Control by Potassium of the Size Distribution of Escherichia coli FtsZ Polymers Is Independent of GTPase Activity

The influence of potassium content (at neutral pH and millimolar Mg2+) on the size distribution of FtsZ polymers formed in the presence of constantly replenished GTP under steady-state conditions was studied by a combination of biophysical methods. The size of the GTP-FtsZ polymers decreased with lower potassium concentration, in contrast with the increase in the mass of the GDP-FtsZ oligomers, whereas no effect was observed on FtsZ GTPase activity and critical concentration of polymerization. Remarkably, the concerted formation of a narrow size distribution of GTP-FtsZ polymers previously observed at high salt concentration was maintained in all KCl concentrations tested. Polymers induced with guanosine 5′-(α,β-methylene)triphosphate, a slowly hydrolyzable analog of GTP, became larger and polydisperse as the potassium concentration was decreased. Our results suggest that the potassium dependence of the GTP-FtsZ polymer size may be related to changes in the subunit turnover rate that are independent of the GTP hydrolysis rate. The formation of a narrow size distribution of FtsZ polymers under very different solution conditions indicates that it is an inherent feature of FtsZ, not observed in other filament-forming proteins, with potential implications in the structural organization of the functional Z-ring.

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Multi-functional regulator MapZ controls both positioning and timing of FtsZ polymerization

Biochemical Journal ◽

10.1042/bcj20190138 ◽

2019 ◽

Vol 476 (10) ◽

pp. 1433-1444 ◽

Cited By ~ 4

Author(s):

Zhang Feng ◽

Jiahai Zhang ◽

Da Xu ◽

Yong-Liang Jiang ◽

Cong-Zhao Zhou ◽

...

Keyword(s):

Critical Concentration ◽

Dynamic State ◽

Protein Z ◽

Gtp Hydrolysis ◽

Intracellular Domain ◽

Precise Positioning ◽

Daughter Cells ◽

Division Site ◽

Z Ring ◽

Key Residues

AbstractThe tubulin-like GTPase protein FtsZ, which forms a discontinuous cytokinetic ring at mid-cell, is a central player to recruit the division machinery to orchestrate cell division. To guarantee the production of two identical daughter cells, the assembly of FtsZ, namely Z-ring, and its precise positioning should be finely regulated. In Streptococcus pneumoniae, the positioning of Z-ring at the division site is mediated by a bitopic membrane protein MapZ (mid-cell-anchored protein Z) through direct interactions between the intracellular domain (termed MapZ-N (the intracellular domain of MapZ)) and FtsZ. Using nuclear magnetic resonance titration experiments, we clearly assigned the key residues involved in the interactions. In the presence of MapZ-N, FtsZ gains a shortened activation delay, a lower critical concentration for polymerization and a higher cooperativity towards GTP hydrolysis. On the other hand, MapZ-N antagonizes the lateral interactions of single-stranded filaments of FtsZ, thus slows down the formation of highly bundled FtsZ polymers and eventually maintains FtsZ at a dynamic state. Altogether, we conclude that MapZ is not only an accelerator to trigger the polymerization of FtsZ, but also a brake to tune the velocity to form the end-product, FtsZ bundles. These findings suggest that MapZ is a multi-functional regulator towards FtsZ that controls both the precise positioning and proper timing of FtsZ polymerization.

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Characterization of GTPase Activity of TrmE, a Member of a Novel GTPase Superfamily, from Thermotoga maritima

Journal of Bacteriology ◽

10.1128/jb.182.24.7078-7082.2000 ◽

2000 ◽

Vol 182 (24) ◽

pp. 7078-7082 ◽

Cited By ~ 40

Author(s):

Kunitoshi Yamanaka ◽

Jihwan Hwang ◽

Masayori Inouye

Keyword(s):

Thermotoga Maritima ◽

Binding Protein ◽

Protein A ◽

Hydrolysis Rate ◽

Gtpase Activity ◽

Gtp Binding Protein ◽

Gene Encoding ◽

Gtp Hydrolysis ◽

Gtp Binding

ABSTRACT A gene encoding a putative GTP-binding protein, a TrmE homologue that is highly conserved in both prokaryotes and eukaryotes, was cloned from Thermotoga maritima, a hyperthermophilic bacterium.T. maritima TrmE was overexpressed in Escherichia coli and purified. TrmE has a GTPase activity but no ATPase activity. The GTPase activity can be competed with GTP, GDP, and dGTP but not with GMP, ATP, CTP, or UTP. Km andk cat at 70°C were 833 μM and 9.3 min−1, respectively. Our results indicate that TrmE is a GTP-binding protein with a very high intrinsic GTP hydrolysis rate. We also propose that TrmE homologues constitute a novel subfamily of the GTPase superfamily.

