MinD directly interacting with FtsZ at the H10 helix suggests a model for robust activation of MinC to destabilize FtsZ polymers

Cell division in bacteria is a highly controlled and regulated process. FtsZ, a bacterial cytoskeletal protein, forms a ring-like structure known as the Z-ring and recruits more than a dozen other cell division proteins. The Min system oscillates between the poles and inhibits the Z-ring formation at the poles by perturbing FtsZ assembly. This leads to an increase in the FtsZ concentration at the mid-cell and helps in Z-ring positioning. MinC, the effector protein, interferes with Z-ring formation through two different mechanisms mediated by its two domains with the help of MinD. However, the mechanism by which MinD triggers MinC activity is not yet known. We showed that MinD directly interacts with FtsZ with an affinity stronger than the reported MinC–FtsZ interaction. We determined the MinD-binding site of FtsZ using computational, mutational and biochemical analyses. Our study showed that MinD binds to the H10 helix of FtsZ. Single-point mutations at the charged residues in the H10 helix resulted in a decrease in the FtsZ affinity towards MinD. Based on our findings, we propose a novel model for MinCD–FtsZ interaction, where MinD through its direct interaction with FtsZ would trigger MinC activity to inhibit FtsZ functions.

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Escherichia coli Division Inhibitor MinCD Blocks Septation by Preventing Z-Ring Formation

Journal of Bacteriology ◽

10.1128/jb.183.22.6630-6635.2001 ◽

2001 ◽

Vol 183 (22) ◽

pp. 6630-6635 ◽

Cited By ~ 89

Author(s):

Sebastien Pichoff ◽

Joe Lutkenhaus

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Direct Interaction ◽

Ring Formation ◽

Ring Assembly ◽

Z Ring ◽

Alternative Fixation ◽

Green Fluorescent

ABSTRACT The min system spatially regulates division through the topological regulation of MinCD, an inhibitor of cell division. MinCD was previously shown to inhibit division by preventing assembly of the Z ring (E. Bi and J. Lutkenhaus, J. Bacteriol. 175:1118–1125, 1993); however, this was questioned in a recent report (S. S. Justice, J. Garcia-Lara, and L. I. Rothfield, Mol. Microbiol. 37:410–423, 2000) which indicated that MinCD acted after Z-ring formation and prevented the recruitment of FtsA to the Z ring. This discrepancy was due in part to alternative fixation conditions. We have therefore reinvestigated the action of MinCD and avoided fixation by using green fluorescent protein (GFP) fusions to division proteins. MinCD prevented the localization of both FtsZ-GFP and ZipA-GFP, consistent with it preventing Z-ring assembly. Consistent with a direct interaction between FtsZ and the MinCD inhibitor, we find that increased FtsZ, but not FtsA, suppresses MinCD-induced lethality. Furthermore, strains carrying various alleles offtsZ, selected on the basis of resistance to the inhibitor SulA, displayed variable resistance to MinCD. These results are consistent with FtsZ as the target of MinCD and confirm that this inhibitor prevents Z-ring assembly.

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Regulation of Virus Neutralization and the Persistent Fraction by TRIM21

Journal of Virology ◽

10.1128/jvi.00728-12 ◽

2012 ◽

Vol 86 (16) ◽

pp. 8482-8491 ◽

Cited By ~ 53

Author(s):

W. A. McEwan ◽

F. Hauler ◽

C. R. Williams ◽

S. R. Bidgood ◽

D. L. Mallery ◽

...

