Protein expression, characterization, crystallization and preliminary X-ray crystallographic analysis of a Fic protein from Clostridium difficile

Abstract

Fic domains in proteins are found in abundance in nature from the simplest prokaryotes to animals. Interestingly, Fic domains found in two virulence factors of Gram-negative bacteria have recently been demonstrated to catalyse the transfer of the AMP moiety from ATP to small host GTPases. This post-translational modification has attracted considerable interest and a role for adenylylation in pathology and physiology is emerging. This work was aimed at the structural characterization of a newly identified Fic protein of the Gram-positive bacterium Clostridium difficile. A constitutively active inhibitory helix mutant of C. difficile Fic was overexpressed in Escherichia coli, purified and crystallized by the vapour-diffusion technique. Preliminary X-ray crystallographic analysis shows that the crystals diffract to at least 1.68 Å resolution at a synchrotron X-ray source. The crystals belonged to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a=45.6, b=80.8, c=144.7 Å, α=β=γ=90°. Two molecules per asymmetric unit corresponds to a Matthews coefficient of 2.37 Å3 Da(-1) and a solvent content of 48%.

Keywords: Clostridium difficile; Fic protein; adenylylation.

Figure 1

Purification and oligomeric state of CdFic^SE/AA. (a) Coomassie-stained 4–15% gradient SDS–PAGE of purified CdFic^SE/AA. Lane M, molecular-weight marker (labelled in kDa). Lane 1, elution from the HisTrap affinity column, 1.8 µg protein. Lane 2, as lane 1 but with 5.4 µg protein. In addition to the dense band corresponding to CdFic^SE/AA next to the 25 kDa band of the marker, there are two bands (bands 1 and 2) just above the 10 kDa band of the marker. These bands correspond to a degradation product of CdFic^SE/AA as determined by mass-spectrometric analysis. (b) Size-exclusion chromatography analysis of the oligomeric state of isolated CdFic^SE/AA using a standard Superdex 75 HR 10/30 column. The arrows at the top indicate the elution volumes of proteins of known mass applied to the same column (indicated in kDa; V _o, void volume). The elution volume of CdFic^SE/AA (9.95 ml) suggests that the molecule is present as a dimer in solution. Comparison with elution volumes of standard proteins suggests a molecular weight of ∼57.5 kDa, while the theoretical molecular weight of the dimer is 56.2 kDa.

Figure 2

Crystals of CdFic^SE/AA. (a) Crystals grown by streak-seeding with a 1:1 drop ratio of protein:reservoir solution. Crystals of similar size diffracted synchrotron X-ray radiation to 3.1 Å resolution (data not shown). (b) Optimization of the drop ratio to 3:1 protein:reservoir solution resulted in significantly larger crystals growing up to 1 mm in size. These larger crystals showed significantly improved diffraction and a 1.68 Å resolution data set was collected as shown in Fig. 3 ▶ and Table 3 ▶.

Figure 3

Synchrotron X-ray diffraction pattern collected from a single cryocooled CdFic^SE/AA mutant crystal corresponding to that shown in Fig. 2 ▶(b). Clear diffraction was visible to approximately 2 Å resolution. The inset shows a close-up view of the reflections.