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Mutation of the Arg191 in FtsZ impairs cytokinetic abscission of Bacillus subtilis cells Hemendra Pal Singh Dhaked, Anusri Bhattacharya, Saroj Yadav, Sarath Chandra Dantu, Ashutosh Kumar, and Dulal Panda Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00493 • Publication Date (Web): 14 Sep 2016 Downloaded from http://pubs.acs.org on September 16, 2016
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Mutation of the Arg191 in FtsZ impairs cytokinetic abscission of Bacillus
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subtilis cells
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Funding source – The work is supported by a grant from Department of Science and
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Technology (DST, India) to DP.
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Hemendra Pal Singh Dhaked1,+, Anusri Bhattacharya1,+, Saroj Yadav2, Sarath Chandra Dantu1,
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Ashutosh Kumar1, & Dulal Panda1,*
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1
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Mumbai, 400076, India
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2
Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay,
IITB-Monash Research Academy, Indian Institute of Technology Bombay, Mumbai, 400076,
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India
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+
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*Corresponding Author - Department of Biosciences and Bioengineering, Indian Institute of
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Technology Bombay, Powai, Mumbai 400076, India. Telephone: 91-22-2576-7838. Fax: 91-22-
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2572-3480. Email:
[email protected].
These authors contributed equally to this work
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Abbreviations
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WT-FtsZ, Wild-type-FtsZ; E. coli, Escherichia coli; M. tuberculosis, Mycobacterium
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tuberculosis; BsFtsZ, Bacillus subtilis FtsZ; S. aureus, Staphylococcus aureus; PIPES,
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piperazine-N,N′-bis(2-ethanesulfonic acid); BSA, bovine serum albumin; GTP, Guanosine 5'-
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Triphosphate; PMSF, Phenylmethanesulfonyl Fluoride; IPTG, Isopropyl-Beta-D-
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Thiogalactoside; FITC, fluorescein isothiocyanate; LB, Luria bertani; HEPES, (4-(2-
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hydroxyethyl)-1-piperazineethanesulfonic acid; SDS-PAGE, sodium dodecyl sulfate
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polyacrylamide gel electrophoresis; CD, Circular dichroism; MD, Molecular dynamics; DSSP,
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Define Secondary Structure of Proteins.
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Abstract
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FtsZ monomers assemble to form a dynamic Z-ring at the mid-cell position in bacteria that co-
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ordinates bacterial cell division. Antibacterial agents, plumbagin, and SB-RA-2001, were found
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to bind to FtsZ and to inhibit Z-ring formation in bacteria. Docking analysis indicated similar
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binding regions for these two inhibitors on FtsZ and the residue R191 was involved in binding
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interaction with both the compounds. In this work, the importance of R191 in FtsZ assembly and
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in bacterial cell division was analyzed. The R191A-FtsZ exhibited significantly reduced
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polymerization ability. Further, the mutant FtsZ could poison the assembly of WT-FtsZ. The
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expression of R191A-FtsZ in Bacillus subtilis strain PL2084 perturbed the Z-ring formation and
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produced filamentous cells indicating that the mutation hindered the division of these cells. The
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results suggested that R191A is a dominant negative mutation of FtsZ. Molecular dynamics
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simulations of R191A-FtsZ and WT-FtsZ revealed a kink in the helices H5 and H7 in the active
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site of R191A-FtsZ as compared to WT-FtsZ, which is required for the FtsZ assembly. The
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findings suggested that R191 is an important residue for FtsZ assembly, which can be targeted
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for designing FtsZ inhibitors.
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In bacteria, the process of cell division is orchestrated by the tubulin homolog FtsZ.1 At the
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center of the dividing cell, FtsZ assembles into a dynamic ring-like structure, called the Z-ring,2
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which initiates the process of cell division. Several accessory proteins are also recruited to the Z-
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ring in a hierarchical manner to form the divisome complex.3-5 A functional FtsZ is indispensable
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for the bacterial cell division. FtsZ consists of two globular domains namely an N-terminal
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domain and a C-terminal domain6 (Fig. 1Aa). The globular domains are connected by a 23 amino
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acid long helical structure, called H7-helix. The N-terminal domain consists of a GTP-binding
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signature sequence (GGGGTS/TG). The role of different domains of FtsZ has been summarized
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in the table S1. The sequence is highly conserved in bacteria and archae. The C-terminal domain
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contains a synergy loop T7, which is thought to be responsible for the hydrolysis of GTP. During
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the formation of the protofilaments, a T7 loop of top FtsZ subunit longitudinal interacts with the
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nucleotide binding pocket of the bottom subunit, which leads to the activation of GTP
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hydrolysis2 (Fig. 1Ab).
