, also at the site of APC/T cell interface, since herewith we consider only those cases in which the molecular associations of class I HLA molecules are supported by unambiguous physical measurements at the surface of live cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The EBV-transformed JY human B lymphoblast cell line (21) was cultured in RPMI 1640 medium supplemented with 10% FCS in 5% CO2 atmosphere, at 37°C.
mAbs and Fab preparation
W6/32 (IgG2a
) mAb with specificity for the heavy chain of
class I HLA A,B,C molecules and L368 (IgG1) mAb specific for
2-microglobulin (2m)
were kindly provided by F. Brodsky (University of California, San
Francisco, CA). Fab were prepared from mAbs using a method described
earlier (22). Briefly, IgG mAbs were dialyzed with
phosphate buffer (100 mM
Na2HPO4, 150 mM NaCl, 1 mM
EDTA, pH 8) and digested with activated papain at 37°C for 10 min.
The enzyme activity was terminated by addition of iodoacetamide. The
reaction mixture was passed through a Sephadex G-100 superfine column.
Collected Fab fractions were further separated from the residual IgG
content by passing through a protein A-Sepharose column.
Conjugation of Fab with fluorescent dyes
Aliquots of Fab (at least at 1 mg/ml concentration) were conjugated, as described earlier (15), with 6-(fluorescein-5-carboxamido)hexanoic acid succinimidyl ester (5-SFX; Molecular Probes, Eugene, OR), or 6-(tetramethylrhodamine-5(and -6)-carboxamido)hexanoic acid succinimidyl ester (5(6)-TAMRA-X, SR). The proteins were transferred into a carbonate-bicarbonate buffer at pH 8.3, mixed with 30- to 100-fold molar excess of freshly prepared fluorescent dyes (in DMSO), and incubated for 45–60 min, at room temperature. Unreacted dye molecules were removed by gel filtration through a Sephadex G-25 column. Final Ab concentration and the dye to protein ratio were determined spectrophotometrically (15). In case of SFX and TAMRA-X, the ratio varied between 0.8 and 1.2 dye/Fab. The fluorescently tagged Fab retained their biological activity as supported by the competition with identical, but unlabeled Fabs.
Labeling of cells with fluorescent Fabs
Freshly harvested cells were washed twice in ice-cold PBS (pH
7.4). The cell pellet was suspended in 100 µl of PBS (1 x
107 cells/ml) and incubated with 
10 µg of
SFX- or TAMRA-X-conjugated Fabs for 60 min on ice. The excess of Fabs
was at least 30-fold above the Kd
during the incubation. To avoid possible aggregation of the Fabs, they
were air fuged (at 5 x 104 x g,
for 30 min) before labeling. Staining of cells was checked under
fluorescence microscope after the labeling procedure. Special care was
taken to keep the cells on ice before FRET analysis to avoid
significant protein internalization. The labeled cells were washed with
excess of cold PBS and then occasionally fixed with 1% formaldehyde.
Data obtained with fixed cells did not differ significantly from those
of unfixed, viable cells. The number of binding sites on the cell
surface was determined from the mean values of flow cytometric
histograms of cells labeled to saturation with SFX-conjugated Fabs. The
mean fluorescence intensities were converted to number of binding sites
by calibration with fluorescent microbeads (Quantum 25; Flow Cytometry
Standards, San Juan, PR).
Labeling of cells with lipid probes
The surface of JY cells was labeled for FRET-based vertical distancemeasurements by 2-(4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocho-line (BODIPY-PC (581/591)) as acceptor for SFX-conjugated Fabs bound either to the heavy or the light chain of class I HLA molecules. When labeling with BODIPY-PC (581/591) (Molecular Probes), a final concentration of up to 5 µg/ml of dye was added to the cell suspension, at a cell density of 4–5 x 106 cells/ml. Labeling was conducted for 20 min on ice, followed by 20 min at 37°C. Cells were washed with ice-cold PBS, labeled with Fabs, and analyzed immediately by flow cytometry. Cells were kept in ice-cold buffer also during the flow cytometric analysis.
Flow cytometric resonance energy transfer measurements
Flow and image cytometric techniques based on FRET, a spectroscopic ruler working on the 2- to 10-nm scale, are very suitable for studying conformation and associations of biomolecules (8, 23, 24, 25, 26, 27). Efficiency (probability) of FRET depends on inverse sixth power of the actual separation distance of donor and acceptor dyes (confined in this study to the V domains of Fab molecules). This holds only if the effect of spatial orientation of donor and acceptor dipoles on FRET efficiency can be neglected (27). In our study, the spacer groups on the dyes allowed a reasonable dynamic averaging of dipole orientations, as confirmed by the low value of fluorescence anisotropy of cell-bound dye-Fab conjugates (r < 0.15).
