Promoting crystallisation of the Salmonella enteritidis fimbriae 14 pilin SefD using deuterium oxide

Abstract

The use of heavy water (D(2)O) as a solvent is commonplace in many spectroscopic techniques for the study of biological macromolecules. A significant deuterium isotope effect exists where hydrogen-bonding is important, such as in protein stability, dynamics and assembly. Here we illustrate the use of D(2)O in additive screening for the production of reproducible diffraction-quality crystals for the Salmonella enteritidis fimbriae 14 (SEF14) putative tip adhesin, SefD.

Figure 1

Representative images of SefD_dscA crystals grown in both 50% and 100% D₂O.

Figure 2

Global arrangement of SefD_dscA molecules within crystals grown in 0% and 100% D₂O. (A) Domain-swapped dimer of SefD_dscA in 0% D₂O. The single molecule of the asymmetric unit is coloured yellow whilst its symmetry mate is coloured olive. The dimer face is composed of a hydrophobic surface and solely backbone hydrogen bonding within the A2-G′-G-A2′ sheets. (B) Crystal packing in the 0% and 100% D₂O crystal forms. Top panel: SefD_dscA molecules (green) form ‘rod-like’ structures and the unit cells are viewed down the crystallographic c-axis. The spacegroup C222₁ (0% D₂O: red box) is a subgroup of P6₅22 (orange box) and is very similar to the P2₁2₁2₁ (100% D2O: red box) packing. Bottom panel: SefD_dscA domain-swapped dimers (red, blue and green) viewed parallel to half the unit cell c-axis. The asymmetric units are represented as dashed boxes.

Figure 3

(A) Effects of deuterium on the inter-domain domain-swapped dimer interface. Top panel: A SefD_dscA dimer from a 100% D₂O crystal (blue) is shown superimposed onto a single chain of a 0% D₂O dimer (yellow) and clearly demonstrates a twisting and displacement of the other subunit of the dimer in the different solvents (blue arrow). The dimer interface which orchestrates these changes is boxed and the secondary structure is labeled. Bottom panel: (left) the box has been blown up and backbone residues in the A2 and G strands are shown as sticks with inter-sheet hydrogen bonds depicted as dashed lines. (Right) This region has been rotated to highlight the distortion in the sheet structure (B) Effect of deuterium on the overall packing in crystals of SefD_dscA. Packing of SefD_dscA in both crystal forms are shown along the a and c unit cell axes with the E (blue) and F (purple) chains in the 100% D₂O crystal and equivalent positions in the 0% D₂O crystal highlighted. The asymmetric units are depicted as dashed boxes. To the right of each is a schematic representation shown as green arrows. In the 0% D₂O crystals, two ‘face-to-face’ dimers are shown (yellow star), however in the 100% D₂O crystal, due to changes within the domain-swapped dimer interface (Fig. 2; Fig. 3; Supplementary Fig. 5; Supplementary Fig. 7) which are propagated down the c-axis, only one of these dimers is seen (yellow star), whilst to maintain lattice integrity, chains E and F do not form the usual dimer (red star).