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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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Intra- and inter-protofilament interactions in ParM filaments(a) Atomic model of one protofilament (strand) of ParM is shown with the residues at the protein:protein interface highlighted in red. See ED Table 2 for a detailed list. (b) A magnified view of the interface. Three residues at the interface have been labelled. (c) The complete ParM filament (i.e. both protofilaments/strands) shown end-on. (d) Atomic model of the ParM filament with the inter-protofilament residues at the protein:protein interface highlighted in orange. (e) A magnified view of d). Salt bridging residues are labelled. (f) An orthogonal view of d). See ED Table 2 for a detailed list of interacting residues.
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Figure 6: Intra- and inter-protofilament interactions in ParM filaments(a) Atomic model of one protofilament (strand) of ParM is shown with the residues at the protein:protein interface highlighted in red. See ED Table 2 for a detailed list. (b) A magnified view of the interface. Three residues at the interface have been labelled. (c) The complete ParM filament (i.e. both protofilaments/strands) shown end-on. (d) Atomic model of the ParM filament with the inter-protofilament residues at the protein:protein interface highlighted in orange. (e) A magnified view of d). Salt bridging residues are labelled. (f) An orthogonal view of d). See ED Table 2 for a detailed list of interacting residues.

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)

Intra- and inter-protofilament interactions in ParM filaments(a) Atomic model of one protofilament (strand) of ParM is shown with the residues at the protein:protein interface highlighted in red. See ED Table 2 for a detailed list. (b) A magnified view of the interface. Three residues at the interface have been labelled. (c) The complete ParM filament (i.e. both protofilaments/strands) shown end-on. (d) Atomic model of the ParM filament with the inter-protofilament residues at the protein:protein interface highlighted in orange. (e) A magnified view of d). Salt bridging residues are labelled. (f) An orthogonal view of d). See ED Table 2 for a detailed list of interacting residues.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493928&req=5

Figure 6: Intra- and inter-protofilament interactions in ParM filaments(a) Atomic model of one protofilament (strand) of ParM is shown with the residues at the protein:protein interface highlighted in red. See ED Table 2 for a detailed list. (b) A magnified view of the interface. Three residues at the interface have been labelled. (c) The complete ParM filament (i.e. both protofilaments/strands) shown end-on. (d) Atomic model of the ParM filament with the inter-protofilament residues at the protein:protein interface highlighted in orange. (e) A magnified view of d). Salt bridging residues are labelled. (f) An orthogonal view of d). See ED Table 2 for a detailed list of interacting residues.
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