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Allosteric modulation of the GTPase activity of a bacterial LRRK2 homolog by conformation-specific Nanobodies

Biochemical Journal ◽

10.1042/bcj20190843 ◽

2020 ◽

Vol 477 (7) ◽

pp. 1203-1218 ◽

Cited By ~ 3

Author(s):

Margaux Leemans ◽

Christian Galicia ◽

Egon Deyaert ◽

Elise Daems ◽

Linda Krause ◽

...

Keyword(s):

Turnover Rate ◽

Kinase Activity ◽

Kinase Inhibitors ◽

Bound State ◽

Allosteric Modulation ◽

Gtpase Activity ◽

Gtp Hydrolysis ◽

Lrrk2 Kinase ◽

Dimeric Form ◽

Novel Strategy

Mutations in the Parkinson's disease (PD)-associated protein leucine-rich repeat kinase 2 (LRRK2) commonly lead to a reduction of GTPase activity and increase in kinase activity. Therefore, strategies for drug development have mainly been focusing on the design of LRRK2 kinase inhibitors. We recently showed that the central RocCOR domains (Roc: Ras of complex proteins; COR: C-terminal of Roc) of a bacterial LRRK2 homolog cycle between a dimeric and monomeric form concomitant with GTP binding and hydrolysis. PD-associated mutations can slow down GTP hydrolysis by stabilizing the protein in its dimeric form. Here, we report the identification of two Nanobodies (NbRoco1 and NbRoco2) that bind the bacterial Roco protein (CtRoco) in a conformation-specific way, with a preference for the GTP-bound state. NbRoco1 considerably increases the GTP turnover rate of CtRoco and reverts the decrease in GTPase activity caused by a PD-analogous mutation. We show that NbRoco1 exerts its effect by allosterically interfering with the CtRoco dimer–monomer cycle through the destabilization of the dimeric form. Hence, we provide the first proof of principle that allosteric modulation of the RocCOR dimer–monomer cycle can alter its GTPase activity, which might present a potential novel strategy to overcome the effect of LRRK2 PD mutations.

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Assembly of an FtsZ Mutant Deficient in GTPase Activity Has Implications for FtsZ Assembly and the Role of the Z Ring in Cell Division

Journal of Bacteriology ◽

10.1128/jb.183.24.7190-7197.2001 ◽

2001 ◽

Vol 183 (24) ◽

pp. 7190-7197 ◽

Cited By ~ 63

Author(s):

Amit Mukherjee ◽

Cristian Saez ◽

Joe Lutkenhaus

Keyword(s):

Cell Division ◽

Critical Concentration ◽

Gtpase Activity ◽

Support Cell ◽

Z Ring ◽

Ftsz Assembly ◽

The Stability

ABSTRACT FtsZ, the ancestral homologue of eukaryotic tubulins, assembles into the Z ring, which is required for cytokinesis in prokaryotic cells. Both FtsZ and tubulin have a GTPase activity associated with polymerization. Interestingly, the ftsZ2 mutant is viable, although the FtsZ2 mutant protein has dramatically reduced GTPase activity due to a glycine-for-aspartic acid substitution within the synergy loop. In this study, we have examined the properties of FtsZ2 and found that the reduced GTPase activity is not enhanced by DEAE-dextran-induced assembly, indicating it has a defective catalytic site. In the absence of DEAE-dextran, FtsZ2 fails to assemble unless supplemented with wild-type FtsZ. FtsZ has to be at or above the critical concentration for copolymerization to occur, indicating that FtsZ is nucleating the copolymers. The copolymers formed are relatively stable and appear to be stabilized by a GTP-cap. These results indicate that FtsZ2 cannot nucleate assembly in vitro, although it must in vivo. Furthermore, the stability of FtsZ-FtsZ2 copolymers argues that FtsZ2 polymers would be stable, suggesting that stable FtsZ polymers are able to support cell division.

