73
Chapter II
compared with the molecular dynamics simulations that were carried out with DR1
molecules lacking the entire peptide (Painter et al., 2008; Rupp et al., 2011), the
observations are different as a small divergence and not a narrowing or partially
collapsing of the helices around the peptide N-terminus was observed. However, it is
also possible that a larger part of the peptide has to be absent for collapse of the groove.
Furthermore, a small conformational change of residue valine β85 was observed
which is a conserved residue forming part of the P1 pocket and exhibits a conserved
rotamer. In the presented structure Valβ85 is rotated outwards opening up part of the P1
pocket, which probably further destabilizes the P1 residue and may facilitate release of
the entire peptide. The altered rotamer of Valß85 was not observed for the DR1/CLIP
structure missing residue P-2 (Gunther et al., 2010) and may be dependent on the
absence of residue P-1.
The absence of two N-terminal peptide residues led to exposure of DR residues
(Alaα52, Serα53, Hisβ81 and Valβ85) that are normally covered by or in contact with
the peptide, raising the question whether DM may interact with these newly exposed
DR residues and may bind to the DR region normally covered by the peptide N-
terminus. To test this hypothesis residue Valβ85, which is usually covered by peptide
residue P-1, was mutated to an aspartic acid introducing a charged residue and SPR
experiments revealed a slower on-rate, but also slower off-rate for DM binding of the
DR mutant compared to DR wild type. To further investigate this hypothesis more SPR
experiments with different mutations in the respective DR region need to be carried out.
Summarizing, we can say that if the two N-terminal peptide residues are missing (P-2,
P-1) and with it three conserved hydrogen bonds between peptide N-terminus and DR,
no major conformational change of the peptide-binding groove is observed. Whether
DM binds to the newly exposed DR region and whether release of the P1 anchor residue
induces a major conformational change of the DR molecule still has to be determined.
However, the presented DR1 structure carrying an HA peptide missing two N-terminal
peptide residues reveals a relevant intermediate state during peptide release contributing
to a detailed understanding of peptide exchange by MHC II molecules.
74
Chapter III
4
Chapter III: Investigating HLA-DM binding to high-
affinity MHC II/peptide complexes
4.1
Introduction
DM plays an important role as peptide editor of MHC II molecules ensuring the
generation
of
high-stability
MHC II/peptide
complexes.
The
presence
of
MHC II/peptide complexes with a long half-life on the cell surface is important for
multiple T cell encounters and potential antigen recognition. Previous studies have
shown that low-stability complexes are good substrates for DM-mediated peptide
release whereas high-stability complexes are poor substrates with some high-affinity
complexes believed to be DM insensitive (Kropshofer et al., 1996). For example,
DR1/HA was believed to be resistant to DM action as no DM-facilitated peptide
exchange could be observed (Kropshofer et al., 1996; Sloan et al., 1995). Experiments
carried out in our lab using an N-terminally truncated peptide revealed that DM binds to
a MHC II conformer with a critical part of the binding groove empty (Anders et al.,
2011). These novel data suggested that spontaneous release of the peptide N-terminus
may be required for initial DM binding and also implied that in contrast to previous
assumptions DM theoretically should bind to any MHC II/peptide complex, even if to a
small extent, as peptide motion is always present.
It had been shown before that peptide exchange in the presence or absence of DM is
dependent on both temperature and pH (Denzin and Cresswell, 1995; Reay et al., 1992;
Sloan et al., 1995). Furthermore, previous data revealed that dissociation of the peptide
N-terminus represents a crucial component of the energy barrier which has to be
overcome explaining the strong temperature dependence of DM binding (Anders et al.,
2011). To test whether DM binding to high-stability MHC II/peptide complexes could
be enhanced by higher temperature also increasing peptide mobility, SPR experiments
were carried out at 25 ºC and 37 ºC. The SPR experiments at 37 ºC were substantially
more challenging than experiments at room temperature and had to be carried out
carefully as the machine used for measuring surface plasmon resonance is very sensitive
and high temperature, for example, can cause spontaneous bubble formation in the flow
chambers.
75
Chapter III
Additionally, to test the functional relevance of the DM binding assay carried out
using surface plasmon resonance, data obtained by SPR were compared to functional
experiments measuring DM catalyzed peptide exchange by fluorescence polarization.
For this purpose, DM binding to a low- and a high-affinity DR/peptide complex was
measured by SPR and compared to DM catalyzed peptide exchange of both complexes
monitored by fluorescence polarization.
4.2
Materials and Methods
4.2.1
Preparation of HLA-DR/peptide complexes
Soluble DR1/CLIP complexes were produced using stably transfected CHO cells in
hollow fiber bioreactors (Day et al., 2003) and purified by affinity chromatography
(L243, American Type Culture Collection). Aggregates were removed by gel filtration
(Superose 6, GE Healthcare) and the linker between peptide and N-terminus of DRβ
chain cleaved with thrombin (20 units thrombin/mg protein, 1.5 h, 25 °C). HA(306-318)
peptide was synthesized by Peptide 2.0 with a DNP label linked to a lysine group at the
peptide C-terminus. For peptide exchange DR1/CLIP was incubated (30 ºC, 8 hours)
with 50 µM HA peptide and 50 µM J10-1 in 50 mM citrate phosphate buffer (pH 5.3),
150 mM NaCl at a protein concentration of 0.3 mg/mL including protease inhibitors.
Unbound peptide was removed by gel filtration (Superose 12, GE Healthcare) and
DR1/HA further purified by anti-DNP chromatography.
DR2/MBP and DR2/CLIP were expressed in Sf9 insect cells after infection with
recombinant Baculovirus (MOI > 5, pAcDB3 plasmid with BaculoGold Baculovirus;
BD Biosciences). Proteins were purified by affinity chromatography (L243, American
Type Culture Collection) and the linker between peptide and DRß chain cleaved with
thrombin (20 units thrombin/mg protein, 1.5 h, 25 °C). The complexes were further
purified by ion exchange chromatography (MonoQ, GE Healthcare) and aggregates
were removed by gel filtration (Superose 6, GE Healthcare). Purified proteins were
buffer exchanged to the final buffer for SPR experiments (50 mM citrate phosphate
buffer, pH 5.3, 150 mM NaCl, 0.06 % C
12
E
9
detergent).
Dostları ilə paylaş: |