Deuterium oxide promotes assembly and bundling of FtsZ protofilaments

Abstract

The assembly and bundling of FtsZ protofilaments play an important role during bacterial cell division. Deuterium oxide (D₂O) is known to have strong stabilization effects on the assembly dynamics of several proteins including tubulin, a homologue of FtsZ. Here, we found that D₂O enhanced the light-scattering intensity of the assembly reaction, increased sedimentable polymer mass, and induced bundling of FtsZ protofilaments. D₂O also increased the stability of FtsZ polymers under challenged GTP conditions and suppressed dilution-induced disassembly of protofilaments. D₂O enhances the assembly parameters of FtsZ and microtubules albeit differently. For example, D₂O induced bundling of FtsZ protofilaments, whereas it did not induce bundling of microtubules in vitro. In addition, D₂O strongly suppressed the GTP hydrolysis rate of microtubules, but it had no effect on the initial rate of GTP hydrolysis of the FtsZ assembly. D₂O (80%) also increased the helical content of FtsZ by 25% compared to the helical content of FtsZ in aqueous buffer. D₂O was shown to reduce the binding of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) to tubulin. In contrast, we found that D₂O strongly enhanced the binding of bis-ANS to FtsZ. The results indicated that D₂O promotes assembly and bundling of FtsZ protofilaments by increasing hydrophobic interactions between the protofilaments. The results also suggest that the phosphate release rather than the on-site GTP hydrolysis is the rate-limiting step of the GTP turnover reaction.