Humidity control as a strategy for lattice optimization applied to crystals of HLA-A*1101 complexed with variant peptides from dengue virus

Abstract

T-cell recognition of the antigenic peptides presented by MHC class I molecules normally triggers protective immune responses, but can result in immune enhancement of disease. Cross-reactive T-cell responses may underlie immunopathology in dengue haemorrhagic fever. To analyze these effects at the molecular level, the functional MHC class I molecule HLA-A*1101 was crystallized bound to six naturally occurring peptide variants from the dengue virus NS3 protein. The crystals contained high levels of solvent and required optimization of the cryoprotectant and dehydration protocols for each complex to yield well ordered diffraction, a process that was facilitated by the use of a free-mounting system.

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 in lane 2 and 3. Lanes 4–7 show purified GTS1.1, GTS2.2, GTS2.1 and GTS3.1, respectively.

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 the fluorescent intensity of cells stained with the PE-conjugated tetrameric pMHC complexes.

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 room temperature within 24 h.

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 a MAR CCD detector on beamline 10.1 of the SRS (Daresbury), with the crystal-to-detector distance set such that the resolution at the edge of the detector was 3.5 Å. The experimental conditions (exposure time, oscillation range etc.) are identical between images, with the exception that the backstop position has altered (the backstop was manually moved out of position to allow access to the crystal for annealing and then repositioned). For subsequent data collection, the detector position was altered to measure data to a somewhat higher resolution limit (3.2 Å, Table 2 ▶).