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It has been reported that some mutations in FtsZ either perturb the Z-ring assembly or lead to the
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improper localization of the Z-ring during the division process.7-11 Feucht et al. identified nine
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mutants of FtsZ, which led to anomalies in cell division leading to the formation of minicells,
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filamentous cells or cells exhibiting twisted divisions.12 In another study, the effect of 16 site-
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directed mutants of E. coli FtsZ on their assembly and ability to complement the temperature-
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sensitive ftsZ84 mutation in E. coli was analyzed. The mutations positioned on the lateral
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interface could not complement ftsZ84 suggesting that the residues involved in the lateral
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interactions of the protofilaments were essential for an efficient cell division. Interestingly, these
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mutants showed either decreased or increased GTPase activity compared to the WT-FtsZ.13
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Earlier studies have introduced several mutations in E. coli which disrupted the interactions
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between the FtsZ subunits at the longitudinal interface.7-11 Specifically, mutations in the T7-loop
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and in the nearby residues that are involved in longitudinal interactions could not support cell
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division.7 In addition, eight out of thirteen mutations located in the GTP binding site and the
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nearby region of FtsZ also failed to carry out cell division.7 In-vitro, these mutants showed lower
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rates of GTP hydrolysis as compared to the WT-FtsZ. Similarly, an alteration of a single C155 of
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M. tuberculosis FtsZ inhibited the assembly of FtsZ.8 Further, E93R mutation in E. coli FtsZ
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enhanced the assembly of FtsZ. Overexpression of E93R mutant inhibited cell division and
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produced elongated bacterial cells. However, it could complement the function of WT-FtsZ in a
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temperature sensitive complementation system.9 Therefore, the mutations of certain amino acids
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of FtsZ had significant effects on its assembly properties.7-13
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Recently, the identification of point mutations in FtsZ has also gained profound importance in
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the field of antibacterial drug discovery.14,15 Mutations have been identified in FtsZ, which
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renders them insensitive towards potent FtsZ targeted inhibitors. The most prominent amongst
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these small molecule inhibitors is PC190723.15 While PC190723 has been reported to be a
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promising FtsZ inhibitor with anti-staphylococcal activity, the activity of the inhibitor has been
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impaired in some mutant forms of FtsZ, which have been identified through in-silico modeling
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of PC190723 into BsFtsZ structure. PC-190723 binds between the H7 helix and C-terminus of S.
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aureus FtsZ which leads to the stabilization of FtsZ. This adversely affects the GTPase activity
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of S. aureus FtsZ.16,17
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A docking analysis of plumbagin and SB-RA-2001 with BsFtsZ showed that they bind in a cleft
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present between H7 helix and C-terminal domain of FtsZ.18,19 Interestingly, both the molecules
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shared a common residue R191 for the binding to FtsZ (Fig. 1B). R191 is located in the H7 helix
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of FtsZ and it forms a hydrogen bond with both the molecules. Since R191 was involved in
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interaction with both the molecules; we investigated the importance of this residue in the
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functionality of FtsZ. Using multiple sequence alignment, R191 has been found to be partially
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conserved in FtsZ across the bacterial species (Fig. 2). The analysis indicates that R191 is
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conserved in B. subtilis, Staphylococcus aureus, and Clostridium botulinum. In E. coli,
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Methanococcus jannaschii,and Salmonella typhi, the arginine residue is replaced by a lysine
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residue. R191 is not conserved in Agrobacterium radiobacter, Campylobacter coli, and
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Mycobacterium tuberculosis. Campylobacter coli has an alanine residue at the position of R191
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and an arginine residue succeeds to the alanine residue in this organism. It is possible that the
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arginine residue functionally mimics R191 in B. subtilis. Further, an anti-staphylococcal
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compound, PC190723, was reported to interact with the R191 residue of S. aureus FtsZ and a
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mutation of R191 residue was found to be lethal to the bacteria.14. Recently, R191P FtsZ has
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been found to be lethal for B. subtilis strain 16820, while a mutation of K190 in E. coli FtsZ did
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not affect the division of E. coli cells21. Thus, R191 of FtsZ appears to be important in the cell
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division of some bacteria such as B. subtilis and Staphylococcus aureus.