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In vitro assembly and GTP hydrolysis by bacterial tubulins BtubA and BtubB

The Journal of Cell Biology ◽

10.1083/jcb.200410027 ◽

2005 ◽

Vol 169 (2) ◽

pp. 233-238 ◽

Cited By ~ 59

Author(s):

Christopher A. Sontag ◽

James T. Staley ◽

Harold P. Erickson

Keyword(s):

Critical Concentration ◽

Three Dimensional ◽

Outer Diameter ◽

Gtpase Activity ◽

Gtp Hydrolysis ◽

Assembly Mechanism ◽

In Vitro Assembly ◽

Cooperative Assembly ◽

Versus Concentration

Arecent study identified genuine tubulin proteins, BtubA and BtubB, in the bacterial genus Prosthecobacter. We have expressed BtubA and BtubB in Escherichia coli and studied their in vitro assembly. BtubB by itself formed rings with an outer diameter of 35–36 nm in the presence of GTP or GDP. Mixtures of BtubB and BtubA formed long protofilament bundles, 4–7 protofilaments wide (20–30 protofilaments in the three-dimensional bundle). Regardless of the starting stoichiometry, the polymers always contained equal concentrations of BtubA and BtubB, suggesting that BtubA and B alternate along the protofilament. BtubA showed negligible GTP hydrolysis, whereas BtubB hydrolyzed 0.40 mol GTP per min per mol BtubB. This GTPase activity increased to 1.37 per min when mixed 1:1 with BtubA. A critical concentration of 0.4–1.0 μM was indicated by light scattering experiments and extrapolation of GTPase versus concentration, thus suggesting a cooperative assembly mechanism.

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Investigation of Regulation of FtsZ Assembly by SulA and Development of a Model for FtsZ Polymerization

Journal of Bacteriology ◽

10.1128/jb.01612-07 ◽

2008 ◽

Vol 190 (7) ◽

pp. 2513-2526 ◽

Cited By ~ 89

Author(s):

Alex Dajkovic ◽

Amit Mukherjee ◽

Joe Lutkenhaus

Keyword(s):

Kinetic Scheme ◽

Sos Response ◽

Ring Structure ◽

Linear Polymers ◽

Gtpase Activity ◽

Gtp Hydrolysis ◽

Z Ring ◽

Ftsz Assembly

ABSTRACT In Escherichia coli FtsZ organizes into a cytoskeletal ring structure, the Z ring, which effects cell division. FtsZ is a GTPase, but the free energy of GTP hydrolysis does not appear to be used for generation of the constriction force, leaving open the question of the function of the GTPase activity of FtsZ. Here we study the mechanism by which SulA, an inhibitor of FtsZ induced during the SOS response, inhibits FtsZ function. We studied the effects of SulA on the in vitro activities of FtsZ, on Z rings in vivo, and on a kinetic model for FtsZ polymerization in silico. We found that the binding of SulA to FtsZ is necessary but not sufficient for inhibition of polymerization, since the assembly of FtsZ polymers in the absence of the GTPase activity was not inhibited by SulA. We developed a new model for FtsZ polymerization that accounts for the cooperativity of FtsZ and could account for cooperativity observed in other linear polymers. When SulA was included in the kinetic scheme, simulations revealed that SulA with strong affinity for FtsZ delayed, but did not prevent, the assembly of polymers when they were not hydrolyzing GTP. Furthermore, the simulations indicated that SulA controls the assembly of FtsZ by binding to a polymerization-competent form of the FtsZ molecule and preventing it from participating in assembly. In vivo stoichiometry of the disruption of Z rings by SulA suggests that FtsZ may undergo two cooperative transitions in forming the Z ring.

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Macrostructure and microphysics of Saturn's E-ring

International Astronomical Union Colloquium ◽

10.1017/s0252921100101691 ◽

1984 ◽

Vol 75 ◽

pp. 607-613 ◽

Cited By ~ 2

Author(s):

Kevin D. Pang ◽

Charles C. Voge ◽

Jack W. Rhoads

Keyword(s):

Size Distribution ◽

Spherical Shape ◽

Mie Scattering ◽

Narrow Size Distribution ◽

Electrostatic Levitation ◽

Volcanic Origin ◽

Micron Diameter

Abstract.All observed optical and infrared properties of Saturn's E-ring can be explained in terms of Mie scattering by a narrow size distribution of ice spheres of 2 - 2.5 micron diameter. The spherical shape of the ring particles and their narrow size distribution imply a molten (possibly volcanic) origin on Enceladus. The E-ring consists of many layers, possibly stratified by electrostatic levitation.

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