51
Chapter II
molecular replacement with the program Phaser (McCoy et al., 2007) using the
previously solved DR1/HA structure (PDB: 1DLH, (Stern et al., 1994)) as a search
model. The structure was refined using Phenix (Adams et al., 2010) and evaluation of
the structure was carried out with MolProbity (Chen et al., 2010). During refinement
manual adjustments of the structure were performed using the software Coot (Emsley
and Cowtan, 2004). To assess the accuracy of the crystal structure the statistical
quantities R
free
and R
work
were used (Brunger, 1992). Figures depicting protein
structures are generated with PYMOL (Schrodinger, 2010).
3.2.7
Eluting covalently linked peptide from HLA-DR1
To release the covalently linked peptide from DR1 the complex was incubated for 1
hour at 25 ºC in the presence of 4 mM DTT. After addition of full length HA peptide,
using twenty times molar excess, the complex was incubated at 25 ºC for an additional
hour. The sample was injected to an α-DNP affinity column and the separated peptide
carrying a DNP-label eluted with 50 mM CAPS buffer (pH 11.5). The eluted peptide
was lyophilized, dissolved in water and submitted for mass spectrometry.
3.2.8
Mass spectrometry
Lyophilized peptide sample was dissolved in water and separated by a nano-liquid
chromatography system (Nano-Acquity UPLC system from Waters Corporation) and
analyzed by an ABI model 4800 TOF/TOF Matrix Assisted Laser Desorption mass
spectrometer and by a Thermo Scientific LTQ Orbitrap XL (Thermo Fisher Scientific).
3.3
Results and discussion
3.3.1
Preparing HLA-DM and HLA-DR1 carrying a truncated HA peptide, both
used for crystallization experiments
DM used for crystallization experiments was expressed in CHO cells and purified
following the scheme described in figure 3.1. First, DM was purified by affinity
chromatography and protein tags were proteolytically removed. After incubation under
reducing conditions, free cysteines of DM were alkylated using iodoacetamide. DM was
further purified by gel filtration and ion exchange chromatography before mixing with
52
Chapter II
DR1 protein. DR1 molecules carrying covalently linked HA peptide variants were
prepared as described in figure 3.2. Initially, DR1 molecules carrying a low-affinity
CLIP peptide were expressed in
Sf9 insect cells and purified by affinity
chromatography. Aggregates were removed by gel filtration and the linker between
peptide and DRβ chain, as well as C-terminal leucine zippers, were proteolytically
cleaved. DR1/CLIP
low
was further purified by ion exchange chromatography. As for
protein crystallization, large amounts of covalently linked DR1/peptide complex are
required, the peptide exchange protocol had to be improved as the yield amounted to
only 5 %. By using a milder reducing agent (glutathione instead of DTT), balancing
redox conditions and longer incubation times favoring the formation of the disulfide
bond between DR1
and peptide, the yield of the peptide exchange step was
improved up
to 30 %. Following the optimized peptide exchange protocol described in 3.2.1, the
low-affinity CLIP peptide was exchanged versus an N-terminally truncated HA peptide.
After peptide exchange under redox conditions excess peptide was removed by gel
filtration. By improving the yield of the peptide exchange reaction it was feasible to
prepare enough protein for crystallization experiments. In the end the covalently linked
MHC II/peptide complex was purified by ion exchange chromatography and the overall
yield was ~ 8 %.
expression in CHO cells (bioreactor)
protein C-affinity chromatography
thrombin digestion
incubation under reducing conditions
dialysis in the presence of iodoacetamide
gel filtration
ion exchange chromatography (MonoQ)
Figure 3.1: Preparation of DM used for crystallization trials. DM was expressed in CHO cells
using hollow fiber bioreactors. After protein purification using protein C-affinity chromatography protein
tags were removed by thrombin digestion. Following incubation under reducing conditions DM was
dialyzed in the presence of iodoacetamide to alkylate free cysteines. DM was further purified by gel
filtration and ion exchange chromatography.
53
Chapter II
expression of DR1/CLIP
low
(SKARMATGALAQA) in
Sf9 insect cells
affinity chromatography (L243)
gel filtration
V8 cleavage
ion exchange c hromatography (MonoQ)
loading of HA peptide variant (P-2, P-1, P1 missing) under redox conditions
gel filtration
affinity chromatography (α-DNP)
ion exchange chromatography (MonoQ)
Figure 3.2: Preparation of DR1 carrying an N-terminally truncated HA peptide. DR1/CLIP
low
was expressed in
Sf9 insect cells as a secreted protein. The protein was purified by L243 affinity
chromatography and aggregates removed by gel filtration chromatography. The linker between peptide
and DRβ chain as well as C-terminal leucine zippers were cleaved by V8 protease. After ion exchange
chromatography the cleaved peptide was exchanged versus an HA peptide variant missing residues P-2,
P-1 and P1 under redox conditions. Excess peptide was removed by gel filtration and the DR1 complex
carrying a covalently linked HA peptide variant further purified by affinity chromatography and ion
exchange chromatography.
3.3.2
Measuring pH-dependent affinity of HLA-DM to HLA-DR1 carrying an N-
terminally truncated HA peptide using surface plasmon resonance
To determine optimal conditions for crystallizing the non-covalently linked complex
of DM and DR1, surface plasmon resonance experiments were carried out at different
pH. As can be seen in figure 3.3 the strongest affinity of DM to the covalent complex of
DR1 and an N-terminally truncated HA peptide was measured at pH 5.5 with a
dissociation constant of ~ 1.6 µM. With increasing pH, DM affinity decreased with a
dissociation constant of 4.7 μM at pH 6.5 and 15.4 µM at neutral pH. To favor
crystallization of the non-covalent complex of DM and DR1 crystallization conditions
with pH lower than 6.5 were preferentially screened.