Minimal conformational plasticity
enables TCR cross-reactivity to different
MHC class II heterodimers
Christopher J. Holland1, Pierre J. Rizkallah1, Sabrina Vollers2, J. Mauricio Calvo-Calle3, Florian Madura1,
Anna Fuller1, Andrew K. Sewell1, Lawrence J. Stern2,3, Andrew Godkin1,4* & David K. Cole1*
Kingdom,2Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01655,3Department of
Biochemistry & Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655,4Department of
Integrated Medicine, University Hospital of Wales, Cardiff, CF14 4XW, United Kingdom.
Successful immunity requires that a limited pool of ab
molecules. Structures of unligated and ligated MHC class-I-restricted TCRs with different ligands,
supplemented with biophysical analyses, have revealed a number of important mechanisms that govern
TCR mediated antigen recognition. HA1.7 TCR binding to the influenza hemagglutinin antigen
(HA306–318) presented by HLA-DR1 or HLA-DR4 represents an ideal system for interrogating pMHC-II
antigen recognition. Accordingly, we solved the structure of the unligated HA1.7 TCR and compared it to
both complexstructures. Despitearelatively rigidbinding mode,HA1.7T-cells couldtolerate mutations in
key contact residues within the peptide epitope. Thermodynamic analysis revealed that limited plasticity
and extreme favorable entropy underpinned the ability of the HA1.7 T-cell clone to cross-react with
HA306–318presented by multiple MHC-II alleles.
in complex with a ‘self’ MHC. The potential array of peptides that can be generated from combinations of the 20
To date, our understanding of the molecular events that occur upon TCR engagement have come, almost
exclusively, from MHC class-I (MHC-I)-restricted TCRs. The structures of several TCR-pMHC-I pairings, in
bound and unbound form, have shown that MHC-I antigen recognition can be very dynamic. In a number of
cases, large CDR loop movements (.5A˚) have been reported during TCR binding4. Early thermodynamic
analyses of these TCR-pMHC-I interactions showed that binding was generally characterized by unfavorable
entropy (i.e. transition from a disordered to an ordered state) which was counteracted by favorable enthalpy (i.e.
an exothermic reaction mediated by a net gain in electrostatic interactions)5. These analyses suggested that
conformational plasticity in the TCR CDR loops was energetically favored and played an important role in T-
cell antigen recognition and crossreactivity6–8. However, more recent studies have shown that the TCR face can
remain more rigid9–13, and MHC-I restricted TCRs can bind using a range of thermodynamic strategies5.
Furthermore, although the surface of pMHC-Is normally remain conformationally fixed during binding, large
instances of MHC-I restricted T-cell antigen recognition14. It is not clear whether such mechanisms extend to
recognition of MHC-II-restricted antigens. Understanding of the molecular events involved in pMHC-II recog-
nition has been hamperedby thefact thatthere areno thermodynamic data forhuman MHC-IIrestricted TCRs,
ab T-cell receptors (TCRs) provide cover for a vast
b T-cell receptor (TCR) binding to peptide-major histocompatibility complex class-II (pMHC-II)
orchestrates the adaptive immune response1. The specificity of this pivotal receptor/ligand interaction is
determined by the antigen-binding variable domains of the TCR. These variable domains include six
18 July 2012
17 August 2012
4 September 2012
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SCIENTIFIC REPORTS | 2 : 629 | DOI: 10.1038/srep00629
and only one human TCR-pMHC-II complex has been determined
with an accompanying unligated TCR structure15. A direct compar-
II ligation15. However, the relationship of how these conformational
changes to the TCR might have influenced the potential degeneracy
of the responding T-cell was not investigated15.
To explore this question further, we examined TCR-pMHC-II
binding degeneracy using the HA1.7 TCR that recognizes an influ-
enza hemagglutinin derived epitope (HA306–318). This influenza-
derived peptide has been shown to bind to HLA-DRa*0101 in com-
plex with either; HLA-DRb*0101 (DR1), DRb*1501, DRb*0401
(DR4), DRb*0404, DRb*0501 or DRb*0701 and is, therefore, a
so called ‘universal’ antigen16–18. Curiously, the HA1.7 CD41T-cell
clone recognized HA306–318presented by both DR1 (DR1-HA) and
DR4 (DR4-HA)19. The differences in amino acid sequence between
notdirectly contactthe TCR.However,other DR1 restricted HA305–320
specific TCRs were unable to recognize HA305–320in the context of
DR420. Therefore,allelic variationsin the peptide-binding groovecan
influence the immunogenicity of the pMHC-II surface. Additionally,
previous investigations have shown that the HA1.7 T-cell clone can
recognize multiple peptide ligands in the context of both DR1 and
DR421,22. Taken together, these data indicate that the HA1.7 T-cell
clone can promiscuously bind to different MHC-IIs presenting a
wide range of distinct peptide epitopes.