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Interestingly, plumbagin bound to R191A-FtsZ with weaker affinity than WT-FtsZ. Further,
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plumbagin could not significantly inhibit the assembly of R191A-FtsZ suggesting the
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involvement of R191 residue in the binding of plumbagin to FtsZ. Therefore, the role of R191A
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residue in the assembly and GTPase activity of FtsZ was examined by comparing the
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polymerization and GTPase activity of the mutant FtsZ with that of the native FtsZ. To
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understand the effect of this mutation on the FtsZ structure in atomic detail, we performed three
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independent 500 ns long molecular dynamics (MD) simulations each, for both WT-FtsZ and
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R191A-FtsZ. R191A-FtsZ displayed weak polymerization ability as compared to the WT-FtsZ.
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In addition, the mutant hindered the polymerization of WT-FtsZ and also caused
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depolymerization of the preformed WT-FtsZ polymers. In B. subtilis cells, the mutant induced
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extensive elongation of the cells. MD simulations results indicated a kinking of the H5 and H7
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helices of R191A-FtsZ as compared to that of the WT-FtsZ, which further leads to the widening
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of the active site. The change in active site can explain the reduced polymerization activity of
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R191A-FtsZ. This study signifies the importance of R191A point mutation on the assembly of
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FtsZ and bacterial cell division.
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Materials and methods
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Materials
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PIPES, BSA, lysozyme, GTP, and PMSF were purchased from Sigma (St. Louis, MO). IPTG
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was obtained from Calbiochem (Darmstadt, Germany). Ni-NTA resin was procured from Qiagen
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(Hilden, Germany) and Bio-Gel-P6 resin was obtained from Bio-Rad (CA, USA). FITC was
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purchased from Sigma. Analytical grade reagents were used for the study.
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Purification of WT-FtsZ and R191A-FtsZ
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E. coli BL21 (DE3) pLysS strain was transformed with the pET16b vector containing the WT-
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FtsZ construct. The transformed cells were grown in LB media at 37 °C containing 100 µg/ml
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ampicillin and 12.5 µg/ml chloramphenicol and induced at late log phase with 1 mM IPTG for 6
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h. The cells were pelleted down and washed with lysis buffer (50 mM NaH2PO4 (pH 8.0) and
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300 mM NaCl). The cells were suspended in ice-cold lysis buffer containing 0.1% β-ME and 2
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mM PMSF, homogenized on ice for 15 min and then, incubated with lysozyme (1 mg/ml) for an
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additional 1 h on ice. Subsequently, the cell suspension was subjected to sonication (10 pulses of
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1 min each) and centrifuged at 26234×g for 45 min at 4 °C. Imidazole (5 mM) was added to the
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clear cell lysate and then, incubated with activated Ni-NTA resin for 1 h at 4 °C. The resin was
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loaded into the column and the flow through was collected. The resin was washed with wash
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buffers (25 mM PIPES (pH 6.8), 300 mM NaCl) containing increasing concentrations of
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imidazole (25 mM and 50 mM). Finally, the protein was eluted with elution buffer (25 mM
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PIPES (pH 6.8), 300 mM NaCl and 250 mM imidazole) and desalted by passing through Biogel
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P-6 resin, pre-equilibrated with 25 mM PIPES (pH 6.8). The desalted protein was concentrated
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and the protein concentration was estimated by Bradford reagent using BSA as a standard22 and
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stored at -80 °C. The final concentration of FtsZ was determined by considering the correction
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factor for the FtsZ/BSA ratio.23 A protocol from the Quikchange site-directed mutagenesis kit
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from Stratagene was used to introduce a single point mutation in WT-FtsZ at position 191 by
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replacing the arginine residue with an alanine. Recombinant FtsZ-pET16b was used as the
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template plasmid and the primers designed for the mutation were: Forward primer:
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5'GCGAAGCGGATAACGTACTTGCCCAAGGGGTTCAAGGTATTTC3'
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Reverse primer: 5'GAAATACCTTGAACCCCTTGGGCAAGTACGTTATCCGCTTCGC3'.
