22
Chapter I
2
Chapter I: Investigating enhanced peptide exchange of
MHC class II molecules by the small molecule catalyst J10
2.1
Introduction
Peptides presented by MHC II molecules on the cell surface play a pivotal role as
they can evoke different immune responses upon interaction with CD4+ T cells which
can lead to antibody production (Vinuesa et al., 2005), cell destruction by cytotoxic T
cells (Behrens et al., 2004) or formation of CD4+ regulatory T cells (Sakaguchi, 2010).
Actively modifying the repertoire of presented peptides is therefore of interest for
various medicinal applications including peptide based vaccination (e.g. therapeutic
cancer vaccination) and treatment with peptide immunomodulators (e.g. Copaxone used
for treatment of multiple sclerosis). However, the efficiency with which administered
peptides are loaded onto MHC II molecules is usually very low, with the therapeutic
peptide often proteolytically degraded before it can be loaded onto a MHC II molecule.
Therefore, enhancing MHC II loading could improve the efficacy of peptide-based
therapeutics.
Our lab previously identified a small molecule (Nicholson et al., 2006) called J10
that significantly accelerates the rate of peptide loading onto DR molecules in the
absence of DM. Different from previously discovered MHC II loading enhancers
(MLE) with activities at millimolar concentrations, J10 is active in the low micromolar
range, which makes it more suitable as a potential adjuvant for peptide vaccination and
immunotherapies. Different from DM, J10 catalyzes peptide exchange also at neutral
pH although it has optimal activity at an acidic pH, like DM. Catalyzing peptide
exchange at neutral pH could be advantageous if J10 is used as adjuvant for peptide
therapeutics as it could load peptides on MHC II molecules present in early endosomes
and on the cell surface. Furthermore, it was shown that J10 enhances peptide
presentation by MHC II molecules in vivo (Call et al., 2009). Extensive medicinal
chemistry has been already carried out which improved the activity of the small
molecule. However, as J10 and its derivatives have high potential as adjuvant for
peptide therapeutics and might reveal a highly peptide receptive DR conformation also
relevant for the peptide exchange mechanism of DM, it is of great interest to understand
how the small molecules catalyze peptide exchange.
23
Chapter I
Two methods were applied to investigate the peptide exchange mechanism of J10;
first, X-ray crystallography to explore the binding site of the small molecule, and
second, NMR spectroscopy to investigate peptide release catalyzed by the small
molecule in solution. At first, attempts to crystallize J10 derivatives (J10-1, J10-12, see
figure 2.1) together with the MHC II/peptide complex DR2/MBP were carried out using
co-crystallization and ligand soaking. Identification of the specific binding site of J10
would help to design small molecules with a higher affinity and therefore higher
potency for potential therapeutic applications. To investigate the effect of J10 binding
on DR2/MBP in solution, NMR experiments were performed. HSQC spectra of isotope
labeled MBP peptide in complex with DR2 were measured before and after addition of
a J10 derivative. By comparing the HSQC spectra chemical shifts and intensity changes
could be detected and together with the peak assignment could provide information
about which part of the peptide is affected by small molecule binding. For isotope
labeling, the peptide had to be expressed separately from DR2 and subsequently
quantitatively exchanged against an already bound lower-affinity peptide (in this case
CLIP peptide). The most active J10 derivative was chosen for small molecule addition
(J10-1, see figure 2.1). Furthermore, to qualitatively measure binding of J10 to low- and
high-affinity MHC II/peptide complexes
19
F-NMR experiments of a J10 derivative were
performed (J10-11, see figure 2.1).
The application of these two complementary methods was utilized to gain a
comprehensive molecular understanding of the MHC II/J10 interactions and their
interplay with peptide-binding. The findings could guide further small molecule design
as well as provide new information about peptide-binding to and release from MHC II
molecules in general.
Figure 2.1: Chemical structures of the most active J10 derivatives enhancing MHC II loading.
From right to left the activity of the small molecules increases. The small molecules J10-1 and J10-12
were used for co-crystallization trials. NMR experiments with isotope-labeled MBP peptide were carried
out using the small molecule J10-1 and
19
F-NMR spectra were collected of the small molecule J10-11.
24
Chapter I
2.2
Materials and Methods
2.2.1
Preparation and crystallization of HLA-DR2/MBP in the presence of J10-1
and J10-12
Soluble DR2/MBP was expressed in Sf9 insect cells using the baculovirus system
(pAcDB3 plasmid with BaculoGold Baculovirus; BD Biosciences). The MBP (85-99)
peptide (ENPVVHFFKNIVTPR) was covalently linked to the N-terminus of the DRß
chain with a thrombin-cleavable linker. The hydrophobic transmembrane regions of
DRα and DRß were replaced with leucine zipper dimerization domains from the
transcription factors Fos and Jun (Kalandadze et al., 1996). The protein was purified by
affinity chromatography using mAb L243 (American Type Culture Collection). The
leucine zipper dimerization domains were cleaved with V8 protease, and for some
DR2/MBP complexes the peptide linker was cut with thrombin. The protein was further
purified by anion exchange chromatography.
For co-crystallization experiments DR2/MBP was concentrated to ~ 10 mg/mL and
incubated with 2 mM J10-1 or 2 mM J10-12 at 25 ºC or 37 ºC. Crystals were obtained
at 18 ºC in 100 mM glycine, pH 5.1-5.4, 14-18% PEG 6000 and 20 mM or 50 mM
sodium acetate using the hanging drop method. For soaking experiments the crystals
were transferred into a solution containing the crystallization conditions and 2 mM J10-
1 or 2 mM J10-12, crystals were incubated at 25 ºC for 4 h. Crystals were flash cooled
in liquid nitrogen using 30 % glycerol or 35 % ethylene glycol as cryoprotectant.
Diffraction data were collected on beamline X25 at the National Synchrotron Light
Source (Brookhaven Laboratories, Upton, NY, USA) with a resolution of up to 3 Å.
Data were processed with HKL2000 (Otwinowski and Minor, 1997). DR2/MBP
crystallized in the hexagonal space group P3
1
with unit cell dimensions a = b = 118.6 Å,
c = 75.4 Å and two molecules per asymmetric unit. The structure was determined by
molecular replacement with the program MOLREP (Vagin and Teplyakov, 2010) using
the already solved DR2/MBP structure (PDB: 1BX2, (Smith et al., 1998)) as a search
model. For structure refinement the program CNS (Brunger, 2007; Brunger et al., 1998)
was used. Manual model building was carried out in O (Jones et al., 1991) and COOT
(Emsley and Cowtan, 2004). To model the small molecules into observable electron
density coordinates and molecular topologies of the small molecules were generated
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