Here, we determined the structure of the unligated HA1.7 TCR in
order to understand the molecular rules that allowed it to bind to
both DR1 and DR4. We proceeded to perform an analysis of HA1.7
CD41T-cell activation against a series of peptides with mutations to
key contact residues within the HA peptide epitope. Finally a ther-
nition. Overall, these data provide important new information con-
cerning the molecular rules that govern antigen recognition by
MHC-II restricted T-cells.
Minimal CDR-loop movement enables HA1.7 TCR binding to
DR1-HA and DR4-HA. To date, only one TCR-pMHC-II com-
plex has been solved in conjunction with the unligated TCR.
Theses structures were for an altered-self–reactive TCR of uncer-
tain degeneracy15. In order to analyze the degree of conformational
plasticity during binding of a degenerate TCR to MHC-II, we
determined the structure of the HA1.7 TCR using diffraction data
extending to 2.4A˚(Table 1). The HA1.7 TCR has previously been
solved in complex with DR1-HA and DR4-HA and was the first
human MHC-II restricted TCR complex to be published39,40. Thus,
this important disease-relevant model system allowed a comparison
between ligated and unligated TCR binding in two distinct systems.
Molecular replacement was successful in space group P1211,
consistent with the presence of one molecule of the complex per
asymmetric unit. The resolution was sufficiently high to show the
conformation of the HA1.7 TCR CDR loops and contained well-
defined electron density throughout the structure. The final model
showed 100% of residues in the preferred, or allowed, regions of the
Ramachandran plot and geometry consistent with the data resolu-
tion. The crystallographic R/Rfree factors were 22.9% and 29.4%,
respectively. The ratio was within the accepted limits shown in the
theoretically expected distribution41. The molecular visualization
software, PyMol, was used to perform a secondary structure-based
alignment of the unligated HA1.7 TCR with the two solved TCR-
pMHC-II complexes, HA1.7-DR1-HA (PDB51FYT)39and HA1.7-
DR4-HA (PDB51J8H)40. This allowed direct visualization of the
conformation of the CDR loops before and after MHC-II associ-
ation (Figure 1A–D). Measurement of individual CDR loop shifts
upon ligation indicated that very little movement (maximum
movement 5 2.28A˚) was required in these regions for association
to occur with either DR1-HA or DR4-HA (Table 2) (calculated as
in4). The average CDR loop movement observed during association
with either DR1-HA or DR4-HA was 1.28A˚and 1.52A˚, respectively.
Comparison of the HA1.7 TCR complex structures demonstrated
that the HA1.7 TCR made 4 hydrogen bonds and 8 salt bridges
with DR1-HA, compared to 3 hydrogen bonds and 7 salt bridges
with DR4-HA (Supplementary Tables 1&2). Analysis of the HA1.7
that the CDR loop movements were required to allow formation of
,50% of the HA1.7 TCR hydrogen bonds and salt bridges upon
complex formation with either DR1-HA, or DR4-HA (Figure 1E&D).
Furthermore, a greater degree of movement was required by the
HA1.7 TCR to associate with DR4-HA compared to DR1-HA,
possibly explaining the difference in binding affinity between the
two complexes42. The largest difference in conformational move-
ment was observed for the CDR1b loop, moving 2.28A˚when in
complex with DR4-HA, and only 0.97A˚when bound to DR1-HA.
This conformational shift allowed 28D of the TCR CDR1b loop to
contact HA-315K forming 2 salt bridges in both complexes
(Figure 1E&F). We then investigated the thermal stability, by
studying the B-factor heat plots, for residues involved in the
binding interface for the ligated and unligated DR1-HA and HA1.7
molecules (Figure 2). In agreement with our structural analyses, the
B-factor heat plots were similar for the ligated and unligated
molecules, indicating minimal conformational adjustments during
binding. Interestingly, DR1-HA (Figure 2A&B) underwent a greater
degree of stabilization, observable by a greater changein the B-factor
heat plot, compared to the HA1.7 TCR (Figure 2C&D). Thus, these
analyses support the notion that the HA1.7 TCR remains relatively
rigid during ligand engagement.