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The plasmid isolated was sequenced (Xcelris, India) and the mutation was confirmed. The
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mutant protein R191A-FtsZ was purified by the procedure described above. Since the histidine
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tag of FtsZ has no discernable effect on the assembly of FtsZ,24-26 all the experiments were
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performed using histidine tag FtsZ.
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Circular dichroism spectroscopy
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The far-UV CD (200-260 nm) spectra of WT-FtsZ and R191A-FtsZ (2 µM) were monitored at
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25 °C using a cuvette of 0.1 cm path length in a JASCO J810 spectropolarimeter (Tokyo, Japan)
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in 4 mM pipes pH 6.8. The CD spectrum of buffer was subtracted from the corresponding data
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sets.9
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Light scattering assay
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Different concentrations of WT-FtsZ and R191A-FtsZ (4, 6 and 8 µM) in buffer A (25 mM
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PIPES (pH 6.8), 50 mM KCl and 5 mM MgCl2) were incubated on ice for 10 min. Then, 1 mM
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GTP was added to each of the reaction mixtures and the cuvette was immediately transferred to
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an FP-6500 JASCO fluorescence spectrophotometer (Tokyo, Japan) connected to a temperature
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controlled (37 °C) water bath. The assembly kinetics of FtsZ was monitored by 90° light
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scattering for 10 min at 500 nm. The light scattering intensity of the only buffer was also
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measured and subtracted from each of the corresponding reaction sets to obtain the corrected
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data.27 Additionally, light scattering of WT-FtsZ and R191A-FtsZ (8 µM) was also performed in
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buffer B (50 mM HEPES, pH 7.5, 50 mM KCl, 2.5 mM MgCl2 and 1 mM GTP) at 37 ºC.
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To study the effect of R191A-FtsZ on the polymerization of WT-FtsZ, WT-FtsZ (8 µM) was
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incubated with R191A-FtsZ (4, 8 and 12 µM) in the presence of buffer A on ice for 10 min. WT-
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FtsZ and R191A-FtsZ were also incubated separately in buffer A on ice for 10 min. 1 mM GTP
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was added to each reaction mixture and the polymerization kinetics was monitored as described
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earlier.
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To test the effect of R191A-FtsZ on preformed WT-FtsZ polymers, 6 µM of WT-FtsZ was
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incubated in buffer A on ice for 10 min. 1 mM GTP was added to the reaction mixture and
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polymerization was observed for 800 s. In a separate set, 6 µM of R191A-FtsZ was added to the
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same reaction mixture after 300 s of polymerization. BSA (6 µM) and buffer were added in equal
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volume at 300 s. Both BSA and buffer were used as controls.
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Sedimentation assay
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Different concentrations of WT-FtsZ and R191A-FtsZ (4, 6 and 8 µM) in buffer A were
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incubated on ice for 10 min. Then, 1 mM GTP was added to each of the reaction mixtures and
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incubated at 37 °C for 10 min. The polymeric suspension was subjected to centrifugation at
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158,000×g for 30 min at 30 °C. The pellet was dissolved in SDS-containing loading dye and run
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on 10% SDS-PAGE. Quantification of the precipitated FtsZ was done using ImageJ software.28
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Transmission electron microscopy
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8 µM of WT-FtsZ and R191A-FtsZ were polymerized in the buffer B at 37 °C for 3 min. The
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polymeric mixtures were then adsorbed onto formvar-carbon coated copper grids (300 mesh) and
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washed using deionised water, and dried at room temperature. Negative staining of the grids was
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done using 2% uranyl acetate solution. The grids were further dried and observed under a JEM
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2100 ultra HRTEM instrument at 200 kV.
