The VP35 IID mutants (R312A, K319A/R322A and K339A) were cloned, expressed and purified to homogeneity as assessed by Coomassie staining of SDS–PAGE gels (Fig. 2
). Analysis of the chromatograms from size-exclusion columns indicated similar elution volumes (12.9 ml for R312A, 12.8 ml for K319A/R322A and 12.6 ml for K339A), suggesting that these proteins retained the hydrodynamic radius of wild-type VP35 IID (Fig. 2
; Leung, Ginder, Nix et al.
). However, ThermoFluor analysis revealed that the mutant VP35 IID proteins have different T
values (Figs. 2
Figure 2 Biophysical analysis of mutant proteins. (a) Representative chromatograms from analytical size-exclusion columns of VP35 IID mutants R312A (blue), K319A/R322A (red) and K339A (green). The inset shows a Coomassie-stained SDS–PAGE gel of the highly (more ...)
Interestingly, each of the three VP35 IID mutants crystallized under different conditions and belonged to a different space group. Bipyramidal crystals of the VP35 IID R312A mutant protein grew in 1.85 M sodium phosphate/0.15 M potassium phosphate pH 4.15 at a protein concentration of 26 mg ml−1 within 2–3 d to dimensions of 100 µm in length and about 60 µm at the thickest area (Fig. 3
a). R312A mutant crystals diffracted to 1.95 Å resolution and belonged to space group P6122 (Table 1). The Matthews coefficient was 2.94 Å3 Da−1, corresponding to 58% solvent content and four molecules per asymmetric unit. Plate-shaped crystals of VP35 IID K319A/R322A mutant protein grew in 0.1 M bis-tris pH 6.75, 0.2 M ammonium sulfate and 28% PEG 3350 at a protein concentration of 7 mg ml−1 after 1 d to dimensions of 100 × 50 × 20 µm (Fig. 3
b). K319A/R322A mutant crystals belonged to space group P212121 and diffracted to 1.7 Å resolution (Table 1). The Matthews coefficient was 2.2 Å3 Da−1, corresponding to 44% solvent content and two molecules per asymmetric unit. Cubic crystals of VP35 IID K339A mutant appeared in 1–2 d after equilibration against 0.1 M sodium citrate pH 2.8, 0.3 M lithium sulfate and 13% PEG 8000 with 10 mg ml−1 protein. The crystals had dimensions of 200 × 200 × 150 µm and diffracted to a resolution of 2.4 Å (Fig. 3
c). These crystals belonged to space group P21 (Table 1). The Matthews coefficient was 2.95 Å3 Da−1, corresponding to 58% solvent content and six molecules per asymmetric unit. Data-collection statistics for all three mutant VP35 IID proteins are summarized in Table 1.
Figure 3 Representative crystals of VP35 IID mutants. (a) R312A (approximate dimensions 30 × 30 × 100 µm), (b) K319A/R322A (approximate dimensions 100 × 50 × 20 µm) and (c) K339A (approximate dimensions (more ...)
Data collection for mutant VP35 IID proteins
All three mutants of VP35 IID displayed diminished dsRNA binding and interferon inhibition without affecting the structural integrity of the protein (Cardenas et al.
; Leung et al.
; Prins et al.
). Interestingly, all four residues that we mutated are surface residues with high degrees of conformational flexibility (Lys and Arg) and their mutation to alanine would result in surface-entropy reduction (Cieślik & Derewenda, 2009
; Derewenda & Vekilov, 2006
). Identification of these residues for further analysis was primarily based on their sequence conservation and their location within the central basic patch, which we have recently shown to be important for interferon inhibition. Analysis of these mutant structures will provide insight into structural and mechanistic aspects of Ebola VP35-mediated circumvention of the host immune response. Additional analysis of the crystal contacts of these structures will provide correlations between surface conformational entropy and crystal lattice formation. These efforts will be reported shortly.