Cysteine 155 plays an important role in the assembly of Mycobacterium tuberculosis FtsZ.
The assembly of FtsZ plays an important role in bacterial cell division. Mycobacterium tuberculosis FtsZ (MtbFtsZ) has a single cysteine residue at position 155. We have investigated the role of the lone cysteine residue in the assembly of MtbFtsZ using different complimentary approaches, namely chemical modification by a thiol-specific reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) or a cysteine-chelating agent HgCl(2), and site-directed mutagenesis of the cysteine residue. HgCl(2) strongly reduced the polymerized mass of MtbFtsZ while it had no detectable effect on the polymerization of Escherichia coli FtsZ, which lacks a cysteine residue. HgCl(2) inhibited the protofilamentous assembly of MtbFtsZ and induced the aggregation of the protein. Further, HgCl(2) perturbed the secondary structure of MtbFtsZ and increased the binding of a hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) with MtbFtsZ, indicating that the binding of HgCl(2) altered the conformation of MtbFtsZ. Chemical modification of MtbFtsZ by DTNB also decreased the polymerized mass of MtbFtsZ. Further, the mutagenesis of Cys-155 to alanine caused a strong reduction in the assembly of MtbFtsZ. Under assembly conditions, the mutated protein formed aggregates instead of protofilaments. Far-UV CD spectroscopy and ANS binding suggested that the mutated MtbFtsZ has different conformation than that of the native MtbFtsZ. The effect of the mutation or chemical modification of Cys-155 on the MtbFtsZ assembly has been explained considering its location in the MtbFtsZ crystal structure. The results together suggest that the cysteine residue (Cys-155) of MtbFtsZ plays an important role in the assembly of MtbFtsZ into protofilaments.
Jaiswal
Richa
R
School of Biosciences and Bioengineering, Indian Institute of Technology, Bombay, Powai, Mumbai 400076, India.
Panda
Dulal
D
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002614:Chelating Agents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004136:Dinitrobenzenes / Q000737:chemistry; D008627:Mercuric Chloride / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D017433:Protein Structure, Secondary / Q000235:genetics
D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins; D002614:Chelating Agents; D003598:Cytoskeletal Proteins; D004136:Dinitrobenzenes; C071525:FtsZ protein, Bacteria; D008627:Mercuric Chloride; C027132:1-anilino-8-naphthalenesulfonate; C010936:2,4-dinitrothiocyanatobenzene; D003545:Cysteine