In their previous analyses of the HA1.7-DR1-HA and HA1.7-
DR4-HA complexes, Hennecke et al. demonstrated that the center
and COOH-terminal half of the HA306–318peptide bound deeper in
the groove and closer to the b1 a-helix of DR4 compared to DR140.
Thus, although only small CDR loop shifts were observed (Table 2),
these movements were essential for binding. Furthermore, it is likely
to tolerate the conformational differences associated with HA306–318
presented by either DR1 or DR4. This specific level of loop plasti-
city, although small, may be important to allow the HA1.7 TCR to
Table 1 | Data collection and refinement statistics (molecular
Data set statistics
Unit cell parameters (A˚,u)
c572.6, b 94.3
No reflections used
Rcryst (no cutoff) (%)
Bond lengths (A˚)
Bond Angles (u)
Mean B value (A˚ 2)
Outliers Ramachandran plot (%)
Overall ESU based on Maximum Likelihood (A˚)
*Values in parentheses are for highest-resolution shell.
SCIENTIFIC REPORTS | 2 : 629 | DOI: 10.1038/srep00629
bind to the range of altered peptide ligands (APLs) described herein
with adequate strength to enable recognition by the HA1.7 T-cell
previously demonstrated that the hemagglutinin specific CD41
T-cell clone, HA1.7, could cross react with the HA306–318epitope
presented by either DR1 or DR419. Furthermore, HA1.7 can
recognize APLs in the context of DR1 and DR421,22demonstrating
the principle of peptide cross reactivity. Structural investigation
by Hennecke et al., into how the HA1.7 clone could achieve
this, suggested that the HA1.7 TCR could tolerate the differing
conformations of the HA306–318peptide, at positions 310K and
311Q, when presented by DR4 compared to DR139. We therefore
set out to determine the degree of functional tolerance of the
HA1.7 T-cell clone for previously identified TCR contact resi-
dues39,40. A set of APLs were generated in which key TCR contacts
in the HA306–318peptide were mutated (307K, 309V, 310K, 312N &
315K), while key MHC contacts were conserved (Figure 3A)20,43.
Importantly, these mutations, that were not at key anchor residue
positions, did not substantially influence peptide-MHC stability
Figure 1 | Comparison of the HA1.7 TCR CDR loop conformations pre and post ligation to DR1 and DR4. (A) Superposition of the unligated HA1.7
loops comparing ligated and unligated structures (colors as in (A)). (C&D) A view of the superimposed CDR loops from the perspective of the TCR
contacting a stick representation of the HA306–318peptide (red). Green loops are the unligated HA1.7 CDR loops, yellow are HA1.7 CDR loops when
ligated to DR1 (C) and blue are HA1.7 CDR loops when ligated to DR4 (D). (E&F) HA1.7 CDR1b contacting the HA306–318residue, 315K, presented by
either DR1 (E) or DR4 (F). Green loops are the unligated HA1.7 CDR loops, yellow are HA1.7 CDR loops when ligated to DR1 and blue are HA1.7 CDR
loops when ligated to DR4. Contact distances were smaller, allowing salt bridge formation, between the TCR and 315K in the TCR ligated forms.
Table 2 | CDR loop movement during HA1.7 TCR binding to DR1-
HA and DR4-HA
TCR pMHC-IICDR loop shift (A˚)
PyMol software was used to overlap and align the unligated HA1.7 TCR with the two ligated
of the CDR loops before and after MHC-II association. Measurement of individual CDR loop shifts
was carried out as previously described4.
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We evaluated the ability of the HA1.7 T-cell clone to tolerate
substitutions in the peptide ligand at TCR contact residues using
a variety of assays. Cell-surface HA1.7 TCR binding of purified
surface MHC-peptide complexes was measured using T-cell prolif-
eration (Supplementary Figure 1A) and IL-2 secretion (Supple-
mentary Figure 1B) assays with peptide-pulsed DR11lymphoblas-
toid cells used as antigen-presenting stimulator cells (see Methods
for details). Each of these assays showed that a range of conservative
(312Q), semi-conservative (312S, 312T) and non-conservative
(312F), substitutions were tolerated by HA1.7 at asparagine 312 in
the centre of the HA306–318epitope, with only substitution for lysine
(312K) completely abolishing recognition (Figure 3C–E, Supple-
mentary Figure 1). Furthermore, the HA306–318epitope contains
three lysine residues at position 307, 310 and 315, which form the
majority of hydrogen bonds/salt bridges with the CDR loops of
the HA1.7 TCR when bound to either DR1 or DR4, and a valine
at position 309 that participates in hydrophobic interactions.
