3.1. Protein production and functional assays
The yields of refolded complex after size-exclusion chromatography purification were 3–5 times higher for nonbiotinylated pMHCs compared with biotinylated pMHCs, indicating typical refolding efficiencies of 12 and 3%, respectively (data not shown), the former being in line with previously reported values (10–20% depending on peptide and MHC heavy chain) for pMHCs refolded for structural studies (Reid et al.
). Gel electrophoresis indicated that the samples were over 98% pure (Fig. 1).
Figure 1 Production of pMHC. pMHCs purified by anion-exchange chromatography were run on 15% SDS–PAGE under reducing conditions. Standard molecular-weight markers (kDa) were run in lane 1, while inclusion bodies of MHC heavy chain and β2m were (more ...)
The conformation of the refolded pMHCs was verified by DEIA using monoclonal antibody W6/32 that recognizes a conformational epitope. Furthermore, the T-cell receptor (TCR) binding ability of the refolded pMHCs was tested by a competitive binding assay as shown in Fig. 2. The refolded pMHCs inhibited PE-tetramers binding to GTS-specific T-cell lines, resulting in reduction of the fluorescent intensity. These demonstrated that the refolded pMHCs retained the functionality of the physiological membrane-bound pMHCs since they had the ability to interact with GTS epitope-specific T-cell lines.
Figure 2 Tetramer competition assay. The GTS-specific T-cell lines were stained with 1 µg PE-conjugated tetrameric pMHC in the presence (blue) or absence (red) of a 100-fold excess of the refolded pMHC and analyzed by FACS. The histograms show (more ...)
672 crystallization conditions were screened in a series of experiments using several batches of the GTS1.1 and GTS3.1 variant pMHCs. Crystallization occurred at room temperature in two major groups of screen conditions; namely, those containing ammonium sulfate or phosphate salts (Table 3). Initial efforts to collect suitable X-ray data focused on optimized crystals from the ammonium sulfate condition (which was the first of the two crystallization precipitants to be identified). These crystals typically diffracted to 7 Å resolution using both in-house and synchrotron sources. Extensive efforts to improve the diffraction quality (varying the cryoprotection and use of dehydration protocols) provided no consistent route forward. Attention was therefore shifted to the second group of crystallization precipitants to be identified: the phosphate salts.
Crystallization conditions that produced pMHC crystals
A reservoir solution consisting of 0.63 M NaH2PO4 and 1.17 M K2HPO4 pH 8.2 was judged on the basis of visual inspection to be the most promising of the phosphate conditions identified in the nanolitre-scale screens. This condition was therefore used as the basis for crystal optimization in standard microlitre-scale sitting drops. The GTS1.1 variant crystallized within 16 h using this condition and showed Bragg diffraction to better than 3.5 Å resolution when tested on the in-house rotating-anode X-ray source (after cryoprotection by transfer to mother liquor containing 20% glycerol).
Having established a potentially suitable condition for the growth of crystals of suitable quality for structure determination, microlitre-scale crystallization optimizations were carried out for all six pMHC variants. For each pMHC a series of conditions were tested: these conditions were centred on 0.63 M NaH2PO4, 1.17 M K2HPO4 pH 8.2, but varied in salt concentration and pH. Identical salt concentrations but slight differences in pH proved to be essential for the crystallization of the full set of pMHC variants (Table 2 and Fig. 3).
Figure 3 GTS pMHC crystals. Purified pMHCs were crystallized in 0.63 M NaH2PO4, 1.17 M K2HPO4 buffer using sitting-drop vapour diffusion. The buffer pH was varied for each variant as described in Table 2. All crystals grew at (more ...)
Crystallization conditions for the MHC class I molecule HLA-A11 have been reported previously by two groups (Li et al.
; Li & Bouvier, 2004
; Blicher et al.
). Bouvier and coworkers reported data for HLA-A11 in complex with two unrelated peptide epitopes from HIV-1 proteins (AIFQSSMTK from reverse transcriptase and QVPLRPMTYK from Nef; Li & Bouvier, 2004
), while Blicher and coworkers (Blicher et al.
) detail crystals of two further complexes, those of HLA-A11 with a peptide (KTFPPTEPK) derived from the SARS nucleocapsid and with AIMPARFYPK, a sequence homologue of an epitope in hepatitis B virus DNA polymerase. The crystallization conditions in all these examples were PEG-based and were closely related (the crystallization precipitant was either PEG MME 5000 or PEG 4000 at a concentration of 20–30%). Indeed, PEG-based conditions are typical for the crystallization of pMHC complexes (Garboczi et al.
