43
Chapter I
N-terminus (Trp α43 to Ser α53) showed involvement in small molecule binding
(unpublished data). Furthermore, J10 and its derivatives show activity on all tested DR
alleles (DR1, DR2, DR4) indicating they might bind to the non-polymorphic DRα
chain. As this region is also known to be involved in DM binding and activity (Doebele
et al., 2000) it is possible that the small molecule and DM might target similar
MHC II/peptide interactions. However, unfortunately, the extra electron density could
not unambiguously be accounted for by the small molecule. Reasons for the absence of
well-defined density for the small molecule could be partial occupancy of the binding
site by the small molecule. It is also possible that the small molecule did not bind to the
DR2 species which crystallized as it might bind to DR2 conformations only available if
the peptide is partially released which has been seen for DM recently (Anders et al.,
2011). As the MBP peptide is a high-affinity peptide for DR2 this species might have
been small especially as the peptide was still covalently linked to the protein which
further increased peptide affinity. In the obtained DR2 structure the MBP peptide was
fully bound and showed no conformational changes. Unfortunately, crystals set up with
DR2/MBP complex having the linker between peptide and DR2 cleaved did not provide
data sets with sufficient resolution, maybe due to higher flexibility. For another group
of MHC II loading enhancers (di-peptides, see table 1.1, (Gupta et al., 2008)) some
evidence points to the P1 pocket as a possible binding site although a co-crystal
structure has not been determined, yet. It is possible that J10 might bind to a similar
site, for which partial peptide release would be necessary. In the future co-
crystallization experiments with the small molecule could be performed using a MHC II
molecule carrying a low-affinity peptide or even an N-terminally truncated peptide (see
chapter II). Screening new crystallization conditions might result in well diffracting
crystals and reveal a structure presenting a partially filled peptide-binding groove or
conformational changes upon small molecule binding.
To investigate the effects of J10 and its derivatives on the
dynamics of peptide bound
to MHC II molecules
1
H-
15
N HSQC spectra of triple-labeled (
2
H,
13
C,
15
N) MBP
peptide in complex with DR2 were measured before and after addition of the small
molecule J10-1. When the spectra were compared no obvious chemical shifts of the
peaks were detected but several changes in peak intensities were observed which could
indicate peptide changes resulting from interactions of the small molecule with the
MHC II/peptide complex. Furthermore, as there were more peaks (~ 29) than expected
for a 15 amino acid long peptide and only three possible overlaps with the spectrum of