In the present study, the FRET efficiency was determined in a Becton
Dickinson (Sunnyvale, CA) FACStarPlus flow
cytometer, by measuring both the extent of donor-quenching and
acceptor sensitization through detecting spectrally selected
fluorescence intensities on cell by cell basis, as described in details
earlier (25, 26, 28). The efficiency of energy transfer
(E) is expressed as percentage of the donor’s (SFX)
excitation energy tunneled to the acceptor (TAMRA-X) molecules. The
mean values of the energy transfer efficiency histograms (determined on
a cell population; see Fig. 1
B) were used and tabulated as
characteristic FRET efficiencies between two epitopes. Intra- and
intermolecular epitope distances as well as the out-of-membrane plane
location of HLA epitopes were determined by FRET, using the strategy
shown in Fig. 1A.
|
In case of protein to lipid FRET measurements, the fluorescent
proteins are surrounded by a large array of acceptor-labeled lipids.
Therefore, the efficiency of FRET in this study will also depend on the
donor to acceptor ratio. In such a situation only, "closest approach
distances of labeled epitopes from the membrane surface"
(perpendicular to the cell membrane) can be estimated according to the
theory and model described by Yguerabide (29, 30). The use
of this approach is justified by the observation that at the applied
concentrations of the lipid probe the Stern-Volmer plots displaying
Ida/Id (ratio of donor
intensities in the presence and absence of acceptor, respectively) vs
the surface density of BODIPY-PC varied linearly. On the basis of the
overlap between absorption and emission spectra of the applied dyes, we
used Ro value of 3.9 nm for the SFX-BODIPY PC
(581/591) donor-acceptor pair. The calculation of closest distance for
Fabs from the cell membrane is described in details in the footnote of
Table I.
|
Three-dimensional molecular models were created from the crystal
structure of the extracellular domain of HLA-A2 (PDB code: 1AKJ) and
that of human Fab B7-15A2 (PDB code: 1AQK). L368-binding epitope was
identified by Asp38, Glu44,
and Arg45 residues of the
2m of HLA (31, 32), and
W6/32-binding epitope was identified by Asp86 and
Lys121 residues of the heavy chain of HLA, close
to the junction of the peptide-binding domains and also to the
2m domain (31, 33).
An automated search sequence (using Fortran programs written by one of
the authors, P. B.) has been generated to find all possible
positions of the Fabs relative to the HLA class I complex in their
multimolecular complex corresponding to the situation when
fluorescence-labeled Fabs are bound to both the W6/32 and L368 epitopes
of the HLA class I molecule (Fig. 2, A and B).
During the process, grid points were assigned to specific locations
within the molecules (Fig. 2, C and D). Fabs were
docked into place by the geometrical center of the surface residues of
their hypervariable regions (Fig. 2D) using a 0.6-nm lower
and 1.6-nm upper limits for distances measured from carbons of the
epitope-building residues of HLA (Fig. 2C). The lower and
upper limits may mimic the contact distances of carbons of the
interacting residues in case of small residue-small residue and bulky
residue-bulky residue contacts, respectively. Fluorescent labels on the
Fab molecules were found to be confined specifically to the V region of
the Fabs (unpublished result: J. T.). FRET-measured interepitope
and epitope-to-cell surface distances represent dynamically averaged
values based on a cylindrical fluorescence distribution model of
fluorescence-labeled cell surface-bound Fab molecules
(34). Accordingly, intermolecular distances were estimated
in the present study always from the geometrical center of the V region
of the Fab molecules (Fig. 2D).
|
E).
This layer was searched for possible dimer configurations using as
criteria the FRET-measured W6/32-W6/32 and L368-L368 intermolecular
distances. The first molecule was allowed to move only in direction of
z-axis in the layer, while the second molecule was
positioned along the Z and Y axes with 0.2-nm steps, and rotation
around its axis perpendicular to the membrane was allowed in the range
of 0–360 degrees with 15-degree steps. Vertical tilting of the class I
HLA molecules was adjusted to position the presented peptide close to
parallel to the membrane surface to provide maximal exposure toward the
surrounding environment. Top view of the HLA molecules situated in the
most populated vertical layer found in this search is shown in Fig. 2F. This figure visualizes the effect of the errors
originated from the uncertainty of FRET measurements and from the
modeling procedure on the accuracy of the in plane (parallel to the
cell membrane) position determination of the second HLA molecule
compared with a fixed one. Based on this procedure, the allowed
distance range between the two HLA molecules is 7–9.4 nm, and for the
rotational angle of second molecule -30 to +45 degrees.
