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Structures of actin-like ParM filaments show architecture of plasmid-segregating spindles.

Bharat TA, Murshudov GN, Sachse C, Löwe J - Nature (2015)

Bottom Line: Growing ParMRC spindles push sister plasmids to the cell poles.The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions.Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.

View Article: PubMed Central - PubMed

Affiliation: Structural Studies Division, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.

ABSTRACT
Active segregation of Escherichia coli low-copy-number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments. ParR links the filaments with centromeric parC plasmid DNA, while facilitating the addition of subunits to ParM filaments. Growing ParMRC spindles push sister plasmids to the cell poles. Here, using modern electron cryomicroscopy methods, we investigate the structures and arrangements of ParM filaments in vitro and in cells, revealing at near-atomic resolution how subunits and filaments come together to produce the simplest known mitotic machinery. To understand the mechanism of dynamic instability, we determine structures of ParM filaments in different nucleotide states. The structure of filaments bound to the ATP analogue AMPPNP is determined at 4.3 Å resolution and refined. The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions. Also using electron cryomicroscopy, we reconstruct ParM doublets forming antiparallel spindles. Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.

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Related in: MedlinePlus

The ParM inter-protofilament interface is small but important(a) Cryo-EM density for the ParM+AMPPNP filament is shown at an isosurface contour level of 2.0 σ from the mean value. Overlaid on the density, refined atomic coordinates from REFMAC are additionally displayed as grey ribbons. Residues forming salt bridges at the inter-protofilament interface are highlighted. (b) The same figure as a), except the cryo-EM density shown at an isosurface contour level of 1.5 σ from the mean. (c) 1.0 σ from the mean. (d-f) A magnified view of the primary salt-bridged interface consisting of charged residues that form the ParM inter-protofilament interface. The cryo-EM density is shown as a mesh at three different contour levels to demonstrate resolved side chain densities. Positively charged residues are highlighted in red while negatively charged residues are highlighted in orange. (g) Two residues (K258 and R262) that were the best resolved (marked with an * in d), were mutated to aspartic acid to test the importance of this inter-protofilament interface. A cryo-EM image of this mutant protein assembled with AMPPNP is shown. A much higher concentration of the protein was required to obtain filaments on cryo-EM grids (Supplementary Methods). This experiment was repeated four times. (h) Randomly selected cryo-EM images of ParM+AMPPNP and ParM(K258D, R262D)+AMPPNP were used to count occurrences of straight and bent filaments by visual inspection. The results of this quantification are shown as a percentage bar diagram. For the ParM protein, 82% of all filaments were classified as straight, while 18% were bent (n=345). Using exactly the same classification criteria, only 15% of the filaments were found to be straight and 85% of the filaments were bent (n=45) for the ParM(K258D, R262D) mutant protein. (i) Reference-free class averages show that most of the ParM(K258D, R262D) filaments are made up of double protofilaments like wild-type ParM. Some class averages show evidence of bending. A few class averages show that single protofilaments were present in the sample (lower panels). However, the double mutation destabilises the entire ParM filament, making filament formation an unfavourable reaction, illustrating that even though the inter-protofilament interface is small, it is critical for ParM filament formation.
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Figure 7: The ParM inter-protofilament interface is small but important(a) Cryo-EM density for the ParM+AMPPNP filament is shown at an isosurface contour level of 2.0 σ from the mean value. Overlaid on the density, refined atomic coordinates from REFMAC are additionally displayed as grey ribbons. Residues forming salt bridges at the inter-protofilament interface are highlighted. (b) The same figure as a), except the cryo-EM density shown at an isosurface contour level of 1.5 σ from the mean. (c) 1.0 σ from the mean. (d-f) A magnified view of the primary salt-bridged interface consisting of charged residues that form the ParM inter-protofilament interface. The cryo-EM density is shown as a mesh at three different contour levels to demonstrate resolved side chain densities. Positively charged residues are highlighted in red while negatively charged residues are highlighted in orange. (g) Two residues (K258 and R262) that were the best resolved (marked with an * in d), were mutated to aspartic acid to test the importance of this inter-protofilament interface. A cryo-EM image of this mutant protein assembled with AMPPNP is shown. A much higher concentration of the protein was required to obtain filaments on cryo-EM grids (Supplementary Methods). This experiment was repeated four times. (h) Randomly selected cryo-EM images of ParM+AMPPNP and ParM(K258D, R262D)+AMPPNP were used to count occurrences of straight and bent filaments by visual inspection. The results of this quantification are shown as a percentage bar diagram. For the ParM protein, 82% of all filaments were classified as straight, while 18% were bent (n=345). Using exactly the same classification criteria, only 15% of the filaments were found to be straight and 85% of the filaments were bent (n=45) for the ParM(K258D, R262D) mutant protein. (i) Reference-free class averages show that most of the ParM(K258D, R262D) filaments are made up of double protofilaments like wild-type ParM. Some class averages show evidence of bending. A few class averages show that single protofilaments were present in the sample (lower panels). However, the double mutation destabilises the entire ParM filament, making filament formation an unfavourable reaction, illustrating that even though the inter-protofilament interface is small, it is critical for ParM filament formation.

