Magnesium-induced Linear Self-association of the FtsZ Bacterial Cell Division Protein Monomer

Abstract

The bacterial cell division protein FtsZ from Escherichia coli has been purified with a new calcium precipitation method. The protein contains one GDP and one Mg 2؉ bound, it shows GTPase activity, and requires GTP and Mg 2؉ to polymerize into long thin filaments at pH 6.5. FtsZ, with moderate ionic strength and low Mg 2؉ concentrations, at pH 7.5, is a compact and globular monomer. Mg 2؉ induces FtsZ self-association into oligomers, which has been studied by sedimentation equilibrium over a wide range of Mg 2؉ and FtsZ concentrations. The oligomer formation mechanism is best described as an indefinite self-association, with binding of an additional Mg 2؉ for each FtsZ monomer added to the growing oligomer, and a slight gradual decrease of the affinity of addition of a protomer with increasing oligomer size. The sedimentation velocity of FtsZ oligomer populations is compatible with a linear singlestranded arrangement of FtsZ monomers and a spacing of 4 nm. It is proposed that these FtsZ oligomers and the polymers formed under assembly conditions share a similar axial interaction between monomers (like in the case of tubulin, the eukaryotic homolog of FtsZ). Similar mechanisms may apply to FtsZ assembly in vivo, but additional factors, such as macromolecular crowding, nucleoid occlusion, or specific interactions with other cellular components active in septation have to be invoked to explain FtsZ assembly into a division ring.The FtsZ protein, which is found in all known eubacteria and archaea as well as in chloroplasts with the exception of the Chlamydiae (1, 2), plays a central role in bacterial cell division (3-5). During the life of a cell, FtsZ molecules remain in the cytoplasm until the time previous to division when they localize at the midcell point, forming a ring at the inner face of the cytoplasmic membrane where the septum will form (6, 7). The interactions of FtsZ with other septator proteins that are functionally relevant, such as PBP3 and FtsA, are being discovered (reviewed in Refs. 4,5,8,and 9). Several regulators of FtsZ ring formation have been found that either prevent/inhibit polymer formation (like the SOS response protein SulA (10), the MinCDE complex (11), and more recently, the ErzA protein from Bacillus subtilis (12)) or stabilize the FtsZ polymer (like the membrane protein ZipA (13, 14)). Despite this information, FtsZ ring structure and assembly are still poorly understood.FtsZ binds guanine nucleotides and has GTPase activity (15-17), which is dependent on protein concentration and probably is linked to self-association equilibria (15,16,18). Multiple sequence alignment and secondary structure predictions indicate that bacterial FtsZ is a homolog of eukaryotic tubulins (19,20). The x-ray crystal structure of the FtsZ from the archaeon Methanococcus jannaschii (21) and the electron crystallographic model structure of the ␣␤-tubulin dimer (22) have similar folds.The nature of the polymers that FtsZ forms in vivo is unknown. FtsZ self-assembles in vitro to form filaments and rings (23-28...

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

“…Careful biophysical characterization has previously allowed the elucidation of isodemic self‐association behavior for several other proteins, including insulin (Jeffrey et al , 1976), FtsZ (Rivas et al , 2000), Phage ϕ29 protein p6 (Abril et al , 1997), chicken deoxy hemoglobin D (Rana & Riggs, 2011), human apolipoprotein C‐II (Yang et al , 2012), tubulin (Frigon & Timasheff, 1975), and for the assembly of heterochromatin protein 1 with nucleosomes in controlling heterochromatin spread (Canzio et al , 2013). Isodesmic self‐association may be a general mechanism for creating large, labile polymers that can disaggregate readily when required.…”

“…Some of these assemblies form via nucleation‐driven polymerization and result in stable complexes, and we are starting to understand the structural basis for their proximity‐enhanced activation (Korennykh et al , 2009; Yin et al , 2009; Li et al , 2012a; Lu et al , 2014). Other assemblies depend on transient interactions and are more labile, generating oligomers with broad size distributions (Rivas et al , 2000; Errington et al , 2012; Canzio et al , 2013). The functions of these more labile higher‐order oligomers are not well understood.…”

FtsZ and the division of prokaryotic cells and organelles

“…Most methods for monitoring FtsZ assembly indicate that the assembly of protofilaments is COOPERATIVE, with a defined critical concentration for assembly 33,34 . However, the presence of only one binding face for a new monomer to attach to an existing protofilament indicates an ISODESMIC ASSEMBLY mechanism 35,36 , as does the appearance of single protofilaments in electron micrographs of assembled FtsZ in the absence of bundling factors. How can the apparent cooperativity of assembly be rationalized with the microscopic evidence of single-stranded protofilaments?…”

Dynamic FtsZ polymerization is sensitive to the GTP to GDP ratio and can be maintained at steady state using a GTP-regeneration system

“…In the presence of GDP (Rivas et al, 2000(Rivas et al, , 2001Sossong et al, 1999) or in the presence of GTP under particular conditions where only single protofilaments are formed (Romberg et al, 2001), polymerization is proposed to be isodesmic. Under conditions where FtsZ forms more complex polymers, polymerization and the GTPase activity are co-operative (Caplan & Erickson, 2003;Mukherjee & Lutkenhaus, 1999;White et al, 2000).…”

“…Since GTP hydrolysis has been shown to occur immediately upon FtsZ polymerization , GDP levels will increase throughout the reaction, as GTP levels decrease. The effects of these changes in nucleotide concentration as the assay progresses are likely to be complex since GDP-bound and GTP-bound FtsZ molecules have different polymerization characteristics (Mukherjee & Lutkenhaus, 1999;Rivas et al, 2000Rivas et al, , 2001Romberg et al, 2001; Sossong et al, 1999). Indeed, previous work has demonstrated that addition of GDP in at least 10 times excess (compared to GTP concentration) causes rapid depolymerization of FtsZ/FtsZ2 co-polymers which are [GDP] ratio reduces both the length of the FtsZ polymerization reaction (which we designate t 0?5 ) and the initial light-scattering maximum (delta).…”