Individual substitutions of these dominant contact residues with
semi-conservative (307S), and some (309R, 310F) but not all non-
conservative (309E, 310V), substitutions were also tolerated by
HA1.7 (Figure 3C–E, Supplementary Figure 1). Interestingly, a
semi-conservative mutation from lysine 315, which forms 4 salt
bridges and 1 hydrogen bond with the HA1.7b CDR loops (Supple-
mentary Table 1&2), to histidine still produced a good receptor
agonist (Supplementary Figure 1), but the affinity of the interaction
was suffciently weak that tetramer staining was barely above back-
ground (Figure 3E, Supplementary Figure 1). Overall, these data
and previous studies21,22suggest that HA1.7 T-cells have the ability
to recognize with a wide range of different peptide ligands presented
by both DR1 and DR4, some with very weak affinities.
Entropic effects drive HA1.7 TCR antigen recognition. The vast
majority of TCR-pMHC interactions reported are characterized by
favorable enthalpy, with entropy playing a more varied role5. These
studies have provided important molecular information concerning
Figure 2 | Analysis of free energy comparing the unligated HA1.7 TCR
and DR1-HA strutures and the HA1.7-DR1-HA complex. Surface
representation of unligated and ligated HA1.7 TCR and DR1-HA colored
in blue. (A) unligated DR1-HA (PDB: 1DLH)58, (B) complexed DR1-HA
(PDB: 1FYT)39, (C) unligated HA1.7 TCR (PDB: 4GKZ) and (D)
complexed HA1.7 TCR (PDB: 1FYT)39.
Figure 3 | Analysis of altered peptide ligand (APL) stability and tetramer staining of the HA1.7 T-cell clone. (A) Schematic of the presentation of the
andnon-conservative mutationsareshownasyellowsticks.(B)IC50APLstabilityassay.Thebaringreyshowsourpreviously publishedobservationthat
the Y308A mutation, that is involved in anchoring the HA peptide into the DR1 binding groove, substantially affects pMHC-II stability31. The black bars
show our new data that demonstrates that all of the APLs used in this study bind to DR1 with similar affinities, discounting the possibility that peptide
stability was the chief factor determining the antigen sensitivity of the HA1.7 T-cell clone. (C–E) HA1.7 T-cell tetramer staining histogram plots using
DR1-APL tetramers. (C) Tetramer staining using DR1 bound to a non-cognate A2 peptide, 310F, 312S, 312F and wild-type HA. (D) Tetramer staining
peptide, 315H, 312K, 310N/312T, 310V, 309E and wild-type HA.
SCIENTIFIC REPORTS | 2 : 629 | DOI: 10.1038/srep00629
the energetic factors that contribute to T-cell antigen recognition.
thermodynamic analyses of HA1.7-DR1-HA and HA1.7-DR4-HA
were performed. The binding equilibrium (KD) was determined at 5
different temperatures for each complex and enthalpy (DHo), en-
tropy (TDSo) and heat capacity (DCpo) were calculated by a non-
linear regression of temperature (K) plotted against the free energy
(DGo) (Figure 4A&B, Supplementary Figure 2). The HA1.7 TCR
exhibited extreme thermodynamic parameters compared to pre-
viously published TCR-pMHC interactions, with the largest unfa-
vourable enthalpic contribution reported upon binding to both
DR1-HA and DR4-HA (DHo5 16 and 18 kcal/mol for DR4-HA
and DR1-HA, respectively) (Figure 4A&B). This large enthalpic
penalty indicated a net loss in the number of electrostatic inter-
actions during complex formation. Thus, the greatest reported
favourable entropic energy (TDSo5 20.9 and 23.8 kcal/mol for
DR4-HA and DR1-HA, respectively) was required to enable
HA1.7 antigen recognition, suggesting a net loss of order during
binding (Figure 4A&B). These characteristics are the opposite of
what was originally proposed to be the TCR thermodynamic signa-
ture (favourable enthalpy and unfavourable entropy) by early
thermodynamic TCR-pMHC-I investigations44–46. Our observation,
that HA1.7 TCR binding to DR1-HA and DR4-HA was entropically
driven, also contradicts the prediction by Hennecke et al that the
binding energy driving the HA1.7-DR1-HA interaction would be
derived from the formation of new electrostatic interactions39,40.
