Summary auto-generated
This study responds to criticisms of previously reported polymerization of MtFtsZ-DC169, a C-terminally truncated mutant of Mycobacterium tuberculosis FtsZ protein containing only the first 210 residues. Critics suggested the polymerization was merely protein aggregation without functional relevance because critical domains were deleted. The authors provide biochemical evidence refuting these contentions. Using GTPase assays and light-scattering measurements, they demonstrate that MtFtsZ-DC169 hydrolyzes GTP at 97±2 nmol GTP h⁻¹(mg protein)⁻¹ and undergoes GTP-induced, GTP-specific polymerization dependent on Mg²⁺ ions, similar to full-length MtFtsZ. The mutant did not polymerize with ATP or without GTP, ruling out non-specific aggregation. These results indicate the truncated protein maintains proper folding including the critical T7 loop required for GTPase activity. The findings suggest that the first 210 amino acids of MtFtsZ are sufficient to confer GTP hydrolysis and magnesium-dependent, GTP-specific polymerization activities, demonstrating that the C-terminal region moderates polymerization kinetics rather than being essential for these enzymatic functions.
Key findings
- MtFtsZ-DC169 truncated mutant exhibits GTPase activity at 97±2 nmol GTP h⁻¹(mg protein)⁻¹, demonstrating the T7 loop maintains proper conformation despite C-terminal deletion
- The mutant shows GTP-induced, GTP-specific polymerization dependent on Mg²⁺ ions, with no polymerization occurring with ATP or without nucleotide
- The first 210 amino acids of MtFtsZ are sufficient to confer both GTPase and polymerization activities, indicating the C-terminal region modulates rather than enables these functions
- Polymerization is true protofilament formation rather than protein aggregation, as evidenced by specific biochemical assays
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Abstract
We recently reported that the deletion mutant MtFtsZ-ΔC169 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).