The HLA dimer of Fig. 3, A and B, is one
representative example from the closely situated dimeric configurations
in Fig. 2F. The intermolecular epitope distances correspond
to the FRET-determined experimental distance data in Table I and
represent a situation when the binding of fluorescent labeled is
allowed to both the L368 and W6/32 epitopes. The closely associated
dimer of Fig. 3, C and D, was created by
superimposing the membrane-proximal domain of HLA class I on the
membrane-proximal domain of the crystal structure of the dimer of HLA
class II heterodimers (PDB code: 1SEB). The best fit among the two
molecules resulted in 0.16-nm root mean square deviation for the 76–76
carbon atoms of the respective domains of the molecules. The
tetrameric arrangement of Fig. 3, E and F, was
created by superimposing class I HLA-A2 monomers from both kinds of
dimers (Fig. 3, A, B, and C,
D).
|
/TCR complex was created from the
crystal structure of HLA-A2/peptide/CD8 complex (PDB code: 1AKJ)
and the crystal structure of HLA-A2/peptide/TCR complex (PDB code:
1AO7) by superimposing the respective HLA molecules. Positions of the
HLA molecules in the tetramer of Fig. 3, G and H,
are the same as those of the HLA molecules in the tetrameric complex of
Fig. 3, E and F. Superposition of the structures
was performed by using Whatif 4.0 (G. Vriend, EMBL, Heidelberg,
Germany) or O 5.8.1 (T. A. Jones, Department of Molecular Biology,
Biomedical Centre, Uppsala, Sweden) programs, and the alignments
of sequences were created in Clustalw 1.7 (35). Molecular
structures were examined on a Silicon Graphics Indigo2 computer running
the program Sybyl version 6.3 (Tripos, St. Louis, MO). The above outlined modeling approach was chosen because the exact primary sequences of the W6/32 and L368 Abs are yet unknown; therefore, traditional force field-based methods could not be used for the optimization of the 3D configuration of the multimolecular complexes investigated.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Fig. 1A illustrates the
strategy of FRET measurements conducted on JY B cells by the flow
cytometric energy transfer method (25, 26). The JY cells
are EBV-transformed B lymphocytes (HLA-A2, B7, DR4,
DQw1.3)+ (21) with a dominance of
the HLA-A2 allelic determinant. Heavy and light chain
(2m) epitopes on class I HLA molecules were
targeted by Fab of W6/32 and L368 mAbs, respectively. The W6/32 Ab was
reported to bind near to the Asp86 and
Lys121 residue of the -chain of HLA
class I molecule, close to the junction of the peptide-binding
1/2
domains and also to the membrane-proximal 3
and 2m domains (31).
Gln2 of 2m at the N-terminal
region was predicted to be involved in the binding of W6/32 Fab in our
computer modeling, due to the close proximity of it to the epitope on
the -chain (33). The antigenic epitope
defined by L368 mAb is localized on the human
2m molecule involving residues
Asp38, Glu44, and
Arg45 of the three-stranded -pleated sheet of
2m (31, 32).
For FRET measurements, these Fab were conjugated with either
succinimidyl-fluorescein (X-SF) dye, serving as energy donor, or
succinimidyl-rhodamine (X-SR) dye, serving as energy acceptor. In
average, Fab molecules carried 1 fluorophore/molecule with a high
degree of motional freedom (indicated by emission anisotropy
values, data not shown) allowing a dynamic orientational averaging
(36). This condition allows a reasonable distance
estimation from FRET efficiencies, since the statistical average of the
orientation factor (otherwise also influencing FRET efficiency),
2 = 2/3, is a good approximation for this
case. For epitope mapping, three kinds of FRET measurements were
conducted on JY cells: 1) intramolecular FRET between donor-L368 and
acceptor-W6/32 (R3 distance), 2) intermolecular
FRET between donor-W6/32 and acceptor-W6/32 (R4
distance), and 3) intermolecular FRET between donor-L368 and
acceptor-L368 (not indicated on Fig. 1A).
The FRET efficiency histograms constructed from cell by cell flow
cytometric measurements (Fig. 1B) show that the distribution
of intramolecular FRET efficiency (distances between the heavy and
light chain epitopes on the same HLA molecule) is narrower than the
distribution of intermolecular FRET efficiencies. This reflects that
the homo-association (oligomerization) of class I HLA molecules is only
partial, and also shows a heterogeneity over the cell population. The
FRET efficiency data (means of the histograms) were converted to
molecular distances, as described in the legends to Table I. The
intramolecular epitope distance between 2m
(L368) and the heavy chain (W6/32) was 6.8 nm, and the average
distance between heavy chain epitopes (W6/32) of two neighboring HLA
molecules was maximum 7.6 nm. The average distance between two
neighboring light chain epitopes was a bit higher, 7.9 nm (Table I).