The expulsion of ordered water molecules has been previously
implicated as an important mechanism enabling TCR binding to
pMHC47. Such a loss of ordered water could also partly explain the
large favourable entropic energy we observed for HA1.7 binding to
DR1-HA and DR4-HA. Ordered water molecules can form a shell
around hydrophobic protein patches. Thus, we analyzed the hydro-
or DR4-HA using the normalized consensus hydrophobicity scale48
(Figure 4C&D). The hydrophobic surface of DR1-HA and DR4-HA
were virtually identical, so DR1-HA is described from here on. The
HA1.7 TCR was slightly hydrophobic, with residues contributing to
21.31 (Figure 4C). DR1-HA was more hydrophobic, with a score of
phobicity was a factor for generating the extreme thermodynamic
signature we observed in this system, we compared the hydrophobic
interfaces of some other MHC-I restricted TCRs that have been
structurally and thermodynamically characterized in previous stud-
ies44,49–53. The JM22 TCR bound to HLA A*0201-GILGFVFTL with
unfavourable entropy and had a relatively low hydrophobic score
(Supplementary Figure 3A&B), whereas the entropically favour-
able interactions between the LC13 TCR and HLA B*0801-
FLRGRAYGL, and the A6 TCR and HLA A*0201-LLFGYPVYV,
where substantially more hydrophobic (Supplementary Figure
3C–F). These observations support the notion that a more hydro-
molecules upon ligand engagement, generating a larger favourable
entropic gain than less hydrophobic interactions. However, the
Figure 4 | Thermodynamic analyses of HA1.7-DR1-HA and HA1.7-DR4-HA interactions. (A&B) The thermodynamic properties of (A) the HA1.7-
DR1-HA and (B) the HA1.7-DR4-HA interactions. Enthalpy (DHu) and entropy (TDSu) at 298 K, are shown in kcal/mol, and were calculated by a non-
linearregression oftemperature (K)plotted againstthe free energy(DGu).(C&D) Hydrophobic analysis ofthe residues involvedinthe bindinginterface
betweenthe HA1.7TCR and DR1-HA (the hydrophobic characteristics ofthe DR4-HA surface was virtually identical toDR1-HA). Hydrophobicity was
calculated using the normalized consensus hydrophobicity scale as previously described48. The TCR and pMHC surfaces are colored according to this
scale with blue being the most hydrophobiic and red being the least hydrophobic. In order to calculate the hydrophobicity for each interface, the
HA peptide is shown as sticks. (C) HA1.7 TCR (PDB: 4GKZ), (D) DR1-HA (cognate pMHC for the HA1.7 TCR) (PDB: 1FYT)39.
SCIENTIFIC REPORTS | 2 : 629 | DOI: 10.1038/srep00629
hydrophobic score for the HA1.7-DR1/4-HA interaction was sub-
stantially lower than for the other entropically favourable interac-
tions involving the LC13 and A6 TCRs, even though the entropic
value for HA1.7 was much more favourable. Thus, hydrophobicity
alone could not explain the extreme thermodynamic signature for
the HA1.7 TCR. Overall, a combination of a rigid binding mode
ing binding), and the expulsion of some ordered solvent probably
explain the extreme thermodynamic parameters that govern HA1.7
TCR ligand engagement and cross reactivity.
TCRs have shown that MHC-I antigen recognition can be very
dynamic including movements in the TCR CDR loops, peptide, or
molecular mechanism utilized by MHC-II restricted TCRs during
the MHC-II restricted HA1.7 TCR to 2.4A˚. It has been shown prev-
iously that the DR1 restricted CD41T-cell clone, HA1.7, can also be
activated by HA306–318in the context of DR440and the structures of
both HA1.7-DR1-HA and HA1.7-DR4-HA complexes have been
solved39,40. Thus, this important disease relevant model system was
recognition of multiple ligands. Surprisingly, our structural invest-
igation showed that the HA1.7 TCR required only small shifts (up to
a maximum of 2.28A˚) in TCR CDR loop conformations to form, on
average, 50% of the hydrogen bonds and salt bridges upon complex
formation, compared to an average of $ 5A˚for TCR-pMHC-I CDR
loop movements4. Furthermore, a greater degree of movement was
required by the HA1.7 TCR to associate with DR4, which could
explain the lower binding affinity between HA1.7 and DR4-HA
compared to HA1.7 binding to DR1-HA. These observations may
I and pMHC-II molecules. For instance, the structural database of
TCR-pMHC-I and TCR-pMHC-II complexes shows that peptides
presented by MHC-I generally assume a central bulged conforma-
tion, potentially requiring greater TCR CDR loop movements for
engagement compared to TCR binding to the much flatter peptide
conformation in the open-ended MHC-II binding groove39,40,54.