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Covalent modification of R191A-FtsZ by FITC
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R191A-FtsZ was covalently modified with FITC. R191A-FtsZ (25 µM) was incubated with 125
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µM FITC for 4 h on ice. The reaction was quenched by the addition of 10 mM Tris-HCl buffer
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(pH 8.0) for 30 min on ice. Unbound FITC was removed by first passing FITC-R191A-FtsZ
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through a gel filtration (Bio-Gel P4 resin) column followed by dialysis in 25 mM PIPES (pH 6.8)
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at 4 °C for 4 h. The concentration of FtsZ bound FITC was determined by measuring absorbance
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at 495 nm and the concentration of R191A-FtsZ was monitored by Bradford method. The
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incorporation ratio of FITC per R191A-FtsZ monomer was estimated to be 0.8-0.9.
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Copolymerization assay
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FITC-R191A-FtsZ (200 nM) was polymerized without and with WT-FtsZ (8 µM) in buffer B at
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37 °C for 5 min in an eppendorf. The polymerized mixture was centrifuged at 26234×g for 30
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min at 30 °C. The pellet was resuspended in warm buffer B. Ten µL of the sample was placed on
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a glass slide (24 mm × 60 mm) and visualized by a confocal laser scanning microscope
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(Olympus, IX 81 with FV-500) at 60x magnification.
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Measurement of the GTPase activity
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The rate of GTP hydrolysis of WT-FtsZ and R191A-FtsZ was determined as described
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previously.9,29 WT-FtsZ and R191A-FtsZ (6 µM) were polymerized in the presence of 1 mM
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GTP in buffer A at 37 °C. At different (1, 3, 5, 10, 20 and 30 min) time intervals, 40 µl aliquots
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were withdrawn from the reaction mixtures and the hydrolysis reaction was quenched by the
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addition of 7 M perchloric acid (10 % v/v). The amount of inorganic phosphate released was
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determined by the standard malachite green-ammonium molybdate assay.29 The amount of
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inorganic phosphate released in the absence of FtsZ was determined at different time intervals
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and subtracted from the respective reaction sets.
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To determine the GTPase activity of each molecule of polymerized FtsZ, two sets of WT-FtsZ
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and R191A-FtsZ (4, 6 and 8 µM) in buffer A were incubated on ice for 10 min. Then, 1 mM
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GTP was added to each of the reaction mixtures and incubated at 37 °C for 10 min. One set of
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the polymeric suspension was subjected to centrifugation at 158,000×g for 30 min at 30 °C. The
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amount of sedimented FtsZ was determined by Bradford assay using BSA as a standard. The
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amount of inorganic phosphate released from the other set of polymeric suspension was
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determined using standard malachite green-ammonium molybdate assay.29
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Molecular dynamics simulation
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MD simulations were performed using GROMACS 4.6 package.30-34 We used the WT-FtsZ x-
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ray structure 2RHL for the MD simulations. To create the R191A mutant, we used the sequence
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from the WT-FtsZ structure and mutated R191 to A191 and using the wild-type structure as a
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template, we created the mutant structure using the SWISS-MODEL server.35-37 Amber99sb
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forcefield was used for the MD simulations.38,39 Each protein was placed in a dodecahedron box,
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centered, with a distance of 1 nm to the box wall from the protein to all sides and the box was
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solvated using TIP3P water.39 The salt concentration of the system was set to 150 mM. Each
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protein system was energy minimized using steepest descent algorithm to remove steric clashes
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until the largest force acting on the system was smaller than 1000 kJ/mol/nm. This was followed
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by 100 ps temperature equilibration at 298K using Berendsen thermostat and 1ns pressure
12
equilibration40,41 at 1 atm using Berendsen barostat. During both the equilibrations, position
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restraints were applied on heavy atoms using a force constant of 1000 kJ/mol/nm2. For both the
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molecular systems, three data collection production run simulations were started from the end
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structure of pressure equilibration run. In production run simulations, temperature and pressure
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were maintained at 298K and 1 atm with v-rescale thermostat and Parinello-Rahman barostat
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with τt and τp of 1 ps and 2 ps. All bonds were constrained using parallel LINCS algorithm42 and
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all angles containing hydrogen atoms were constrained using the virtual sites implementation of
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the GROMACS package to enable us a use of 4 fs time step. A cut-off of 1.4 nm and 1 nm was
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used for short range Lennard-Jones and Coulomb interaction. Particle-mesh-Ewald method43
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with a spacing of 0.12 nm was used for long-range electrostatic interactions. Each production run
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trajectory was simulated for 500 ns and structures were written every 40 ps. A total of 1.5 µs
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data was collected each for R191A-FtsZ and WT-FtsZ separately.
