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Screening for FtsZ Dimerization Inhibitors Using Fluorescence Cross-Correlation Spectroscopy and Surface Resonance Plasmon Analysis.

Mikuni S, Kodama K, Sasaki A, Kohira N, Maki H, Munetomo M, Maenaka K, Kinjo M - PLoS ONE (2015)

Bottom Line: Subsequently, in the second round of screening by FCCS, 71 candidates were also chosen from 888 chemicals selected via an in silico structural similarity search of the chemicals screened in the first round of screening.Moreover, the dissociation constants between the highest inhibitory chemicals and Staphylococcus aureus FtsZ were determined by SPR.Finally, by measuring the minimum inhibitory concentration, it was confirmed that the screened chemical had antibacterial activity against Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus (MRSA).

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.

ABSTRACT
FtsZ is an attractive target for antibiotic research because it is an essential bacterial cell division protein that polymerizes in a GTP-dependent manner. To find the seed chemical structure, we established a high-throughput, quantitative screening method combining fluorescence cross-correlation spectroscopy (FCCS) and surface plasmon resonance (SPR). As a new concept for the application of FCCS to polymerization-prone protein, Staphylococcus aureus FtsZ was fragmented into the N-terminal and C-terminal, which were fused with GFP and mCherry (red fluorescent protein), respectively. By this fragmentation, the GTP-dependent head-to-tail dimerization of each fluorescent labeled fragment of FtsZ could be observed, and the inhibitory processes of chemicals could be monitored by FCCS. In the first round of screening by FCCS, 28 candidates were quantitatively and statistically selected from 495 chemicals determined by in silico screening. Subsequently, in the second round of screening by FCCS, 71 candidates were also chosen from 888 chemicals selected via an in silico structural similarity search of the chemicals screened in the first round of screening. Moreover, the dissociation constants between the highest inhibitory chemicals and Staphylococcus aureus FtsZ were determined by SPR. Finally, by measuring the minimum inhibitory concentration, it was confirmed that the screened chemical had antibacterial activity against Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus (MRSA).

No MeSH data available.


Related in: MedlinePlus

The structure and fragmentation of FtsZ.A. The crystal structure of the S. aureus FtsZ monomer bound to GTP-γS (Protein Data Bank: 3WGN). B. FtsZ polymerizes into tubulin-like protofilaments by head-to-tail association with GTP. C. FtsZ is shown schematically. The black arrowhead indicates the α-helix in the middle of FtsZ as shown in Fig 1A. D. For FCCS, FtsZ protein was fragmented. The N- and C-terminal FtsZ were fused with EGFP and mCherry (red fluorescent protein), respectively. The mutation at K175D in the N-terminal fragment was inserted to prevent further bundling FtsZ. E. To avoid interaction independent of GTP addition, the α-helix was retained in both the N- and C-terminal fragments (black arrowheads in A and C) in the middle of FtsZ structure. F. By binding to GTP, FtsZK175D_N-terminal-EGFP and FtsZ_C-terminal-mCherry could be dimerized in a head-to-tail manner.
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pone.0130933.g001: The structure and fragmentation of FtsZ.A. The crystal structure of the S. aureus FtsZ monomer bound to GTP-γS (Protein Data Bank: 3WGN). B. FtsZ polymerizes into tubulin-like protofilaments by head-to-tail association with GTP. C. FtsZ is shown schematically. The black arrowhead indicates the α-helix in the middle of FtsZ as shown in Fig 1A. D. For FCCS, FtsZ protein was fragmented. The N- and C-terminal FtsZ were fused with EGFP and mCherry (red fluorescent protein), respectively. The mutation at K175D in the N-terminal fragment was inserted to prevent further bundling FtsZ. E. To avoid interaction independent of GTP addition, the α-helix was retained in both the N- and C-terminal fragments (black arrowheads in A and C) in the middle of FtsZ structure. F. By binding to GTP, FtsZK175D_N-terminal-EGFP and FtsZ_C-terminal-mCherry could be dimerized in a head-to-tail manner.

Mentions: Cytokinesis in bacteria is achieved via protein assembly initiated by polymerization of the tubulin homologue filamenting temperature-sensitive mutant Z (FtsZ, Fig 1A) into the Z-ring, a ring-like structure that lies close to the cytoplasmic membrane at the prospective division site [1–3]. By binding to GTP, FtsZ polymerizes into tubulin-like protofilaments in head-to-tail association of individual units consisting of the C-terminal domain and N-terminal GTPase activation domain (Fig 1B) [4, 5]. Therefore, FtsZ could be a target for new antibiotics because it is the key protein of bacterial cell division. Chemical screening has been performed by filter-trapping [6] and monitoring the turbidity [7–11] and viability of bacteria [12–15] to evaluate the polymerization activity of FtsZ. One of the earliest-identified and well-investigated antibacterial agents against S. aureus is PC190723 [16], many derivatives of which have been synthesized to improve its antibacterial activity [17–20]. Moreover, the antibacterial mechanism of PC190723 functions via impairment of the recycling of FtsZ because the polymer of FtsZ is stabilized by PC190723 [21, 22]. However, it is important to develop new antibiotics from the viewpoint of destabilizing the polymer of FtsZ.