The small TCR CDR loop movements required for the HA1.7 T-
cell degeneracy8. In order to examine whether the degenerate nature
of the HA1.7 T-cell clone extended beyond the recognition of the
DR1-HA and DR4-HA ligands, we altered key TCR contact residues
in the HA306–318epitope. Our initial analysis demonstrated that the
HA1.7 T-cell clone was able to recognize a number of APLs that
contained mutations at key TCR contact residues within the
HA306–318epitope. Importantly, the HA1.7 T-cell clone was extre-
too weak to enable tetramer staining using cognate multimerized
pMHC-II, and were beyond the detection limits of SPR (data not
demonstrating that the HA1.7 T-cell clone is highly degenerate and
can recognize multiple peptide ligands21,22. Therefore, our structural
analysis indicated that minimal TCR CDR loop movements were
sufficient to enable cross-recognition of different ligands by the
HA1.7 T-cell clone.
Early thermodynamic analyses of TCR-pMHC-I interactions
showed that binding was generally characterized by unfavorable
entropy (i.e. transition from a disordered to ordered state) which
was counteracted by favorable enthalpy (i.e. exothermic reaction
mediated by a net gain in electrostatic interactions)5, although more
recent studies have shown that the TCR can bind using a range of
thermodynamic strategies5. Importantly, there are no other reports
of TCR-pMHC-II thermodynamics currently in the literature. We
performed a thermodynamic analysis of the HA1.7-DR1-HA and
HA1.7-DR4-HA interactions in order to further dissect the molecu-
lar basis for HA1.7 antigen recognition. This identified an extreme
and unusual thermodynamic signature (the largest unfavourable
enthalpy and the largest favourable entropy reported). Impor-
tantly, the HA1.7 TCR used a virtually identical thermodynamic
strategy to bind to both DR1-HA and DR4-HA.
A more in depth analysis of the binding interface demonstrated
tion were moderately hydrophobic, compared to other entropically
favourable TCR-pMHC-I interactions. Previous structural investi-
gations have shown that the LC13 and A6 TCRs undergo conforma-
tional changes when engaging different ligands. For example, the
differentligands56.Similarly, theA6TCRhasrecentlybeen shownto
undergo conformational melding upon binding to different
ligands7,57. Thus, compared to the rigid binding mode of the HA1.7
TCR, the CDR loops of the LC13 and A6 TCRs may require greater
stabilisation during binding. This transition from disorder to order
(entropically unfavourable) upon ligand engagement could offset
any energetically favourable expulsion of ordered solvent in these
systems, compared to the ‘lock and key’ binding of the HA1.7 TCR.
Thus, it is likely that a combination of rigid ‘lock and key’ binding,
and the expulsion of ordered solvent explain the extreme ther-
modynamic parameters observed for the HA1.7 TCR. Although
the structures of the APLs reported here are not known (and could
include large CDR loop movements), this rigid TCR binding mode
probably enabled the HA1.7 T-cell clone to cross-react with a range
In summary, we have shown that small TCR CDR loop move-
ments and an extreme thermodynamic signature (largest observed
unfavourable enthalpy and largest reported favourable entropy)
drive the interaction of a MHC-II presented universal haemaggluti-
nin antigen with the HA1.7 TCR. These observations are in contrast
to some previously published data for MHC-I restricted TCRs in
which large conformational changes in the TCR were deemed
important for antigen discrimination and T-cell degeneracy6,8. Our
results show that MHC-II-restricted TCR binding can occur as the
result of minimal conformational plasticity and favorable entropy.
Although further examples of ligated and unligated MHC-II-
restricted TCR structures with different ligands will be required in
order to determine whether this is a common theme for TCRs inter-
acting with pMHC-II ligands, we suggest that MHC-II restricted
TCRs may employ a distinct binding mode compared to MHC-I
restricted TCRs to engage multiple different ligands, perhaps per-
taining to the flatter antigenic landscape of pMHC-II molecules
compared to pMHC-I.
Generation of expression plasmids. The extracellular constructs of the HA1.7 TCR
were designed to incorporate an engineered disulphide link to produce the soluble
domains (variable and constant) for both the a and b chains23. Sequences for the
HA1.7 TCR were cloned and inserted into the pGMT7 expression plasmid allowing
protein expression under the control of the T7 RNA polymerase promoter within a
Rosetta DE3 Escherichia coli system. Plasmid integrity was confirmed by automated
DNA sequencing (Central Biotechnology Services, Cardiff University). Expression
plasmids encoding extracellular domains of DR1 and DR4 have been described24.