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Cloning of WT ftsZ and MT R191A ftsZ in B. subtilis expression vector pHT01
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Two different constructs of pHT01 containing WT ftsZ and R191A ftsZ were prepared. The
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pET16b containing WT ftsZ and R191A ftsZ were used as templates for polymerase chain
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reaction. Restriction sites for BamHI and SmaI were incorporated into the forward
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(CGCGGATCCATGTTGGAGTTCGAAACA) and reverse
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(TCCCCCGGGTTAGCCGCGTTTATTACG) primers, respectively. BamHI and SmaI treated
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PCR amplicons and pHT01 vector were ligated and transformed into DH-5 α E.coli cells. The
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pHT01-WT ftsZ and pHT01-R191A ftsZ clones were selected on Ampicillin-LA plates and
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confirmed by DNA sequencing. The correct clones were then transformed into B. subtilis
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PL2084 competent cells (a kind gift from Dr. Levin P).
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Transformation of recombinant plasmids (pHT01-WT ftsZ and pHT01-R191A ftsZ) into B.
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subtilis strain PL2084
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Cloning of WT ftsZ and R191A ftsZ was done in B. subtilis expression vector pHT01. The
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competent cells of B. subtilis strain PL2084 were prepared. This strain is resistant to
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spectinomycin. This strain has the native ftsz under xylose inducible promoter at the thrC locus.
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This strain grows normally in the presence of xylose, which regulates the expression of
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chromosomal FtsZ.44 The recombinant plasmids (pHT01-WT ftsZ and pHT01-R191A ftsZ) were
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transformed into competent B. subtilis PL2084 cells and the transformants were selected on
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chloramphenicol plates. In the end, two separate recombinants B. subtilis strains PL2084 were
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obtained; one strain had pHT01-WT ftsZ and other strain had pHT01-R191A ftsZ. The
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expression level of WT ftsz and R191A ftsz from pHT01 was also estimated using IPTG in both
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recombinant B. subtilis strains PL2084 using SDS-PAGE. The gel analysis showed expression
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level of R191A-FtsZ was similar to WT-FtsZ (data not shown).
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Effects of the expression of R191A-FtsZ and WT-FtsZ on the length of B. subtilis cells-
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The recombinant B. subtilis strains PL2084 (having pHT01-WT ftsZ and pHT01-R191A ftsZ)
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were grown at 37 °C in LB media containing 15 µg/ml spectinomycin, 5 µg/ml chloramphenicol
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and 0.5% xylose for over-night. The B. subtilis strain PL2084 in LB media containing 15µg/ml
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spectinomycin and 0.5% xylose was grown under similar conditions as a control. The cells were
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harvested, washed three times and resuspended in LB media. The individual culture was
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processed in following way –
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B. subtilis strain PL2084 – LB tubes (containing 15µg/ml spectinomycin) were inoculated with
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B. Subtilis PL2084 cells (OD600 = 0.1), grown in the absence and presence of 0.5% xylose or
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0.5% xylose + 0.1 mM IPTG respectively.
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Recombinant B. subtilis strains PL2084 (having pHT01-WT ftsZ and pHT01-R191A ftsZ )-
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Different FtsZ constructs were inoculated in LB tubes (containing 15 µg/ml spectinomycin and 5
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µg/ml chloramphenicol) and grown in the absence and presence of 0.1 mM IPTG. IPTG is the
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inducer for the expression of the gene cloned in a pHT01 plasmid. The cultures were grown at 37
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°C for 4 h. The cells were harvested, washed three times with PBS and resuspended in PBS, pH
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7.4. The fixing of cells was performed using 2.8% formaldehyde and 0.04% glutaraldehyde. The
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cells were collected by centrifugation, washed three times with PBS, resuspended in PBS and the
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cell morphology was analyzed using a differential interference contrast (DIC) microscope.