; Reid et al.
). The nonstandard conditions required for the crystallization of our HLA-A11–peptide complexes indicate that a broad crystallization screen should be considered if initial attempts centred on PEG-based conditions fail to yield pMHC crystals. In particular, the 1.8 M
phosphate conditions that yielded useful crystals came from the Hampton Quik Screen (block 5 in our standard set of 480 conditions and usually one of the less successful screens; Walter et al.
3.3. Crystal lattice-order optimization and X-ray data collection
It was difficult to establish cryo methods for the collection of well ordered Bragg diffraction. Optimal diffraction data for each complex were obtained using different protocols (Table 2). Two cryoprotectants proved useful, glycerol and sodium malonate, but X-ray data to a suitable resolution for structure determination could only be collected directly from cryoprotected crystals for the GTS1.1 variant pMHC (2.6 Å; Table 2). The crystals of all five other complexes required annealing/fine control of humidity to optimize the diffraction quality. The results of a simple annealing experiment carried out at station BM 10.1 of the SRS (Daresbury, UK) by blocking off the cryostream for 2 s first alerted us to the significant potential for improvement in diffraction quality (Fig. 4). In this experiment, the resolution limit for Bragg diffraction from a crystal of the GTS2.4 variant complex was improved dramatically from ~7 to 3.2 Å. Subsequently, the diffraction quality for crystals of the four remaining complexes, GTS2.1, GTS2.2, GTS2.3 and GTS3.1, was systematically characterized at defined humidity levels using a combination of FMS (Kiefersauer et al.
) and in-house X-ray source/detector. The FMS is still a relatively new device, the principle of which is explained in §2.4
. Essentially, by maintaining the crystal in an air stream of defined humidity, the crystal can be dehydrated or rehydrated over time. The FMS goniometer head can be mounted onto standard X-ray equipment so that the diffraction at each humidity level can be recorded.
Figure 4 Improvement of X-ray diffraction after annealing experiment. The cooling flow from the cryostream to the GTS2.4 crystal was blocked off for 2 s between the measurement of the X-ray diffraction patterns shown in (a) and (b). The images are from (more ...)
In this case, we performed full dehydration analyses on only a few crystals and then used fresh crystals to repeat the dehydration steps which resulted in the best diffraction. Crystals were then frozen directly in a standard cold nitrogen-gas stream without testing their diffraction at room temperature and were only exposed to X-rays once they had been frozen (suitable crystals were then stored in liquid nitrogen for subsequent synchrotron data collection). This approach avoids any of the radiation damage which the crystals experience when they are exposed to X-rays at room temperature. Typically, out of ten crystals tested, four showed improved diffraction. This rather low success rate may partly be a consequence of the difficulty in reproducing the freezing protocol exactly (for example, in covering the crystal with the freezing solution and transferring it to the cold gas stream always within the same time interval). It is clear that even with the FMS there is scope for improving the crystal-handling procedures in order to achieve greater reproducibility for sensitive crystals such as these. From Table 2, it can be seen that for two of the complexes (GTS2.1 and GTS2.3) the optimal humidity level is the start humidity (i.e. that of the crystallization mother liquor); thus, the use of the FMS appears to have allowed us to circumvent residual problems with cryoprotection. For the other two complexes (GTS2.2 and GTS3.1), the optimal humidity is 1% below the start humidity, implying that a slight dehydration of the crystal promotes a beneficial lattice rearrangement.
As expected given their closely related crystallization conditions, the crystals of all six pMHC complexes had essentially identical lattices. The crystals belong to space group C
2 and contain three pMHC complexes in the crystallographic asymmetric unit plus 68–70% solvent (the Matthews coefficients are 3.83–4.16 Å3
; further details including unit-cell parameters are given in Table 2). It is noteworthy that the percentage of solvent in the crystals does indeed correlate with the protocols applied before data collection. The three crystals subjected to annealing or controlled dehydration all show lower solvent content than those simply cryoprotected or flash-frozen at start humidity on the FMS. In all cases the solvent content is relatively high for a protein crystal, particularly when compared with the values of 48–58% for other peptide–HLA-A11 complexes (Li & Bouvier, 2004
; Blicher et al.
) and pMHC complex crystals in general (Garboczi et al.
; Reid et al.
). The diffraction data reported here have resulted in successful structure determinations (phased by molecular replacement) for all six variant complexes. A full analysis of the crystal structures and their implications for the immune response to dengue virus is ongoing and will be reported elsewhere.