Mentions: Surprisingly, the two protofilaments (strands) making up the double-helical ParM filament are held together only by salt bridges (Fig. 2a-b, ED Fig. 2-3 and ED Table 2). The ParM inter-protofilament interface is small (calculated interface area 371 Å2) and does not resemble a canonical protein-protein interface containing a hydrophobic core. To demonstrate the validity of this assessment we mutated two positively charged residues within the inter-protofilament interface to aspartic acids (K258D, R262D) and tested what effect this has on the stability of ParM filaments. Filament formation (with AMPPNP) from the resulting mutant protein ParM (K258D, R262D) was inefficient (ED Fig. 3g). The few filaments that were formed were unstable, and tended to be bent (Fig. 2c, S3h). Reference-free class averaging of these filaments showed that even though the majority of the few observed filaments were double helical like wild-type ParM, some single-helical filaments were also present (Fig. 2d, S3i). These observations indicate that although the interface between protofilaments in ParM is surprisingly small, it is sufficient for double filament assembly since many identical contacts along the filament contribute to the overall binding energy. Different actin-like proteins show very different filament arrangements, from single (crenactin, possibly 11) to parallel double helical (left-handed: ParM, right-handed: actin and non-staggered: MamK 12) and antiparallel, double straight (MreB). We propose that small and simple inter-protofilament contacts could have made it possible to change inter-protofilament arrangements relatively easily during evolution since all these actin-like filaments show similar longitudinal contacts 13.


Structures of actin-like ParM filaments show architecture of plasmid-segregating spindles.

Bharat TA, Murshudov GN, Sachse C, Löwe J - Nature (2015)

The ParM inter-protofilament interface is small but important(a) Cryo-EM density for the ParM+AMPPNP filament is shown at an isosurface contour level of 2.0 σ from the mean value. Overlaid on the density, refined atomic coordinates from REFMAC are additionally displayed as grey ribbons. Residues forming salt bridges at the inter-protofilament interface are highlighted. (b) The same figure as a), except the cryo-EM density shown at an isosurface contour level of 1.5 σ from the mean. (c) 1.0 σ from the mean. (d-f) A magnified view of the primary salt-bridged interface consisting of charged residues that form the ParM inter-protofilament interface. The cryo-EM density is shown as a mesh at three different contour levels to demonstrate resolved side chain densities. Positively charged residues are highlighted in red while negatively charged residues are highlighted in orange. (g) Two residues (K258 and R262) that were the best resolved (marked with an * in d), were mutated to aspartic acid to test the importance of this inter-protofilament interface. A cryo-EM image of this mutant protein assembled with AMPPNP is shown. A much higher concentration of the protein was required to obtain filaments on cryo-EM grids (Supplementary Methods). This experiment was repeated four times. (h) Randomly selected cryo-EM images of ParM+AMPPNP and ParM(K258D, R262D)+AMPPNP were used to count occurrences of straight and bent filaments by visual inspection. The results of this quantification are shown as a percentage bar diagram. For the ParM protein, 82% of all filaments were classified as straight, while 18% were bent (n=345). Using exactly the same classification criteria, only 15% of the filaments were found to be straight and 85% of the filaments were bent (n=45) for the ParM(K258D, R262D) mutant protein. (i) Reference-free class averages show that most of the ParM(K258D, R262D) filaments are made up of double protofilaments like wild-type ParM. Some class averages show evidence of bending. A few class averages show that single protofilaments were present in the sample (lower panels). However, the double mutation destabilises the entire ParM filament, making filament formation an unfavourable reaction, illustrating that even though the inter-protofilament interface is small, it is critical for ParM filament formation.
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Related In: Results  -  Collection