These approximate molecular distances, considered as average distances
between the mass centers of the V regions of Fab molecules, were used
further on in the computer modeling, in which the error arising from
the uncertainty of dye localization within the V
(VH and VL) domains of Fab
was also taken into account.
Mapping of the vertical distances of heavy and light chain epitopes from the surface of the plasma membrane by FRET
The relative position of class I HLA molecule to the exofacial
surface of the plasma membrane has been estimated by FRET measurements
between the heavy chain or the 2m epitopes of
the molecule labeled by fluorescent Fabs and a fluorescent
phosphatidylcholine analogue, BODIPY-PC, localized in the polar, head
group (surface) region of the plasma membrane. The BODIPY-PC molecules,
however, may form a multiple acceptor array around donor Fab-labeled
HLA molecules in the plane of the membrane, depending on their
concentration. Thus, in this study, the actual acceptor density may
also largely influence the FRET efficiency (29, 30).
Therefore, in this case, FRET measurements were conducted at different
donor/acceptor ratios (Fig. 1C). The FRET efficiencies were
analyzed in terms of distances of closest approach from the
Stern-Volmer plots of fluorescence quenching (Fig. 1C),
according to Yguerabide (30) (see legend to Table I). The
light chain (L368) epitope had a closest approach distance
(R2, Fig. 1A) of 7.5 ± 0.2 nm from
the membrane surface, while the heavy chain (W6/32) epitope was found
to be located closer (R1: 6.2 ± 0.2 nm). In the
further molecular modeling, these distance estimates, measured on
intact live cells, were used in steric positioning of the
x-ray-resolved HLA-A2 structures relative to the exofacial surface of
the plasma membrane.
Docking of Fab to the crystallographic structure of HLA-A2
Fig. 2, A and
B, demonstrates a model of the docking of fluorescent Fabs
during the labeling procedure by selected amino acid residues in their
hypervariable regions to the antigenic epitopes on the
2m and heavy chain domains of the class I HLA
molecule. A crystal structure of human Fab B7-15A2 (PDB code: 1AQK) was
used as a model of W6/32 and L368 Fabs (see Materials and
Methods). The relative positions of the geometrical centers of
their V domains have been set to match the distances estimated from
FRET measurements using donor- and acceptor-labeled Fabs (Table I). The
coordinate system is fixed to the plasma membrane surface (not
shown).
The positions of the labels were confined to the geometrical center of
V domain of the Fab molecules (red dot in Fig. 2D), since
the Fabs seemed to be labeled preferentially at the same lysyl residue
at 1:1 dye:Fab ratio, shown by sequencing and mass-spectrometric
analysis of fragmented Abs conjugated with fluorescent dyes
(Tzsér et al., unpublished data). The high reproducibility
of our FRET efficiency data obtained with independently labeled Fabs
also confirms this observation. The Fab molecules are undergoing a
restricted rotation around their binding sites in a cone fashion;
therefore, the positions of the Fab molecules can be regarded as
dynamic motional averages (34). The possible positions of
the fluorescent Fabs relative to the class I HLA molecule have been
determined by an automated screening procedure, as described above,
throughout molecular modeling.
3D molecular models of dimeric and tetrameric forms of HLA-A2
The mean FRET efficiency measured between HLA molecules on JY B cells (and also on a number of transformed human B and T cell lines by Matkó et al. (12, 13)) ranges from 15 to 30%, indicating that a significant portion of HLA-I molecules is present in multimeric forms (e.g., dimers, trimers, tetramers, or higher oligomers) at the cell surface. FRET data alone are not able to precisely measure the extent of oligomerization or the fractional amount of different multimers, but clearly indicate their existence, since purely monomeric form of HLA class I at the cell surface is inconsistent with such an efficient FRET as measured in the present study. Accordingly, we searched for multimeric forms of the HLA class I molecules by molecular modeling.
Following the logic of pair-wise FRET measurements between different epitopes of the HLA class I molecule and also considering that free space must be allocated for fluorescent Fabs attached to both epitopes in a dimeric or higher order arrangement of HLA, an automated computer search sequence has been set up to find all possible relative positions of the two HLA class I molecules in their dimeric complex (for details of the procedure, see Materials and Methods). The identification for an allowed dimeric configuration was done by superimposing FRET-measured interepitope and epitope-cell surface distance data on the corresponding intermolecular distances in the 3D molecular modeling system. Intermolecular distances were measured in the 3D model between the geometrical centers of the V regions of the Fab molecules attached to the HLA class I epitopes and between those and the fluorescent dyes confined to the plane of the plasma membrane.
The first result of the above search procedure was the vertical
localization of the HLA molecules in a 1.8-nm-thick layer above the
plane of the plasma membrane, as displayed in Fig. 2E.
