Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India
Correspondence
Parthasarathi Ajitkumar
(ajit{at}mcbl.iisc.ernet.in)
We recently reported that the deletion mutant MtFtsZ-C169 of Mycobacterium tuberculosis FtsZ (MtFtsZ), which lacked 169 C-terminal residues and retained only the first 210 residues, formed long polymers in about 30 s after the addition of GTP, like the FtsZ of Escherichia coli (EcFtsZ) (Anand et al., 2004
). Both of these FtsZs were purified under denaturing conditions and refolded. By contrast, the full-length MtFtsZ, prepared under identical conditions of denaturation and refolding, formed equivalent-type long polymers in 10 min after the addition of GTP. These observations prompted us to suggest that the residues in the C-terminal region of the MtFtsZ protein might be responsible for imposing slow polymerization on MtFtsZ (Anand et al., 2004
).
Modelling the probable structure of the MtFtsZ-C169 mutant protein (Borhani & White, 2004
) based on the crystal structure data of the full-length MtFtsZ protein (Leung et al., 2004
), and showing exposure of a large hydrophobic patch, which normally remains buried in the native full-length protein, Borhani & White (2004)
raise the following contentions against our observations. (i) Polymerization of the C-terminally truncated MtFtsZ-
C169 mutant is unlikely to be physiologically relevant since the molecule has lost the conserved C-terminal domain and the non-conserved C-terminal tail that is believed to be interacting with other cell-division proteins; (ii) the polymerization elicited by the mutant could be aggregation and not true protofilament formation owing to the exposure of the large hydrophobic patch; and (iii) GTPase activity of the mutant should have been determined as the critical T7 loop, which is required for the activity, would be of uncertain conformation in the mutant owing to the loss of helix H11 that links the stabilizing C-terminal domain.
Disproving these contentions, with qualitative and quantitative biochemical evidence, we demonstrate here that the MtFtsZ-C169 mutant does hydrolyse GTP, and elicits Mg2+-dependent, GTP-induced, GTP-specific polymerization in vitro. Examination of the MtFtsZ-
C169 mutant for GTP hydrolysis revealed that it hydrolysed GTP at the rate of 97·2 nmol GTP h1 (mg protein)1 (Fig. 1
). By contrast, the negative control sample namely, the deletion mutant MtFtsZ-ZN, which lacked the C-terminal 211 residues and therefore the T7 loop critical for GTPase activity did not show GTPase activity (Fig. 1
). Recombinant full-length MtFtsZ and EcFtsZ, which were also purified under denaturing and refolding conditions that are identical to those used for the purification of the mutant, were used as the positive control samples. Full-length MtFtsZ elicited GTP hydrolysis at the rate of 35·8 nmol GTP h1 (mg protein)1 (Fig. 1
), while EcFtsZ showed 1 µmol GTP h1 (mg protein)1 (data not shown).
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It may also be mentioned here that the MtFtsZ-C169 mutant was originally obtained as a spontaneous deletion mutant when the full-length MtftsZ clone in vector pET-20b+ was accidentally maintained in the recA-positive Escherichia coli BL21(DE3) C41 strain (Miroux & Walker, 1996
). As a case of serendipity and to our pleasant surprise, this mutant gave clues on fast polymerization and GTPase activities in vitro (Anand, 2001
). In order to authenticate the spontaneous mutant, we recreated the clone using recombinant DNA methods in order to study biochemical properties of the recombinant mutant protein in vitro; part of this study was reported in our previous article (Anand et al., 2004
). Since our sole interest was to study fast polymerization of the mutant in vitro, we were not concerned about the physiological relevance of the mutant. For the same reason, we did not create an equivalent deletion mutant of EcFtsZ, a control sample suggested by Borhani & White (2004)
, as full-length EcFtsZ in any case polymerizes rapidly and there was no point in creating a faster-polymerizing mutant EcFtsZ protein! Nevertheless, from our studies, it is interesting to note that the first 210 residues of MtFtsZ, forming a Mini FtsZ molecule, are sufficient to confer GTPase and Mg2+-dependent, GTP-induced and GTP-specific polymerization activities on the molecule in vitro.
Acknowledgements
This work was supported by the research grant BT/R&D/15/35/94 from the Department of Biotechnology, Government of India, to P. A. Infrastructure support received from the ICMR-funded Centre for Advanced Study for Molecular Medical Microbiology at the Department of Microbiology and Cell Biology is gratefully acknowledged. The authors gratefully acknowledge the immense help received from Mr S. D. Shibu, Service Engineer, M/s Laser-spectra services, Bangalore, for setting up the parameters for 90° light-scattering experiments in the Jobin Yvon Horiba Spectrofluorimeter FluorMax at Molecular Biophysics Unit, Indian Institute of Science, Bangalore. The authors express immense gratitude to the Chairman, Molecular Biophysics Unit, Indian Institute of Science, Bangalore for the permission to use the spectrofluorimeter and the graduate student Ms K. Beena of the same department for help at the initial stages of the light-scattering experiments.
REFERENCES
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