In vitro assembly studies of FtsZ/tubulin-like proteins (TubZ) from Bacillus plasmids: evidence for a capping mechanism

Abstract

Proteins with a weak sequence similarity to tubulin and FtsZ are expressed from large plasmids of Bacillus anthracis and Bacillus thuringiensis and are probably involved in plasmid segregation. Previously designated RepX and TubZ, we designate them here as TubZ-Ba and TubZ-Bt. We have expressed and purified the proteins for in vitro studies. TubZ-Ba and TubZ-Bt share only 21% amino acid identity, but they have remarkably similar biochemical properties. They both assemble into two-stranded filaments and larger bundles above a critical concentration, and they hydrolyze GTP at a very high rate, approximately 20 GTP min(-1) TubZ(-1). Assembly is also supported by GTPgammaS. A tiny amount of GTPgammaS stabilizes polymers assembled in GTP and inhibits the GTPase by a mechanism involving cooperativity. The nucleotide in the polymers is almost 100% GDP, which is similar to microtubules but very different from the 20-30% GDP in FtsZ polymers. This suggests that the TubZ polymers have a capping mechanism that may be related to the GTP cap that produces dynamic instability of microtubules.

FIGURE 1.

GTP hydrolysis at increasing concentrations of TubZ-Ba. This experiment was in HMK100 buffer at pH 7.7, and gave a hydrolysis rate of 24 GTP TubZ-Ba^–1 min^–1.

FIGURE 2.

TubZ-Ba filament structure observed by negative stain EM. a and b show TubZ-Ba filaments assembled in 100 μm GTP. The filaments mostly appear as wavy two-stranded helices, but occasional polymers have three strands (bottom 2b). c, shows TubZ-Ba filaments assembled in 20 μm GTPγS. Filaments are longer and tend to associate into bundles. In the upper right, a two-stranded filament is seen separating into single strands, d shows TubZ-Ba filaments assembled in 100 μm GTP plus 1 μm GTPγS. e shows TubZ-Ba assembled in GTP in the presence of 10% polyvinyl alcohol, a crowding agent. The TubZ-Ba concentration was 5 μm in all measurements. The scale bar is 100 nm.

FIGURE 3.

Light scattering and GTPase activity of TubZ-Ba measured in the presence of non-hydrolyzable GTP analogues, GTPγS or GMPCPP. a, assembly measured by light scattering of 5 μm TubZ-Ba in 100 μm GTP; in 1.5 μm GTPγS, with 100 μm GTP added at 400 s; and in 1.5 μm GTPγS plus 100 μm GTP added together at time 0. b, inhibition of GTPase activity of TubZ-Ba by GTPγS. Data are shown for 5 μm TubZ-Ba and increasing GTPγS, plus 20, 100, and 500 μm GTP. c, inhibition of GTPase activity by GMPCPP. d, plot of 1/GTPase activity versus GMPCPP concentration, showing that the relationship is not linear. The curvature indicates that the inhibition is cooperative. e, plot of 1/GTPase activity versus GMPCPP concentration for 5 μm FtsZ and 100 μm GTP. In contrast to TubZ, this plot is linear. f, Hill plot of the GTPase inhibition by GTPγS and GMPCPP. The five lines on the left are for TubZ-Ba, with Hill coefficients of 1.7–1.9, and the line on the right is for FtsZ, with a Hill coefficient of 1.

FIGURE 4.

Excess GDP can depolymerize TubZ-Ba assembled in GMPCPP (a) or GTPγS (b).

FIGURE 5.

The amount of GDP in polymer was plotted as a function of total protein for TubZ-Ba and E. coli FtsZ. 95% of the nucleotide in TubZ-Ba was GDP, while only 30% was GDP in FtsZ.

FIGURE 6.

Biochemical properties of TubZ-Bt. a–c, images of negatively stained polymers assembled in 1 mm GTP (a), 1 μm GTPγS(b), and a mixture of 100 μm GTP and 1 μm GTPγS(c). The bar is 100 nm. d, GTPase activity of TubZ-Bt showed a critical concentration of 0.8 μm, and above this a linear activity of 18 GTP TubZ-Bt^–1 min^–1. e, assembly of 3 μm TubZ-Bt with different combinations of nucleotides. In 0.1 mm GTP, rapid assembly was followed by disassembly. In 1 μm GTPγS assembly occurred more slowly and after 100 s remained at the plateau. In 100 μm GTP plus 1 μm GTPγS assembly rose much higher, and then continued a slower rise after 100 s. This second phase is probably due to bundling seen in c. f, Hill plot for inhibition of GTPase by GTPγS gives a Hill coefficient of 1.9.