Screening for FtsZ Dimerization Inhibitors Using Fluorescence Cross-Correlation Spectroscopy and Surface Resonance Plasmon Analysis.

Mikuni S, Kodama K, Sasaki A, Kohira N, Maki H, Munetomo M, Maenaka K, Kinjo M - PLoS ONE (2015)

The structure and fragmentation of FtsZ.A. The crystal structure of the S. aureus FtsZ monomer bound to GTP-γS (Protein Data Bank: 3WGN). B. FtsZ polymerizes into tubulin-like protofilaments by head-to-tail association with GTP. C. FtsZ is shown schematically. The black arrowhead indicates the α-helix in the middle of FtsZ as shown in Fig 1A. D. For FCCS, FtsZ protein was fragmented. The N- and C-terminal FtsZ were fused with EGFP and mCherry (red fluorescent protein), respectively. The mutation at K175D in the N-terminal fragment was inserted to prevent further bundling FtsZ. E. To avoid interaction independent of GTP addition, the α-helix was retained in both the N- and C-terminal fragments (black arrowheads in A and C) in the middle of FtsZ structure. F. By binding to GTP, FtsZK175D_N-terminal-EGFP and FtsZ_C-terminal-mCherry could be dimerized in a head-to-tail manner.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4496089&req=5

pone.0130933.g001: The structure and fragmentation of FtsZ.A. The crystal structure of the S. aureus FtsZ monomer bound to GTP-γS (Protein Data Bank: 3WGN). B. FtsZ polymerizes into tubulin-like protofilaments by head-to-tail association with GTP. C. FtsZ is shown schematically. The black arrowhead indicates the α-helix in the middle of FtsZ as shown in Fig 1A. D. For FCCS, FtsZ protein was fragmented. The N- and C-terminal FtsZ were fused with EGFP and mCherry (red fluorescent protein), respectively. The mutation at K175D in the N-terminal fragment was inserted to prevent further bundling FtsZ. E. To avoid interaction independent of GTP addition, the α-helix was retained in both the N- and C-terminal fragments (black arrowheads in A and C) in the middle of FtsZ structure. F. By binding to GTP, FtsZK175D_N-terminal-EGFP and FtsZ_C-terminal-mCherry could be dimerized in a head-to-tail manner.
Mentions: Cytokinesis in bacteria is achieved via protein assembly initiated by polymerization of the tubulin homologue filamenting temperature-sensitive mutant Z (FtsZ, Fig 1A) into the Z-ring, a ring-like structure that lies close to the cytoplasmic membrane at the prospective division site [1–3]. By binding to GTP, FtsZ polymerizes into tubulin-like protofilaments in head-to-tail association of individual units consisting of the C-terminal domain and N-terminal GTPase activation domain (Fig 1B) [4, 5]. Therefore, FtsZ could be a target for new antibiotics because it is the key protein of bacterial cell division. Chemical screening has been performed by filter-trapping [6] and monitoring the turbidity [7–11] and viability of bacteria [12–15] to evaluate the polymerization activity of FtsZ. One of the earliest-identified and well-investigated antibacterial agents against S. aureus is PC190723 [16], many derivatives of which have been synthesized to improve its antibacterial activity [17–20]. Moreover, the antibacterial mechanism of PC190723 functions via impairment of the recycling of FtsZ because the polymer of FtsZ is stabilized by PC190723 [21, 22]. However, it is important to develop new antibiotics from the viewpoint of destabilizing the polymer of FtsZ.

Bottom Line: Subsequently, in the second round of screening by FCCS, 71 candidates were also chosen from 888 chemicals selected via an in silico structural similarity search of the chemicals screened in the first round of screening.Moreover, the dissociation constants between the highest inhibitory chemicals and Staphylococcus aureus FtsZ were determined by SPR.Finally, by measuring the minimum inhibitory concentration, it was confirmed that the screened chemical had antibacterial activity against Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus (MRSA).

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.

ABSTRACT
FtsZ is an attractive target for antibiotic research because it is an essential bacterial cell division protein that polymerizes in a GTP-dependent manner. To find the seed chemical structure, we established a high-throughput, quantitative screening method combining fluorescence cross-correlation spectroscopy (FCCS) and surface plasmon resonance (SPR). As a new concept for the application of FCCS to polymerization-prone protein, Staphylococcus aureus FtsZ was fragmented into the N-terminal and C-terminal, which were fused with GFP and mCherry (red fluorescent protein), respectively. By this fragmentation, the GTP-dependent head-to-tail dimerization of each fluorescent labeled fragment of FtsZ could be observed, and the inhibitory processes of chemicals could be monitored by FCCS. In the first round of screening by FCCS, 28 candidates were quantitatively and statistically selected from 495 chemicals determined by in silico screening. Subsequently, in the second round of screening by FCCS, 71 candidates were also chosen from 888 chemicals selected via an in silico structural similarity search of the chemicals screened in the first round of screening. Moreover, the dissociation constants between the highest inhibitory chemicals and Staphylococcus aureus FtsZ were determined by SPR. Finally, by measuring the minimum inhibitory concentration, it was confirmed that the screened chemical had antibacterial activity against Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus (MRSA).

No MeSH data available.


Related in: MedlinePlus