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Figure 7: The ParM inter-protofilament interface is small but important(a) Cryo-EM density for the ParM+AMPPNP filament is shown at an isosurface contour level of 2.0 σ from the mean value. Overlaid on the density, refined atomic coordinates from REFMAC are additionally displayed as grey ribbons. Residues forming salt bridges at the inter-protofilament interface are highlighted. (b) The same figure as a), except the cryo-EM density shown at an isosurface contour level of 1.5 σ from the mean. (c) 1.0 σ from the mean. (d-f) A magnified view of the primary salt-bridged interface consisting of charged residues that form the ParM inter-protofilament interface. The cryo-EM density is shown as a mesh at three different contour levels to demonstrate resolved side chain densities. Positively charged residues are highlighted in red while negatively charged residues are highlighted in orange. (g) Two residues (K258 and R262) that were the best resolved (marked with an * in d), were mutated to aspartic acid to test the importance of this inter-protofilament interface. A cryo-EM image of this mutant protein assembled with AMPPNP is shown. A much higher concentration of the protein was required to obtain filaments on cryo-EM grids (Supplementary Methods). This experiment was repeated four times. (h) Randomly selected cryo-EM images of ParM+AMPPNP and ParM(K258D, R262D)+AMPPNP were used to count occurrences of straight and bent filaments by visual inspection. The results of this quantification are shown as a percentage bar diagram. For the ParM protein, 82% of all filaments were classified as straight, while 18% were bent (n=345). Using exactly the same classification criteria, only 15% of the filaments were found to be straight and 85% of the filaments were bent (n=45) for the ParM(K258D, R262D) mutant protein. (i) Reference-free class averages show that most of the ParM(K258D, R262D) filaments are made up of double protofilaments like wild-type ParM. Some class averages show evidence of bending. A few class averages show that single protofilaments were present in the sample (lower panels). However, the double mutation destabilises the entire ParM filament, making filament formation an unfavourable reaction, illustrating that even though the inter-protofilament interface is small, it is critical for ParM filament formation.
Mentions: Surprisingly, the two protofilaments (strands) making up the double-helical ParM filament are held together only by salt bridges (Fig. 2a-b, ED Fig. 2-3 and ED Table 2). The ParM inter-protofilament interface is small (calculated interface area 371 Å2) and does not resemble a canonical protein-protein interface containing a hydrophobic core. To demonstrate the validity of this assessment we mutated two positively charged residues within the inter-protofilament interface to aspartic acids (K258D, R262D) and tested what effect this has on the stability of ParM filaments. Filament formation (with AMPPNP) from the resulting mutant protein ParM (K258D, R262D) was inefficient (ED Fig. 3g). The few filaments that were formed were unstable, and tended to be bent (Fig. 2c, S3h). Reference-free class averaging of these filaments showed that even though the majority of the few observed filaments were double helical like wild-type ParM, some single-helical filaments were also present (Fig. 2d, S3i). These observations indicate that although the interface between protofilaments in ParM is surprisingly small, it is sufficient for double filament assembly since many identical contacts along the filament contribute to the overall binding energy. Different actin-like proteins show very different filament arrangements, from single (crenactin, possibly 11) to parallel double helical (left-handed: ParM, right-handed: actin and non-staggered: MamK 12) and antiparallel, double straight (MreB). We propose that small and simple inter-protofilament contacts could have made it possible to change inter-protofilament arrangements relatively easily during evolution since all these actin-like filaments show similar longitudinal contacts 13.

Bottom Line: Growing ParMRC spindles push sister plasmids to the cell poles.The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions.Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.

View Article: PubMed Central - PubMed

Affiliation: Structural Studies Division, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.

ABSTRACT
Active segregation of Escherichia coli low-copy-number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments. ParR links the filaments with centromeric parC plasmid DNA, while facilitating the addition of subunits to ParM filaments. Growing ParMRC spindles push sister plasmids to the cell poles. Here, using modern electron cryomicroscopy methods, we investigate the structures and arrangements of ParM filaments in vitro and in cells, revealing at near-atomic resolution how subunits and filaments come together to produce the simplest known mitotic machinery. To understand the mechanism of dynamic instability, we determine structures of ParM filaments in different nucleotide states. The structure of filaments bound to the ATP analogue AMPPNP is determined at 4.3 Å resolution and refined. The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions. Also using electron cryomicroscopy, we reconstruct ParM doublets forming antiparallel spindles. Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.

Show MeSH
Related in: MedlinePlus