Keyword(s):

Single Point ◽

Point Mutations ◽

Direct Interaction ◽

Neutralizing Antibody ◽

Adenovirus Type ◽

Antibody Neutralization ◽

Human Virus ◽

Single Point Mutations ◽

Adenovirus Type 5 ◽

Insight Into

Despite a central role in immunity, antibody neutralization of virus infection is poorly understood. Here we show how the neutralization and persistence of adenovirus type 5, a prevalent nonenveloped human virus, are dependent upon the intracellular antibody receptor TRIM21. Cells with insufficient amounts of TRIM21 are readily infected, even at saturating concentrations of neutralizing antibody. Conversely, high TRIM21 expression levels decrease the persistent fraction of the infecting virus and allows neutralization by as few as 1.6 antibody molecules per virus. The direct interaction between TRIM21 and neutralizing antibody is essential, as single-point mutations within the TRIM21-binding site in the Fc region of a potently neutralizing antibody impair neutralization. However, infection at high multiplicity can saturate TRIM21 and overcome neutralization. These results provide insight into the mechanism and importance of a newly discovered, effector-driven process of antibody neutralization of nonenveloped viruses.

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New Temperature-Sensitive Alleles of ftsZ in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.187.1.358-365.2005 ◽

2005 ◽

Vol 187 (1) ◽

pp. 358-365 ◽

Cited By ~ 18

Author(s):

Stephen G. Addinall ◽

Elaine Small ◽

Duncan Whitaker ◽

Shane Sturrock ◽

William D. Donachie ◽

...

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Point Mutations ◽

Ring Formation ◽

Single Amino Acid ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Nonpermissive Temperature ◽

Z Ring

ABSTRACT We isolated five new temperature-sensitive alleles of the essential cell division gene ftsZ in Escherichia coli, using P1-mediated, localized mutagenesis. The five resulting single amino acid changes (Gly109→Ser109 for ftsZ6460, Ala129→Thr129 for ftsZ972, Val157→Met157 for ftsZ2066, Pro203→Leu203 for ftsZ9124, and Ala239→Val239 for ftsZ2863) are distributed throughout the FtsZ core region, and all confer a lethal cell division block at the nonpermissive temperature of 42°C. In each case the division block is associated with loss of Z-ring formation such that fewer than 2% of cells show Z rings at 42°C. The ftsZ9124 and ftsZ6460 mutations are of particular interest since both result in abnormal Z-ring formation at 30°C and therefore cause significant defects in FtsZ polymerization, even at the permissive temperature. Neither purified FtsZ9124 nor purified FtsZ6460 exhibited polymerization when it was assayed by light scattering or electron microscopy, even in the presence of calcium or DEAE-dextran. Hence, both mutations also cause defects in FtsZ polymerization in vitro. Interestingly, FtsZ9124 has detectable GTPase activity, although the activity is significantly reduced compared to that of the wild-type FtsZ protein. We demonstrate here that unlike expression of ftsZ84, multicopy expression of the ftsZ6460, ftsZ972, and ftsZ9124 alleles does not complement the respective lethalities at the nonpermissive temperature. In addition, all five new mutant FtsZ proteins are stable at 42°C. Therefore, the novel isolates carrying single ftsZ(Ts) point mutations, which are the only such strains obtained since isolation of the classical ftsZ84 mutation, offer significant opportunities for further genetic characterization of FtsZ and its role in cell division.

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FtsZ from Divergent Foreign Bacteria Can Function for Cell Division in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00647-06 ◽

2006 ◽

Vol 188 (20) ◽

pp. 7132-7140 ◽

Cited By ~ 31

Author(s):