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Biochemistry
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Image-Pro Plus software was used to measure the cell length (Media Cybernetics, Silver Spring,
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MD, U.S.A.).
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Visualization of the Z-rings in B. subtilis cells -
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Both recombinant strains PL2084 (pHT01-WT ftsZ and pHT01-R191A ftsZ) were grown at 37
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°C in LB media containing 15 µg/ml spectinomycin, 5 µg/ml chloramphenicol and 0.5% xylose
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for over-night. The cells were harvested, washed 3 times, suspended in LB media containing 0.1
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mM IPTG and then, grown for 4 h at 37 °C. The cells were processed as described earlier.45 The
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cytoplasmic FtsZ was stained with a polyclonal FtsZ antibody (1:50), developed in a rabbit. A
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Cy3-conjugated goat anti-rabbit antibody was used as the secondary antibody (1:200). A
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confocal laser scanning microscope (Olympus, IX 81 with FV-500) was used for capturing the
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images.
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Results
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Identification of R191 residue on FtsZ
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A docking analysis of FtsZ-targeted antibacterial agents plumbagin and SB-RA-2001, led to the
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identification of R191 residue as a potential interaction site for anti-FtsZ agents.18,19 An analysis
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of the location of R191 residue on FtsZ showed that the residue was positioned in the lower half
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of the H7 helix on FtsZ. R191 residue on FtsZ was found to be involved in hydrogen bond
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interactions with both plumbagin and SB-RA-2001(Fig. 1B). To investigate the importance of
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this arginine residue in the assembly of FtsZ, the residue was mutated to alanine (R191A). First,
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the binding affinity of plumbagin to R191A-FtsZ and WT-FtsZ was determined as described
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previously18. Plumbagin bound to WT-FtsZ and R191A-FtsZ with a dissociation constant of 6 ±
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4 µM and 23 ± 5 µM, respectively, suggesting that the binding of plumbagin to R191A-FtsZ is
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Biochemistry
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~4 times weaker than WT-FtsZ (Fig. S1). In addition, plumbagin (20 and 40 µM) did not
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significantly inhibit the assembly of R191A-FtsZ (Fig. S2) indicating the importance of R191
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residue in the binding of plumbagin to FtsZ. Further, the effect of R191A mutation on the
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secondary structure of FtsZ was analyzed by far-UV CD spectroscopy and molecular dynamics
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simulations. The far-UV CD spectra of WT-FtsZ and R191A-FtsZ revealed that the secondary
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structure of R191A-FtsZ was unchanged from that of WT-FtsZ (Fig. 3). Additionally, MD
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simulations, based on DSSP analysis46,47 of WT-FtsZ and R191A-FtsZ also showed similar
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secondary structures indicating that the R191A mutation did not have any effect on the
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local/global secondary structures of FtsZ (Fig. S3).
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R191A-FtsZ showed weaker polymerization ability as compared to WT-FtsZ
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The polymerization ability of R191A-FtsZ was remarkably lower than WT-FtsZ (Fig. 4). The
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light scattering traces of the R191A-FtsZ and WT-FtsZ revealed that the assembly of WT-FtsZ
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increased in a concentration-dependent manner while R191A-FtsZ did not polymerize efficiently
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(Fig. 4A). R191A-FtsZ displayed weak light scattering signals under similar conditions (Fig.
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4A). The assembly of WT-FtsZ and R191A-FtsZ was also monitored by collecting FtsZ
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polymers through high-speed centrifugation. The sedimentation assay was performed at
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158,000×g to ensure the sedimentation of small polymers of FtsZ and R191A-FtsZ (Fig. 4B).
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The amount of FtsZ pelleted was much higher for WT-FtsZ than R191A-FtsZ (Fig. 4B). The
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polymerization kinetics of WT-FtsZ and R191A-FtsZ was also examined in HEPES buffer pH
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7.5 (Fig. 4C). The light scattering traces suggested that R191A-FtsZ polymerizes weakly than
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WT-FtsZ (Fig. 4C). The results suggested that the polymerization ability of FtsZ was strongly
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reduced after the replacement of the arginine residue by an alanine residue.