Within this layer, one HLA class I molecule was fixed and used as
reference, and the other molecule in the dimeric configuration was
moved around in 0.2-nm steps, corresponding to the accuracy of the FRET
measurements, and rotated around its vertical axis in a range of 0–360
degrees in 15-degree steps. Based on the above procedure, the distance
between the two HLA class I molecules in their dimer is 7–9.4 nm, and
the second molecule is rotated compared with the other in a range of
-30 to +45 degrees. Fig. 2F nicely demonstrates the
accuracy of the modeling procedure and shows that despite the
relatively wide ranges of relative intermolecular distance and
rotational angles, the HLA class I molecules are fairly well located in
space. The relatively large number of sterically allowed dimeric
configurations is consistent with the assumption that these dimers are
not rigid entities, but they represent a dynamical molecular assembly
embedded in the plasma membrane.
As a result of the above-described search procedure for sterically
allowed and FRET- supported dimeric configurations, a representative
sample was selected for further examination (Fig. 3, A and B). Fig. 3, C and D, shows another sterically allowed
dimeric configuration, in which the class I HLA monomers labeled with
both heavy chain- and 2m-bound fluorescent
Fabs can get to close proximity of each other. This 3D model of the
dimeric configuration has been constructed on separate grounds from the
one described previously (in Fig. 3, A and B). In
this study, an analogy to the hypothetical model of cooligomerization
of CD4, class II MHC, and TCR (37, 38) was assumed. In
this case, the estimated distance between the fluorescent labels is far
beyond the upper limit of the FRET measurements regardless of their
exact position on the Fab molecules. Therefore, such arrangements can
practically be excluded at physiological conditions.
We found that the elements of the previous two dimeric models can be
used in combination to generate a likely tetrameric arrangement. Fig. 3, E and F, shows the 3D configuration of a class
I HLA tetramer serving as reference points for FRET measurements. In
this model of tetrameric molecular complex, the two external HLA
molecules can bind both types of Fabs simultaneously, while the other
two HLA can bind only anti-heavy chain Fab (W6/32), if one takes
into account the steric limitations enforced by the two Fabs binding to
the class I HLA molecule (see Fig. 2B). In this sterically
allowed tetrameric configuration, an efficient FRET signal is expected
between W6/32 and L368 Fabs attached to the same monomer and between
two W6/32 Fabs attached to two different monomers, while no FRET is
expected between two L368 Fabs attached to different monomers. This
configuration matches well FRET data, as described in the discussion
section. The lack of Fab on two of the four 2m
molecules is justified by the steric hindrance.
Fig. 3, G and H, represents a 3D model of
cooligomerization of CD8, class I HLA, and the TCR molecules. This
hypothetical model is based on FRET-defined intermolecular distances
and crystallographic structural data, and uses the model of tetrameric
arrangement of class I HLA molecules (Fig. 3, E and
F). It was constructed by considering the structural
features of contact sites reported for the HLA-A2-TCR complex
(39) and for MHC-CD8 interaction (20, 40).
This model represents a possible 3D organization of a multimolecular
complex consistent with the present FRET data, the former x-ray
crystallographic structures, and signal transduction studies on
triggering of T cell activation (4). This structural
arrangement can also serve as a functionally significant unit to form a
network of CD8-class I HLA multimolecular complexes to concentrate and
clusterize the TCR with its associated signal transduction components
at the site of the T cell-APC interface. This might be especially
important at low level of MHC-peptide challenge.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-helical segment of the HLA
molecule and interactions with neighboring molecules can tilt or
displace the extracellular domain of class I HLA molecules relative to
the membrane surface. With the help of the present, novel molecular modeling approach, we fitted the available x-ray structure of HLA-A2 molecules to in situ biophysical proximity data measured on live cells. The basic goal of the present computer-modeling work was to test whether sterically allowed dimeric and tetrameric arrangements of 3D HLA-A2 structures could be fitted to the FRET-based lateral and vertical epitope-mapping data measured on live cells.
The modeling was based on the following experimental data. Structural
details of the HLA-A2 molecule have been studied by x-ray
crystallography, and the structure of its complex with CD8 has recently
been described (20). Antigenic epitopes, defined by mAbs
L368 and W6/32, used in FRET measurements, have been localized on the
2m and heavy chain domains of class I HLA
molecule, respectively (31, 32). Binding domains of the
contact area in the CD8-MHC complex have also been
identified (40). The above information plus data from a
FRET-based vertical distance mapping of HLA-I heavy and light chain
epitopes from the surface of the plasma membrane of B cells, as well as
data reporting on oligomeric forms of HLA-I on the cell surface, were
all considered throughout the molecular modeling.
