82
Final
discussion and outlook
conformer present in solution which may be exposed after further peptide release, e.g.
release of the P1 anchor. That peptide-binding induces conformational changes of
MHC II molecules has been observed before (Zarutskie et al., 1999) and also
indications of alterations in the P1 pocket (Chou and Sadegh-Nasseri, 2000). Recently, a
structure was published of a DR1 mutant carrying a full length CLIP peptide showing
increased susceptibility to DM and revealing conformational alterations near the N-
terminus of the bound peptide involving a reorientation of a helical region of the DRα
chain for one of the two molecules in the asymmetric unit (Painter et al., 2011). Similar
as discussed above the problem exists not knowing whether the crystallized DR form
with the bound peptide binds to DM or whether part of the peptide first leaves the
binding groove before DM binding occurs. As the mutation alters the P1 pocket and
thereby destabilizes the bound peptide apparent by an increased intrinsic rate of peptide
release (Painter et al., 2011) enhanced DM susceptibility could be also partially due to
increased peptide mobility. To further address the receptive MHC II conformation
which seems to be challenging to crystallize due to its short-lived state and potential
structural diversity other methods might be necessary like NMR spectroscopy to
investigate the dynamic state of the complex in solution. For example, the system
established during this study involving isotope labeled peptide bound to MHC II
molecule can be further developed and used to track the bound peptide N-terminus upon
DM binding in solution. In addition, further crystallization experiments should be
performed with a DR1 molecule carrying an N-terminally truncated HA peptide variant
with a glycine at P2 position and no P1 anchor residue which showed enhanced DM
binding during this study and could reveal whether conformational changes occur once
the P1 anchor is absent.
The co-crystallization experiments of DR2 with the small molecule J10, a MHC
loading enhancer, carried out in this study may involve a similar challenge as the small
molecule may bind to and stabilize a different DR conformation than the form that
crystallized. Functional experiments in this study already revealed a higher affinity of
the small molecule to low-stability MHC II/peptide complexes and co-crystallization
experiments should be repeated with a low-affinity DR/peptide complex. Especially,
DR complexes with N-terminally truncated peptides should be analyzed for J10 binding
and used for co-crystallization experiments as the small molecule may function by
stabilizing the empty peptide-binding groove and a partially empty groove may expose
84
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