Protein expression, refolding, and purification. Competent Rosetta DE3
Escherichia coli cells were induced with 0.5 mM isopropyl b-D-thiogalactoside to
and the DRb1*0401 chain, in the form of inclusion bodies (IBs) as described
were purified into 8 M urea buffer (8 M urea, 20 mM TRIS pH8.1, 0.5 mM EDTA,
30 mM DTT) by ion exchange using a HiTrapTMcolumn (GE Healthcare, UK) to
remove bacterial impurities. For a 1 L TCR refold, 30 mg of TCRa chain IBs were
incubated for 15 min at 37uC with 10 mM DTT and added to cold refold buffer
(50 mM TRIS pH8.1, 2 mM EDTA, 2.5 M urea, 6 mM cysteamine hydrochloride
SCIENTIFIC REPORTS | 2 : 629 | DOI: 10.1038/srep00629
and 4 mM cystamine). After 15 mins, 30 mg of TCRb chain IBs, also incubated for
15 mins at 37uC with 10 mM DTT, were added. For a 1 L pMHC-II refold, 2 mg of
DRa1*0101 chain IBs were mixed with 2 mg of either the DRb1*0101 chain or the
DRb1*0401 chain IBs and 0.5 mg of peptide for 15 min at 37uC with 10 mM DTT24.
The HA306–318peptide (PKYVKQNTLKLAT) (generated by Peptide Protein
Research Ltd., Southampton, UK) was used in the refold. This mixture was then
added dropwise to cold refold buffer (25% glycerol, 20 mM TRIS pH8.1, 1 mM
EDTA, 2 mM glutathione reduced, 0.2 mM glutathione oxidized). Refolds were
incubated for72 hrat4uC.Dialysiswascarriedoutagainst10 mMTRISpH8.1until
the conductivity of the refolds was ,2 mS/cm. The refolds were then filtered, ready
for purification steps. Refolded TCR and pMHC-II proteins were then purified
initially by ion exchange using a Poros50HQ column and then gel filtrated using a
Superdex 200HR column.
pMHC biotinylation. Biotinylated pMHC-II was prepared either by BirA-mediated
enzymatic addition of biotin to bsp-tagged refolded MHC-II-peptide complexes26, or
by chemical biotinylation using biotin-PEG-maleimide to modify refolded MHC-II-
peptide complexes carrying a C-terminal cysteine residue as described previously27.
T-cells. The HA1.7 T-cell clone28provided by Jonathan Lamb, was maintained by
biweekly stimulation with HA peptide-pulsed irradiated DR11antigen presenting
cells as described29, and rested for 8–10 days before use in activation or tetramer
T-cell proliferation assay. Proliferation was determined by incorporation of
[3H]-thymidine in peptide-stimulated T-cells using irradiated (4900 rads) DR11
cells were washed 3 times and re-suspended in cRPMI (RPMI 1640 supplemented
with 10% FBS, 2 mM L-glutamine, 50 U/ml Penicillin, 50 mg/ml Streptomycin, non-
essential amino acids,1 mMsodium pyruvate and 5310252-Mercapthoethanol). To
peptide were added in 200 ml final volume to 96 well round bottom plates. After
24 hours of incubation, 1 mCi/well [3H]-thymidine was added, and after additional
24 hours incubation, tritium incorporation was determined by scintillation counting
(1450 Microbeta TriLux Scintillation counter, Perkin-Elmer, Shelton, CT).
IL-2 assay. IL-2 in supernatants was determined by using CTLL-2 cells (TIB-214,
ATCC Manassas, VA) as previously described30. Briefly, 50 ml of culture medium
from stimulated HA1.7 T-cells or controls were transferred to 96 well plates and
13104CTLL-2 cells added in medium to bring the final volume to 200 ml. After
24 hours the cells were pulsed with 1 mCi/well [3H]-thymidine and incubated for
additional 17 hours. Finally, plates were harvested and3H incorporation determined
by liquid scintillation counting as above. An IL-2 standard curve was used to relate
CTLL-2 proliferation to IL-2 concentration.
Tetramer staining. Tetramer staining was performed as described previously27.
Briefly, streptavidin-PE was added step-wise to DR1-HA peptide complexes in 4
aliquots incubating for 2 minutes before addition of next aliquot to obtain a final
molar ratio was 451. HA1.7 T-cells were incubated with tetramers in PBS13% BSA
for 4 hours at 37uC and for 20 minutes for anti-CD4-APC antibody on ice. Tetramer
and antibody binding was determined using a 4 color FACScalibur (BD Biosciences).
