Crystallization and preliminary X-ray data analysis of the pXO1 plasmid-partitioning factor TubZ from Bacillus cereus

Abstract

TubZ is a structural homologue of tubulin and FtsZ GTPases, which are involved in the type III plasmid-partitioning system. TubZ assembles into polymers in a GTP-dependent manner and drives plasmid segregation as `cytomotive' filaments. In this study, C-terminally truncated TubZ from Bacillus cereus was crystallized in the presence or absence of GDP by the hanging-drop vapour-diffusion method. The crystal of TubZ in complex with GDP belonged to the monoclinic space group P2(1), with unit-cell parameters a=67.05, b=84.49, c=67.66 Å, β=92.92°, and was non-isomorphous with GDP-bound TubZ previously crystallized in the presence of the slowly hydrolysable GTP analogue GTPγS. TubZ was also crystallized in the free form and the crystal belonged to space group P2(1), with unit-cell parameters a=53.91, b=65.54, c=58.18 Å, β=106.19°. Data were collected to 1.7 and 2.1 Å resolution for the free and GDP-bound forms, respectively.

Figure 1

Limited proteolysis of Bc-TubZ. Digested samples were resolved by 12% SDS–PAGE with Coomassie Blue staining. The major fragment is labelled with an arrow. The sizes of the molecular-mass marker (lane M) are indicated on the left in kDa. Lane 1, intact Bc-TubZ; lane 2, Bc-TubZ digested with trypsin; lane 3, Bc-TubZ digested with chymotrypsin; lane 4, Bc-TubZ digested with thermolysin.

Figure 2

GTP hydrolysis of Bc-TubZ with or without GTP analogues. The competing nucleotides used were GDP, GMPPNP and GTPγS.

Figure 3

Crystals of Bc-TubZΔ in the free form (a) and the GDP-bound form (b). The scale bars are 0.1 mm in length.