The modeling approach resulted in several 3D models of possible dimeric
and tetrameric arrangements of class I HLA molecules matching well the
FRET data measured on the surface of APCs (Fig. 3). A further advantage
of the combined FRET-crystallographic structural modeling approach was
pointed out by the possibility of positioning the Fab-binding epitopes
on the light (2m) or heavy chains relative to
the membrane surface, as demonstrated by Fig. 2, A and
B. According to the crystallographic model of HLA-A2, the
position of the 2m was expected to be more
membrane proximal because it was attached to the
3-domain of the heavy chain.
However, FRET data clearly showed that the Fab bound to the L368
epitope was located farther from the plane of the membrane than the Fab
binding to the W6/32 epitope on the heavy chain.
Regarding the dimer models, a relatively large number of sterically
allowed configurations could be constructed from molecular associations
of class I HLA and Fab molecules similar to the one shown on Fig. 2, A and B (see Fig. 2F). These
configurations, however, were based on the assumption of random
orientation of monomeric units in the dimers after collisions limited
by the lateral and rotational motional freedom of cell surface HLA-I
molecules (41). However, by choosing the equilibrium
distance and rotational angle as defined in the modeling procedure and
allowing for the binding of both L368 and W6/32 Fabs, the number of
possible, sterically, and FRET-allowed dimeric configurations could be
reduced to one due to the steric constraints introduced by the bound
Fabs (see model in Fig. 3, A and B). The other
dimeric configuration shown on Fig. 3, C and D,
was constructed by supposing an analogy to the lattice model of class
II HLA superdimers (37, 38). In this configuration, the
two HLA molecules were closer to each other, and the docked Fabs were
pointing apart from each other (compare to Fig. 2B);
thereby, the fluorescent labels were located far beyond the maximal
distance probed by FRET. Therefore, the contribution of this
configuration to the cell surface dimeric class I HLA population must
be small, because the highly efficient FRET observed contradicts the
large interepitope distance predicted in this study by the 3D molecular
modeling.
The hypothetical, sterically allowed model proposed for HLA-I tetramers
(Fig. 3, E and F), constructed from the two kinds
of dimeric configurations, was consistent with a number of experimental
observations on live B cells. 1) The mean intermolecular FRET
efficiency between two anti-heavy chain Fabs was consistently
higher than between two anti-2m
Fabs. 2) A systematically lower number of binding sites was found on
these (and also on other) cells for L368 Fab than for W6/32 Fab using a
flow cytometric Scatchard analysis (see in Ref. 14),
consistent with a possible shielding of L368 Fab binding site in a
tetrameric (or higher oligomeric) arrangement. 3) Surprisingly, the
efficiencies of intramolecular FRET between the
2m (L368) and heavy chain (W6/32) epitopes
were different in the opposite directions. If the donor label was
located on L368, the FRET efficiency was consistently higher
(33.0 ± 1.5%) than in the other direction, when the donor dye was
on the W6/32 Fab (26.4 ± 2.2%). Since the distance of these
epitopes was fixed within a monomeric unit, the same FRET efficiency
was expected in either direction. Indeed, this might be the case for
monomeric and dimeric forms of HLA-I molecules. However, assuming
existence of tetramers corresponding to the model in Fig. 3, E and F, the situation is different. When L368
Fab carries the donor dye, there is always a proximal acceptor-labeled
W6/32 Fab within the FRET distance (10 nm), while if the donor dye
is on the W6/32 Fab, only a part of the W6/32 Fabs could contribute to
FRET, because another part did not have acceptor-L368 Fab within the
FRET-measuring range. Thus, a part of donor-W6/32 Fabs would contribute
to FRET with the same efficiency as do the donor-L368 Fabs toward
acceptor-W6/32 Fabs, while the rest would contribute with zero
efficiency. Therefore, from the measured FRET efficiency values one
could even estimate the maximal possible fraction of tetramers among
the cell surface MHC-I molecules using the following train of thoughts.
The lower FRET efficiency (26.4%) is consistent with a picture in
which 80% of donor-W6/32 Fabs participated in FRET, while 20% did
not. Since in the tetramer two of the four 2m
still can bind L368 Fab, we could say that at most 40% of the total
cell surface 2m is located in tetrameric
forms, while at least 60% of 2m exists in
monomeric or dimeric forms. If higher oligomers (n >
4) were also present, then on the basis of FRET data we could say that
the monomers and dimers together had larger proportions than 60% of
the total class I HLA molecules.
