19
Introduction
Nicholson et al., 2006). By tracking fluorescence polarization of a labeled peptide
bound to soluble DR2 in the presence of soluble DM, four small molecules were found
that substantially enhanced peptide exchange (10
-6
– 10
-4
M). Two of the small
molecules also showed activity in the absence of DM although to a smaller extent.
Microdialysis experiments indicated direct binding to DM for one of the small
molecules (M19, see table 1.1) being active only in the presence of DM. Binding to
both proteins, DM and DR2, was observed for a small molecule (F15, see table 1.1) that
showed activity even without DM. Furthermore, three of the four small molecules were
sensitive to DM mutations localized at the hydrophobic ridge at the top of the concave
side of DM, which is very likely part of the interaction surface of DM and DR (see
figure 1.7), indicating that some of the small molecules may support the DM-DR
interface by contacting residues of both proteins.
J10 and its analogs
Among the most potent loading enhancers of MHC II molecules acting in the
absence of DM are J10 and its analogs discovered by Wucherpfennig and colleagues by
a high-throughput screening (Call et al., 2009). The J10 series of molecules is active on
all tested DR alleles (DR1, DR2, DR4) and showed no sensitivity to polymorphism
involving the P1 pocket, suggesting the peptide exchange mechanism of J10 may be
distinct from the mechanism of the di-peptides and the adamantyl compounds described
above. Extensive medicinal chemistry improved the activity of
the small molecules used
at a concentration of 10
-5
– 10
-4
M and enhancement of peptide presentation has been
demonstrated both
in vitro in
a cellular assay and in vivo.
In general, MLE are remarkably diverse and might use distinct mechanisms to
influence peptide exchange. However, it is likely that the small molecules interfere
either directly or allosterically with one of the two main interactions between peptide
and MHC II molecules, i.e. conserved hydrogen bond network between peptide and
MHC II residues and occupancy of a series of deep pockets formed by MHC II residues.
During these studies, the peptide exchange mechanism of J10 and its analogs was
investigated in the absence of DM applying X-ray crystallography and NMR
spectroscopy as can be seen in chapter I. The J10 series of small molecules has a strong
potential for use as adjuvant for peptide vaccination and in therapeutics due to the low
concentration needed for activity and showed
already promising results in vivo.
21
Introduction
1.9
Scopes and objectives of this study
This doctoral work aimed to elucidate molecular details of the peptide exchange
mechanism of MHC II molecules catalyzed by the protein DM and the synthetic small
molecule J10. To address structural and dynamic aspects of this complex process
various methods were applied including surface plasmon resonance (SPR), X-ray
crystallography and NMR spectroscopy.
Surface plasmon resonance experiments were carried out to investigate DM binding
to high-affinity MHC II/peptide complexes. Recent studies have shown that for DM
binding to occur, the N-terminal part of the peptide has to leave the binding groove.
This partial peptide release could be due to spontaneous peptide motion which should
be enhanced at higher temperature and implies DM binding to all MHC II/peptide
complexes even if to a small extent. To test this hypothesis SPR experiments were
carried out at different temperatures investigating whether DM binding can be induced
to high-affinity MHC II/peptide complexes previously exhibiting no or only little DM
susceptibility.
Crystallographic studies were applied to reveal the MHC II conformation with a
partially empty peptide-binding groove, which could determine whether release of the
peptide N-terminus induces conformational changes of the MHC II structure. Knowing
how the MHC II conformation adapts to loss of crucial interactions between peptide and
MHC II molecule could display important molecular details of the peptide exchange
mechanism. Furthermore, using X-ray crystallography we aimed to identify the binding
site of the small molecule J10, which would allow structure based design to further
improve affinity and activity of the MHC loading enhancer J10.
Applying NMR spectroscopy, the dynamics of the peptide bound to MHC II
molecule were explored in solution. To date, the molecular understanding of the
MHC II/peptide complex is mainly deduced from the static image given by crystal
structures. Investigating MHC II/peptide complexes in solution could advance this
understanding by taking the dynamics of the complex and especially peptide mobility
into account. Furthermore, using the NMR approach, peptide release facilitated by the
small molecule J10 was followed in solution to explore the peptide exchange
mechanism catalyzed by J10.