MHC-II-peptide binding analysis. The MHC-II-peptide binding affinity was
estimatedby competition assay as described31.Briefly, 5 nM biotinylated HApeptide
and 5 nM DR1, refolded in the absence of peptide24, were incubated together with
varying concentrations of unlabelled test peptide at 37uC for four days, before
measurement of DR1-bioHA bya sandwich microplate immunoassayusing the anti-
DR1 monoclonal LB3.1 capture and streptavidin-Eu with delayed fluorescence
detection. Relative binding affinities are reported as the concentration of test peptide
required to inhibit 50% of DR1-bioHA formation, with IC50values determined by
fitting to sigmoidal dose-response curve with Hill coefficient 51. Under these
conditions the IC50values approximate the equilibrium dissociation constant KD
Crystallization, structure determination, and refinement. Refolded HA1.7 TCR
was concentrated to 10 mg/ml in 10 mM TRIS pH 8.1 and 10 mM NaCl. Screens
were set up in 96 well Intelli-plates (Art Robbins Instruments- ARI) using a crystal
phoenix robot (ARI) applying the sitting drop vapour diffusion technique. 0.2 ml
HA1.7 TCR and 0.2 ml crystallisation buffer were dispensed into the small reaction
well and 60 ml crystallisation buffer dispensed into the large reservoir. Intelli-plates
were then sealed and incubated at 18uC in a crystallisation incubator (Molecular
dimensions) and analyzed for crystal formation. HA1.7 TCR crystals appeared in
0.1 M Bis-Tris propane pH 8.5, 0.2 M sodium bromide, 20% w/v PEG 3350. Crystals
selected for further analysis werecryoprotected withethylene-glycol to25% and then
flash cooled in liquid nitrogen in Litho loops (Molecular dimensions). Diffraction
with the MOSFLM package32and the data were scaled, reduced and analyzed with
SCALA and the CCP4 package33. The structure was solved with Molecular
Replacement using PHASER34. The model sequence was adjusted with COOT35and
the model was refined with REFMAC536. Graphical representations were prepared
with PYMOL37. The reflection data and final model coordinates for the HA1.7 TCR
were deposited in the PDB database and assigned accession code: 4GKZ.
Thermodynamic investigation. Thermodynamic analyses were performed using a
BIAcore T100 equipped with a CM5 sensor chip as previously described38. 500
chemically linked to the chip surface. Equilibrium binding analysis was preformed
5uC, 15uC, 25uC, 32uC, and 37uC. Experiments were performed in triplicate.
Representative data are shown. The binding response was determined by subtraction
of the response measured on a control flow cell, which contained non-cognate
pMHC-II, from the response measured in flow cells containing cognate pMHC-II.
Results were analyzed using BIAevaluation 3.1, Microsoft Excel, and Origin 6.1. The
equilibrium-binding constant (KD) values were calculated using a nonlinear curve fit
(y 5 (P1x)/(P21 x)). The thermodynamic parameters were calculated according to
the Gibbs-Helmholtz equation (DGu5 DH 2TDSu).The binding freeenergies, DGu
(DGu 5 2RTlnKD) were plotted against temperature (K) using nonlinear regression
to fit the three-parameter equation, (y5dH1dCp*(x-298)-x*dS-x*dCp*ln(x/298)).
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We thank the staff at Diamond Light Source for providing facilities and support. DKC is a
WellcomeTrust Research Career Development Fellow(WT095767).PJR wassupported by
a RCUK Fellowship. We thank Jennifer Cochran for measurement of MHC-peptide IC50
values. Supported by NIH U19-57319 (LJS) and R01 AI38996 (LJS).
D.K.C., L.J.S., A.G., A.F., C.J.H, S.V., J.M.C.C., P.J.R., and F.M. performed experiments,
analyzed data and critiqued the manuscript. D.K.C., A.G. and L.J.S. conceived and directed
the project. D.K.C., L.J.S., A.K.S., A.G., C.J.H, and P.J.R., wrote the manuscript.
Supplementary information accompanies this paper at http://www.nature.com/
Competing financial interests: The authors declare no competing financial interests.
License: This work is licensed under a Creative Commons Attribution 3.0 Unported
License. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/
How to cite this article: Holland, C.J. etal. Minimal conformationalplasticity enables TCR
cross-reactivity to different MHC class II heterodimers. Sci. Rep. 2, 629; DOI:10.1038/
SCIENTIFIC REPORTS | 2 : 629 | DOI: 10.1038/srep00629