Earlier x-ray data directly revealed superdimers of class II HLA
molecules, but failed to predict such structures for class I HLA
molecules to date. In contrast, a large number of physical measurements
on proximity (by FRET), rotational and lateral mobility, and
cell surface distribution (by scanning near-field optical microsopy) of
HLA-I strongly indicated the existence of such supramolecular
structures in the plasma membrane of live human cells (10, 12, 13, 14, 15, 42, 43). Our FRET data measured on JY B cells were also
in good accordance with these observations. Thus, the 3D models
presented in this work could be considered as highly likely
supramolecular structures formed from HLA-I molecules at the surface of
potential APCs. Besides the momoneric, dimeric, trimeric, and
tetrameric forms, larger clusters (n > 4) might also
exist on the cell surface, as indicated by rotational mobility
(42) and scanning near-field optical microscopy
(43) data. Such clusters were supposed to be initiated
and/or stabilized by 2m-free heavy chains
(12, 44, 45).
These oligomeric structures may have functional significance, as well. Using agonist and partial antagonist peptide-loaded soluble murine class I MHC (Ek) and soluble TCR, Reich et al. (46) reported on a ligand-specific oligomerization of TCR in solution. Moreover, using soluble monomers, dimers, trimers, and tetramers of class I MHC (Ek) ligands to stimulate intact T cells, they found the monomers of agonist peptide-loaded MHC nonstimulatory, the dimers weakly stimulatory, while the trimers or tetramers proved to be highly potent stimuli (5). These observations support the likeliness of a ligand-driven TCR clustering through multivalent engagement, also proposed earlier for human cells (12). Our models based also on in situ physical measurements underline the reality of such multivalent engagement. Such HLA-I oligomers may also appear in the contact areas of APC-T cell conjugates serving as key elements of immunological synopses (3, 47, 48).
Conflicting data and theories have been reported about the necessary
triggering threshold of T cell activation with respect to the necessary
number of HLA-peptide ligands. Delon et al. (4)
demonstrated that soluble monomers of murine MHC-peptide complexes were
able to trigger calcium signals, but only in CD8+
T cells. It was also demonstrated that CD8 molecules could increase the
apparent affinity of MHC-peptide ligands for the TCR (49, 50). Earlier works definitely supported the close cooperation
and molecular proximity of peptide-MHC and CD8 (51), also
at the interface of target and effector cells probed directly by
intercellular FRET measurements between Ag-presenting and effector
cells (47). Considering the CD8-class I HLA interaction,
the present model of HLA tetramer was further expanded with the 3D
structural entities of the connecting CD8 and TCR molecules. A
multimolecular complex involving the molecular entities of dimeric CD8,
class I HLA, and the TCR could directly be generated from the above
tetrameric HLA model without any steric hindrance (see Fig. 3, G and H). Such supramolecular assembly of class I
HLA oligomers with CD8 molecules and TCRs can be considered as a
possible protein cluster existing in Ag-presenting-effector cell
conjugates and comprising several functional advantages: 1) the
possibility of multivalent engagement of TCRs; 2) TCR clustering may be
promoted by the supramolecular complex displayed in Fig. 3, E and F or its expanded CD8/MHC-peptide network;
3) these multimolecular clusters together with other accessory
molecules (e.g., adhesion proteins) may also promote formation of an
immunological synapse (48) at the contact site.
In conclusion, the newly introduced computer-modeling approach based on the combination of x-ray-resolved 3D structures and in situ FRET data measured on live cells offers the advantage of constructing realistic 3D structural models of supramolecular protein complexes in the plasma membrane of cells. By expanding the 3D model of HLA-I oligomers via connecting CD8 dimers and TCRs to it, the existence of a supramolecular complex is predicted that may contribute with an enhanced efficiency to T cell stimulation by multivalent engagement/clustering of TCRs with a cooperating CD8 molecule essential to initialize T cell signaling. This model is compatible with the data of both Boniface et al. (5) and Delon et al. (4); thus, it can partly resolve their apparent contradiction, especially at low level of MHC-peptide stimulus. Similarly, T cell activation threshold has been found recently to depend on the oligomerization degree of chemically defined MHC class II oligomers (52). The presented 3D models are in good accordance with the results of in situ physical measurements of HLA-I distribution/proximity and mobility on live cells. Although throughout this work B cells and not professional APCs (e.g., dendritic cells) were used, we believe that our models help to understand the yet unclear principles of molecular organization of Ag-presenting MHC molecules on the surface of APCs. The present physical analysis can be further extended to APC-CTL conjugates where these molecules are in situ interacting with TCRs and accessory molecules. With the growing number of available x-ray structures of important cell surface macromolecules, the applicability of the method can easily be extended to other significant questions of immunology.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Rezs Gáspár, Department of Biophysics and Cell Biology, University of Debrecen, Medical and Health Science Center, P.O. Box 39, 4012 Debrecen, Hungary.
