Energetics of the Cooperative Assembly of Cell Division Protein FtsZ and the Nucleotide Hydrolysis Switch

Abstract

FtsZ is the first protein recruited to the bacterial division site, where it forms the cytokinetic Z ring. We have determined the functional energetics of FtsZ assembly, employing FtsZ from the thermophilic Archaea Methanococcus jannaschii bound to GTP, GMPCPP, GDP, or GMPCP, under different solution conditions. FtsZ oligomerizes in a magnesium-insensitive manner. FtsZ cooperatively assembles with magnesium and GTP or GMPCPP into large polymers, following a nucleated condensation polymerization mechanism, under nucleotide hydrolyzing and non-hydrolyzing conditions. The effect of temperature on the critical concentration indicates polymer elongation with an apparent heat capacity change of ؊800 ؎ 100 cal mol ؊1 K ؊1 and positive enthalpy and entropy changes, compatible with axial hydrophobic contacts of each FtsZ in the polymer, and predicts optimal polymer stability near 75°C. Assembly entails the binding of one medium affinity magnesium ion and the uptake of one proton per FtsZ. Interestingly, GDP-or GMPCP-liganded FtsZ cooperatively form helically curved polymers, with an elongation only 1-2 kcal mol ؊1 more unfavorable than the straight polymers formed with nucleotide triphosphate, suggesting a physiological requirement for FtsZ polymerization inhibitors. This GTP hydrolysis switch should provide the basic properties for FtsZ polymer disassembly and its functional dynamics.Both prokaryotic and eukaryotic cells use dynamic protein assemblies to fulfill central roles in DNA segregation, cell division, cell shape, and polarity (1-3). Several of these are cytoskeletal assemblies made of nucleotide-binding proteins, which share homologous frameworks, indicating that they have evolved from common ancestors. Thus tubulin (4), the main component of the microtubules of the mitotic spindle that segregates chromosomes, and FtsZ (5), which forms the Z-ring directing bacterial cell division, share the same fold in two of their domains and constitute a distinct family of assembling GTPases (6). Actin (7), which assembles into microfilaments, is a homologue of MreB (8), which assembles into similar filaments determining bacterial cell shape. Other prokaryotic members of the actin family of ATP-binding proteins include ParM, which assembles into actin-like filaments responsible for plasmid segregation (9), and FtsA, which is thought to anchor FtsZ to the membrane and recruit downstream cell division proteins (10).To understand how each of the bacterial protein machines works (11,12), in addition to their in vivo partner proteins, their structures and those of their polymers, a better knowledge is required of the functional energetics, the kinetics and mechanism of their assembly, and of how these may be modulated in the cytosolic environment. FtsZ, the homologue of eukaryotic tubulin, is ubiquitous in Eubacteria, Archaea, and chloroplast. Green fluorescent protein-fused FtsZ has a rapid assembly dynamics in the bacterial Z-ring, comparable to tubulin in a mitotic spindle (13,14). Purified FtsZ forms a number of different pol...

FtsZ and the division of prokaryotic cells and organelles

“…By contrast, microtubule protofilaments consist mostly of GDP-tubulin with a GTP cap and are susceptible to rapid depolymerization once the cap is hydrolysed 30 . Interestingly, GDP can allow FtsZ assembly as well, resulting in curved polymers 31,32 . The potential significance of such polymers in vivo is unclear, especially if nucleotide exchange from the large available pool of GTP is sufficiently rapid to saturate most FtsZ with GTP.…”

Mapping Flexibility and the Assembly Switch of Cell Division Protein FtsZ by Computational and Mutational Approaches

“…Protein and FtsZ-bound nucleotide were spectrophotometrically quantified (13). All FtsZs contained bound nucleotide (mutants, 0.22-0.88 guanine per FtsZ; wild type, 0.26 -0.44).…”

“…The FtsZ mutants were overproduced in Escherichia coli BL21(DE3)pLys, and purified by fast protein liquid chromatography using anionic exchange and subsequent hydrophobic and desalting chromatographies as described (11,13) with yields of 3-15 mg of protein/liter of culture. Protein and FtsZ-bound nucleotide were spectrophotometrically quantified (13).…”

“…This indicates the existence of molecular switches in their assembly-disassembly cycle triggered by the presence/absence of the nucleotide ␥-phosphate, which permits polymer regulation. FtsZ polymerizes into tubulin-like protofilaments (10,11) that can be straight or curved in relation to the GTP-or GDP-bound nucleotide (12)(13)(14). GTP binding displaces the balance to the straight filaments and sheet and bundle condensates, whereas GDP-FtsZ filaments tend to be curved and eventually depolymerize.…”

Cooperative behavior of Escherichia coli cell-division protein FtsZ assembly involves the preferential cyclization of long single-stranded fibrils

“…Centrifugation and light-scattering assays carried out by using FtsZ from different organisms indicate that FtsZ assembly proceeds as a highly cooperative process analogous to a first-order transition or condensation (6,8,10,21,22). According to these assays, there exists a critical protein concentration below which all of the protein is soluble; protein present in excess of this critical concentration is present in a distinct fraction that scatters light more intensely and is substantially more sedimentable than the soluble fraction.…”