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Biochemistry
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R191A-FtsZ polymers exhibited a different morphology than WT-FtsZ polymers
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Electron microscopic studies revealed that the polymer morphology of R191A-FtsZ was
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distinctly different from that of WT-FtsZ. Interestingly, the R191A polymers exhibited short
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protofilaments as compared to the long filamentous polymers of WT-FtsZ (Fig. 4D). The
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difference in polymerization pattern of wild-type and mutant FtsZ may be attributed to the
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mutation in FtsZ.
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R191A-FtsZ inhibited the assembly of WT-FtsZ
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The effect of R191A-FtsZ on the assembly of WT-FtsZ was also analyzed using 90° light
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scattering. R191A-FtsZ inhibited the rate and extent of the assembly of WT-FtsZ in a
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concentration-dependent manner (Fig. 5A). The extent of polymerization of WT-FtsZ was
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reduced by 23 ± 3, 48 ± 5% and 74 ± 10% in the presence of 4, 8 and 12 µM of R191A-FtsZ,
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respectively.
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R191A-FtsZ disassembled preformed polymers of WT-FtsZ
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To monitor the effect of R191A-FtsZ on the preformed polymers of WT-FtsZ, R191A-FtsZ was
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added to the assembly suspension after the assembly of WT-FtsZ reached a plateau level. The
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light scattering signal of WT-FtsZ polymers diminished continuously in the presence of R191A-
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FtsZ. BSA and buffer were taken as controls. Initially, the light scattering signal of WT-FtsZ
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polymers were reduced probably due to the mechanical perturbation but subsequently the
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polymerization signal achieved a similar level as that of the control (Fig. 5B). The results
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indicated that R191A-FtsZ was able to depolymerize preformed FtsZ polymers.
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FITC-R191A-FtsZ copolymerized with WT-FtsZ
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To determine the mode of binding of R191A-FtsZ on WT-FtsZ polymers, a copolymerization
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experiment was performed using unlabelled WT-FtsZ (8 µM) and FITC-R191A-FtsZ (200 nM).
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FITC-R191A-FtsZ was found to decorate WT-FtsZ filaments while speckles were observed
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when only FITC-R191A-FtsZ was incubated. The results suggested that R19A-FtsZ interacted
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with WT-FtsZ and copolymerized to form polymers (Fig. 6).
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The GTPase activity of polymerized R191A-FtsZ was similar to that of WT-FtsZ
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FtsZ is a GTPase and the GTP hydrolysis is known to modulate the assembly dynamics of
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FtsZ48,49. R191A-FtsZ exhibited much lesser GTPase activity as compared to WT-FtsZ. For
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example, the amount of inorganic phosphate released after 30 min of the assembly of 6 µM WT-
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FtsZ and R191A-FtsZ was 29.4 ± 1.0 and 7.1 ± 0.5, moles of Pi, respectively. Further, the rate of
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GTP hydrolysis of WT-FtsZ and R191A-FtsZ was estimated to be 1.2 ± 0.2 and 0.3 ± 0.04 moles
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of Pi/mole of FtsZ/min, respectively (Fig. 7A). The reduction of the GTPase activity of FtsZ
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could either be due to the reduction in the amount of polymerized FtsZ or due to the intrinsic
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inability of the R191A-FtsZ to hydrolyze GTP. To distinguish between the possibilities, the
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GTPase activity was expressed with respect to the amount of polymerized FtsZ (Fig. 7B). The
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GTPase activity of polymerized WT-FtsZ and R191A-FtsZ was found to be similar (Fig. 7B).
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Therefore, the reduced GTPase activity of R191A-FtsZ could be attributed to the inability of the
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mutant to polymerize efficiently rather than a reduction in the GTPase activity of FtsZ.
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R191A induces conformational change in the active site of FtsZ
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The stability of WT-FtsZ and R191A-FtsZ structures were monitored by MD simulations. The
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average root mean square deviation of WT-FtsZ and R191A-FtsZ with respect to the X-ray
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structure backbone of less than