3 Abbreviations used in this paper: FRET, fluorescence resonance energy transfer; 3D, three-dimensional; 2m, 2-microglobulin; BODIPY-PC, 2-(4,4-difluoro-5-(4-phenyl-1, 3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine; Fab, Ag-binding fragment; SFX, 6-(fluorescein-5-carboxamido)hexanoic acid succinimidyl ester; TAMRA-X, 6-(tetramethylrhodamine-5(and -6)-carboxamido)hexanoic acid succinimidyl ester.
Received for publication January 21, 2000. Accepted for publication January 23, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
si, L. Trón. 1992. Transmembrane signaling in lymphocytes. Immunol. Today 13:A12.[Medline]
si, S. Damjanovich. 1994. Lateral organization of the ICAM-1 molecule at the surface of human lymphoblasts: a possible model for its co-distribution with the IL-2 receptor, class I and class II HLA molecules. Eur. J. Immunol. 24:2115.[Medline]
si, J., L. Bene, V. Horejsi, P. Angelisova, S. Damjanovich. 1996. Supramolecular complexes of MHC class I, MHC class II, CD20, and tetraspan molecules (CD53, CD81, and CD82) at the surface of a B cell line JY. J. Immunol. 157:2939.[Abstract]
and HLA-A2. Nature 387:630.[Medline]
si, M. Balázs, L. Mátyus, Jr R. Gáspár, M. Ameloot, R. E. Dale, S. Damjanovich. 1997. Major histocompatibility complex class I protein conformation altered by transmembrane potential changes. Cytometry 27:353.[Medline]
si, J., L. Trón, S. Damjanovich, S. H. Helliwell, D. J. Arndt-Jovin, T. M. Jovin. 1984. Fluorescence energy transfer measurements on cell surfaces: a critical comparison of steady state fluorimetric and flow cytometric methods. Cytometry 5:210.[Medline]
si, S. Damjanovich, S. H. Helliwell, D. J. Arndt-Jovin, T. M. Jovin. 1984. Flow cytometric measurements of fluorescence resonance energy transfer on cell surfaces: quantitative evaluation of the transfer efficiency on a cell-by-cell basis. Biophys. J. 45:939.[Medline]
si, L. Trón, S. Damjanovich. 1988. Luminescence spectroscopic approaches of cell surface dynamics. Q. Rev. Biophys. 21:479.[Medline]
si, J., L. Mátyus, L. Trón, M. Balázs, I. Ember, M. J. Fulwyler, S. Damjanovich. 1987. Flow cytometric measurements of fluorescence energy transfer using single laser excitation. Cytometry 8:120.[Medline]
2-microglobulin map and immunodominant epitope within the three-stranded -pleated sheet. Transplantation 64:640.[Medline]
2-microglobulin affects binding of class I MHC molecules by the W6/32 antibody. Immunogenetics 49:312.[Medline]
-receptors on the surface of mast cells probed by fluorescence resonance energy transfer. Biophys. J. 64:110.[Medline]
relative to MHC class I. J. Immunol. 162:846.2-microglobulin-free MHC class I molecules in T cell immunoregulation. Int. J. Clin. Lab. Res. 23:61.[Medline]
This article has been cited by other articles:
|
|
 |
|
  R. Wang, K. Natarajan, and D. H. Margulies Structural Basis of the CD8{alpha}{beta}/MHC Class I Interaction: Focused Recognition Orients CD8{beta} to a T Cell Proximal Position J. Immunol., August 15, 2009; 183(4): 2554 - 2564. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-M. Segura, P. Guillaume, S. Mark, D. Dojcinovic, A. Johannsen, G. Bosshard, G. Angelov, D. F. Legler, H. Vogel, and I. F. Luescher Increased Mobility of Major Histocompatibility Complex I-Peptide Complexes Decreases the Sensitivity of Antigen Recognition J. Biol. Chem., August 29, 2008; 283(35): 24254 - 24263. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Cebecauer, P. Guillaume, S. Mark, O. Michielin, N. Boucheron, M. Bezard, B. H. Meyer, J.-M. Segura, H. Vogel, and I. F. Luescher CD8+ Cytotoxic T Lymphocyte Activation by Soluble Major Histocompatibility Complex-Peptide Dimers J. Biol. Chem., June 24, 2005; 280(25): 23820 - 23828. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. L. Medintz, J. H. Konnert, A. R. Clapp, I. Stanish, M. E. Twigg, H. Mattoussi, J. M. Mauro, and J. R. Deschamps A fluorescence resonance energy transfer-derived structure of a quantum dot-protein bioconjugate nanoassembly PNAS, June 29, 2004; 101(26): 9612 - 9617. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
)
epitopes of HLA-I to the cell surface lipid probe BODIPY-PC. The extent
of FRET is displayed as a function of surface density of the
acceptor-conjugated phospholipid.