Masaki Osawa ◽

Harold P. Erickson

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Point Mutations ◽

Loss Of Function ◽

Mycoplasma Pulmonis ◽

E Coli ◽

Z Ring ◽

Ftsz Assembly

ABSTRACT FtsZs from Mycoplasma pulmonis (MpuFtsZ) and Bacillus subtilis (BsFtsZ) are only 46% and 53% identical in amino acid sequence to FtsZ from Escherichia coli (EcFtsZ). In the present study we show that MpuFtsZ and BsFtsZ can function for cell division in E. coli provided we make two modifications. First, we replaced their C-terminal tails with that from E. coli, giving the foreign FtsZ the binding site for E. coli FtsA and ZipA. Second, we selected for mutations in the E. coli genome that facilitated division by the foreign FtsZs. These suppressor strains arose at a relatively high frequency of 10−3 to 10−5, suggesting that they involve loss-of-function mutations in multigene pathways. These pathways may be negative regulators of FtsZ or structural pathways that facilitate division by slightly defective FtsZ. Related suppressor strains were obtained for EcFtsZ containing certain point mutations or insertions of yellow fluorescent protein. The ability of highly divergent FtsZs to function for division in E. coli is consistent with a two-part mechanism. FtsZ assembles the Z ring, and perhaps generates the constriction force, through self interactions; the downstream division proteins remodel the peptidoglycan wall by interacting with each other and the wall. The C-terminal peptide of FtsZ, which binds FtsA, provides the link between FtsZ assembly and peptidoglycan remodeling.

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An oscillating MinD protein determines the cellular positioning of the motility machinery in archaea

10.1101/2020.04.03.021790 ◽

2020 ◽

Cited By ~ 3

Author(s):

Phillip Nußbaum ◽

Solenne Ithurbide ◽

James C. Walsh ◽

Megha Patro ◽

Floriane Delpech ◽

...

Keyword(s):

Cell Division ◽

Point Mutations ◽

Sister Group ◽

Atpase Domain ◽

Homologous Proteins ◽

Cell Axis ◽

E Coli ◽

Self Organized ◽

Z Ring ◽

The Mind

MinD proteins are well studied in rod-shaped bacteria such as E. coli, where they display self-organized pole-to-pole oscillations that are important for correct positioning of the Z-ring at mid-cell for cell division. Archaea also encode proteins belonging to the MinD family, but their functions are unknown. MinD homologous proteins were found to be widespread in Euryarchaeota and form a sister group to the bacterial MinD family, distinct from the ParA and other related ATPase families. We aimed to identify the function of four archaeal MinD proteins in the model archaeon Haloferax volcanii. Deletion of the minD genes did not cause cell division or size defects, and the Z-ring was still correctly positioned. Instead, one of the mutations (ΔminD4) reduced swimming motility, and hampered the correct formation of motility machinery at the cell poles. In ΔminD4 cells, there is reduced formation of the motility structure and chemosensory arrays, which are essential for signal transduction. In bacteria, several members of the ParA family can position the motility structure and chemosensory arrays via binding to a landmark protein, and consequently these proteins do not oscillate along the cell axis. However, GFP-MinD4 displayed pole-to-pole oscillation and formed polar patches or foci in H. volcanii. The MinD4 membrane targeting sequence (MTS), homologous to the bacterial MinD MTS, was essential for the oscillation. Surprisingly, MinD4 ATPase domain point-mutations did not block oscillation, but they failed to form pole-patches. Thus, MinD4 from H. volcanii combines traits of different bacterial ParA/MinD proteins.

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Cupin Variants as Macromolecular Ligand Library for Stereoselective Michael Addition of Nitroalkanes

10.26434/chemrxiv.11481834.v1 ◽

2019 ◽

Author(s):

Nobutaka Fujieda ◽

Miho Yuasa ◽

Yosuke Nishikawa ◽

Genji Kurisu ◽

Shinobu Itoh ◽

...

Keyword(s):

Michael Addition ◽

In Silico ◽

Addition Reaction ◽

Single Point ◽

Point Mutations ◽

Unsaturated Ketone ◽

Michael Addition Reaction ◽

Beta Barrel ◽

Cupin Superfamily ◽

Single Point Mutations

Cupin superfamily proteins (TM1459) work as a macromolecular ligand framework with a double-stranded beta-barrel structure ligating to a Cu ion through histidine side chains. Variegating the first coordination sphere of TM1459 revealed that H52A and H54A/H58A mutants effectively catalyzed the diastereo- and enantio-selective Michael addition reaction of nitroalkanes to an α,β-unsaturated ketone. Moreover, in silico substrate docking signified C106N and F104W single-point mutations, which inverted the diastereoselectivity of H52A and further improved the stereoselectivity of H54A/H58A, respectively.

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